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Frontiers in Endocrinology
1
March 2020 | Vitamin D Binding Protein
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ISSN 1664-8714
ISBN 978-2-88963-578-8
DOI 10.3389/978-2-88963-578-8
Frontiers in Endocrinology
2
March 2020 | Vitamin D Binding Protein
VITAMIN D BINDING PROTEIN, TOTAL AND
FREE VITAMIN D LEVELS IN DIFFERENT
PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL
CONDITIONS
Topic Editors:
Daniel David Bikle, University of California, San Francisco, United States
Zhongjian Xie, Central South University, China
Xiangbing Wang, Rutgers, The State University of New Jersey, United States
Citation: Bikle, D. D., Xie, Z., Wang, X., eds. (2020). Vitamin D Binding Protein,
Total and Free Vitamin D Levels in Dierent Physiological and Pathophysiological
Conditions. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-578-8
Frontiers in Endocrinology
3
March 2020 | Vitamin D Binding Protein
04 Editorial: Vitamin D Binding Protein, Total and Free Vitamin D Levels in
Different Physiological and Pathophysiological Conditions
Zhongjian Xie, Xiangbing Wang and Daniel D. Bikle
07 Association of Vitamin D Metabolites With Embryo Development and
Fertilization in Women With and Without PCOS Undergoing Subfertility
Treatment
Thomas Keith Cunningham, Victoria Allgar, Soha R. Dargham, Eric Kilpatrick,
Thozhukat Sathyapalan, Stephen Maguiness, Haira R. Mokhtar Rudin,
Nour M. Abdul Ghani, Aishah Latiff and Stephen L. Atkin
14 The Role of Vitamin D Binding Protein, Total and Free 25-Hydroxyvitamin
D in Diabetes
Rolf Jorde
21 25-Hydroxyvitamin D and Vitamin D Binding Protein Levels in Patients
With Primary Hyperparathyroidism Before and After Parathyroidectomy
Xiangbing Wang, Zhifeng Sheng, Lingqiong Meng, Chi Su, Stanley Trooskin
and Sue A. Shapses
27 Vitamin D Binding Protein, Total and Free Vitamin D Levels in Different
Physiological and Pathophysiological Conditions
Daniel David Bikle and Janice Schwartz
39 The Vitamin D Binding Protein and Inflammatory Injury: A Mediator or
Sentinel of Tissue Damage?
Richard R. Kew
49 Vitamin D Binding Protein and the Biological Activity of Vitamin D
Rene F. Chun, Albert Shieh, Carter Gottlieb, Vahe Yacoubian, Jeffrey Wang,
Martin Hewison and John S. Adams
64 Vitamin D Binding Protein: A Historic Overview
Roger Bouillon, Frans Schuit, Leen Antonio and Fraydoon Rastinejad
Table of Contents
EDITORIAL
published: 11 February 2020
doi: 10.3389/fendo.2020.00040
Frontiers in Endocrinology | www.frontiersin.org 1February 2020 | Volume 11 | Article 40
Edited and reviewed by:
Jonathan H. Tobias,
University of Bristol, United Kingdom
*Correspondence:
Zhongjian Xie
zhongjian.xie@csu.edu.cn
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 13 January 2020
Accepted: 22 January 2020
Published: 11 February 2020
Citation:
Xie Z, Wang X and Bikle DD (2020)
Editorial: Vitamin D Binding Protein,
Total and Free Vitamin D Levels in
Different Physiological and
Pathophysiological Conditions.
Front. Endocrinol. 11:40.
doi: 10.3389/fendo.2020.00040
Editorial: Vitamin D Binding Protein,
Total and Free Vitamin D Levels in
Different Physiological and
Pathophysiological Conditions
Zhongjian Xie 1
*, Xiangbing Wang 2and Daniel D. Bikle3
1Hunan Provincial Key Laboratory of Metabolic Bone Diseases, National Clinical Research Center for Metabolic Diseases,
Department of Metabolism and Endocrinology, The Second Xiangya Hospital of Central South University, Changsha, China,
2Division of Endocrinology, Metabolism and Nutrition, Rutgers University-Robert Wood Johnson Medical School,
New Brunswick, NJ, United States, 3Endocrine Unit, Veterans Affairs Medical Center, University of California, San Francisco,
San Francisco, CA, United States
Keywords: vitamin D binding protein, total 25-hydroxyvitamin D, free 25-hydroxyvitamin D, 1,25-dihydroxyvitamin
D, bioavailable 25-hydroxyvitamin D
Editorial on the Research Topic
Vitamin D Binding Protein, Total and Free Vitamin D Levels in Different Physiological and
Pathophysiological Conditions
Vitamin D binding protein (DBP) is a major plasma carrier for vitamin D and its metabolites.
In recent years, there has been growing interest in understanding the physiological functions and
attributes of DBP. The current issue is comprised of five review articles and two original research
papers concerning the physiology of DBP and its role in different disorders.
Poor vitamin D status is highly prevalent in many different countries (1–4), but the
exact definition of vitamin D status is controversial. The plasma concentration of total
25-hydroxyvitamin D [25(OH)D] is currently used as an indicator of vitamin D status. In the
past decades, however, there has been argument as to whether just measuring total 25(OH)D is
appropriate for the assessment of vitamin D status in different physiological and pathophysiological
conditions (5,6). About 85% of the total circulating 25(OH)D is bound to DBP, and 15% is
bound to albumin. About 0.03% of 25(OH)D circulates in free form. Since 25(OH)D is weakly
bound to albumin and dissociates from it during tissue perfusion, the sum of the free and the
albumin-bound 25(OH)D represents the bioavailable 25(OH)D, which may be readily available for
metabolic function. In contrast, the DBP-bound vitamin D is relatively unavailable to target tissue,
with the exception of a few tissues such as the kidney that express a megalin/cubilin transport
system for DBP-bound 25(OH)D. The concept that it is the free hormone and not the DBP-
bound hormone that enters cells is known as the free hormone hypothesis. In the review by Bikle
and Schwartz it is highlighted that the DBP level is regulated by estrogen, glucocorticoids, and
inflammatory cytokines but not by vitamin D itself, and therefore, these regulators would affect
levels of total 25(OH)D. The review by Bikle and Schwartz focuses on the biological importance
of DBP with emphasis on its regulation of total and free vitamin D metabolite levels in various
clinical conditions. They also point out that attempts to calculate the free level using affinity
constants generated in a normal individual along with measurement of DBP and total 25(OH)D
have not accurately reflected directly measured free levels in a number of clinical conditions. The
authors examine the impact of different clinical conditions as well as different DBP alleles on
the relationship between total and free 25(OH)D, using only data in which the free 25(OH)D
4
Xie et al. Vitamin D Binding Protein
level was directly measured. Following their previous review (7),
the review by Chun et al. discussed a number of important
questions including the following. Is the total 25(OH)D (bound
plus free) or the unbound free 25(OH)D the crucial determinant
of the non-classical actions of vitamin D? While DBP-bound
25(OH)D is important for renal handling of 25(OH)D and
endocrine synthesis of 1,25(OH)2D, how does DBP impact extra-
renal synthesis of 1,25(OH)2D and subsequent 1,25(OH)2D
actions? Are there pathophysiological contexts where total
25(OH)D and free 25(OH)D would diverge in value as a marker
of vitamin D status? This review aims to introduce the concept
of free 25(OH)D and the molecular biology and biochemistry
of vitamin D and DBP, which provides the context for free
25(OH)D, and surveys in vitro, animal, and human studies taking
free 25(OH)D into consideration.
Low DBP levels in patients with primary hyperparathyroidism
(PHPT) were first reported in 2013 by Wang’s group (8) and
confirmed by Battista et al. (9). In the paper by Wang et al.,
the authors recruited 75 patients with PHPT and 75 healthy
control subjects. In addition, 25 PHPT patients underwent
parathyroidectomy and had a 3-month follow up visit. The
results showed that serum DBP levels were lower in patients with
PHPT but that parathyroidectomy restored DBP levels. Lower
DBP levels may be one of the contributing factors of low total
25(OH)D level in PHPT patients, and the total 25(OH)D levels
might not reflect true vitamin D status in patients with PHPT.
In the comprehensive review by Bouillon et al. it was noted
that DBP was originally discovered as a highly polymorphic
protein useful for population studies and originally called Group-
specific Component (GC). It is now known that DBP and
GC are the same protein and appeared early in the evolution
of vertebrates. DBP is genetically the oldest member of the
albuminoid family (which includes albumin, α-fetoprotein, and
afamin, all involved in the transport of fatty acids or hormones).
DBP has a single binding site for all vitamin D metabolites
with a high affinity for 25(OH)D, thereby creating a large
pool of circulating 25(OH)D, which prevents rapid vitamin D
deficiency. The review also highlighted the roles of DBP in
preventing the urinary loss of 25(OH)D and the formation of
polymeric actin fibrils in the circulation after tissue damage.
DBP also plays a minor role in transporting fatty acids. Based
on the fact that the total concentrations of 25(OH)D and
1,25(OH)2D in DBP null mice or humans are extremely low but
calcium and bone homeostasis remain normal, the “free hormone
hypothesis” appears to apply to the vitamin D hormones,
25(OH)D or 1,25(OH)2D, as it does to other steroid hormones
and thyroid hormone.
Vitamin D is important for bone health but may also
have extra-skeletal effects. Cunningham et al. examined vitamin
Dmetabolites in serum samples from age- and weight-
matched women with and without PCOS and reported results
in their paper. The authors found that 25-hydroxy-3epi-
Vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and
24,25-dihydroxyvitamin D3, but not 1,25-dihydroxyvitamin D3
[1,25(OH)2D3], were associated with embryo parameters. The
data suggest that vitamin D metabolites other than 1,25(OH)2D3
are important in fertility. Kew, in his review assesses the
fundamental role of DBP in neutrophilic inflammation and
injury. As highlighted by Kew, DBP induces selective recruitment
of neutrophils. DBP is also an extracellular scavenger for actin
released from damaged/dead cells, and formation of DBP-actin
complexes is an immediate host response to tissue injury. DBP
bound to G-actin functions as an indirect but essential cofactor
for neutrophil migration.
Vitamin D and DBP have immunological effects and may
be important in the development of type 1 diabetes (T1DM).
Moreover, low total 25(OH)D levels are associated with the
development of type 2 diabetes (T2DM). However, there are no
convincing data showing that vitamin D supplementation has
an effect on the prevention of T2DM (10). The review by Jorde
discusses the relations between DBP and total and free 25(OH)D
in T1DM and T2DM.
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication.
FUNDING
This study was supported by National Natural Science
Foundation of China (81672646, 81471055).
REFERENCES
1. Xie Z, Xia W, Zhang Z, Wu W, Lu C, Tao S, et al. Prevalence
of vitamin D inadequacy among chinese postmenopausal women: a
nationwide, multicenter, cross-sectional study. Front Endocrinol. (2007) 9:782.
doi: 10.3389/fendo.2018.00782
2. Hypponen E, Power C. Hypovitaminosis D in British adults at age 45 y:
nationwide cohort study of dietary and lifestyle predictors. Am J Clin Nutr.
(2007) 85:860–8. doi: 10.1093/ajcn/85.3.860
3. Byun EJ, Heo J, Cho S, Lee HJD, Kim HS. Suboptimal vitamin D
status in Korean adolescents: a nationwide study on its prevalence,
risk factors including cotinine-verified smoking status and association
with atopic dermatitis and asthma. BMJ Open. (2017) 7:e016409.
doi: 10.1136/bmjopen-2017-016409
4. Acherjya GK, Ali M, Tarafder K, Akhter N, Chowdhury MK, Islam DU, et al.
Study of vitamin D deficiency among the apparently healthy population in
Jashore, Bangladesh. Mymensingh Med J. (2019) 28:214–21.
5. Youselzadeh P, Shapese SA, Wang X. Vitamin D binding protein impact
on 25-hydroxyvitamin D levels under different physiologic and pathologic
conditions. Int J Endocrinol. (2014) 2014:981581. doi: 10.1155/2014/
981581
6. Jassil N, Sharma A, Bikle DD, Wang X. Vitamin D binding protein and 25-
hydroxyvitamin D levels: emerging clinical applications. Endocr Pract. (2017)
23:605–61. doi: 10.4158/EP161604.RA
7. Chun RF, Peercy BE, Orwoll ES, Nielson CM, Adams JS, Hewison M.
Vitamin D and DBP: the free hormone hypothesis revisited. J Steroid
Biochem Mol Biol. (2014) 144 Pt A:132–7. doi: 10.1016/j.jsbmb.2013.
09.012
Frontiers in Endocrinology | www.frontiersin.org 2February 2020 | Volume 11 | Article 40
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8. Wang X, Shapses SA, Wei S, Sukumar D, Ghosh J. Vitamin D-binding protein
levels in female patients with primary hyperparathyroidism. Endocr Pract.
(2013) 19:609–13. doi: 10.4158/EP12371.OR
9. Battista C, Guarnieri V, Carnevale V, Baorda F, Pileri M, Garrubba M,
et al. Vitamin D status in primary hyperparathyroidism: effect of genetic
background. Endocrine. (2017) 55:266–72. doi: 10.1007/s12020-016-0974-x
10. Pittas AG, Dawson-Hughes B, Sheehan P, Ware JH, Knowler WC,
Aroda VR, et al. Vitamin D Supplementation and Prevention of Type
2 Diabetes. N Engl J Med. (2019) 381:520–30. doi: 10.1056/NEJMoa19
00906
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Xie, Wang and Bikle. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms.
Frontiers in Endocrinology | www.frontiersin.org 3February 2020 | Volume 11 | Article 40
6
ORIGINAL RESEARCH
published: 29 January 2019
doi: 10.3389/fendo.2019.00013
Frontiers in Endocrinology | www.frontiersin.org 1January 2019 | Volume 10 | Article 13
Edited by:
Daniel David Bikle,
University of California, San Francisco,
United States
Reviewed by:
Carol L. Wagner,
Medical University of South Carolina,
United States
Michael F. Holick,
Boston Medical Center, United States
*Correspondence:
Stephen L. Atkin
sla2002@qatar-med.cornell.edu
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 02 October 2018
Accepted: 10 January 2019
Published: 29 January 2019
Citation:
Cunningham TK, Allgar V,
Dargham SR, Kilpatrick E,
Sathyapalan T, Maguiness S, Mokhtar
Rudin HR, Abdul Ghani NM, Latiff A
and Atkin SL (2019) Association of
Vitamin D Metabolites With Embryo
Development and Fertilization in
Women With and Without PCOS
Undergoing Subfertility Treatment.
Front. Endocrinol. 10:13.
doi: 10.3389/fendo.2019.00013
Association of Vitamin D Metabolites
With Embryo Development and
Fertilization in Women With and
Without PCOS Undergoing
Subfertility Treatment
Thomas Keith Cunningham 1,2 , Victoria Allgar 3, Soha R. Dargham 4, Eric Kilpatrick 5,
Thozhukat Sathyapalan 2, Stephen Maguiness 1, Haira R. Mokhtar Rudin 6,
Nour M. Abdul Ghani 6, Aishah Latiff 6and Stephen L. Atkin 4
*
1Hull IVF Unit, Women and Children’s Hospital, Hull Royal Infirmary, Hull, United Kingdom, 2Centre for Diabetes and
Metabolic Research, Hull York Medical School, University of Hull, Hull, United Kingdom, 3Department of Statistics, Hull York
Medical School, University of Hull, Hull, United Kingdom, 4Weill Cornell Medicine Qatar, Doha, Qatar, 5Sidra Medical and
Research Centre, Doha, Qatar, 6Antidoping Laboratory Qatar, Doha, Qatar
Objective: The relationship between fertilization rates and 1,25-dihydroxyvitamin D
(1,25(OH)2D3), 25-hydroxyvitamin D2 (25(OH)D2), 25-hydroxyvitamin D3 (25(OH)D3),
24,25-dihydroxyvitamin D (24,25(OH)2D3), and 25-hydroxy-3epi-Vitamin D3
(3epi25(OH)D3) concentrations in age and weight matched women with and without
PCOS was studied.
Methods: Fifty nine non-obese women, 29 with PCOS, and 30 non-PCOS undergoing
IVF, matched for age and weight were included. Serum vitamin D metabolites were taken
the menstrual cycle prior to commencing controlled ovarian hyperstimulation.
Results: Vitamin D metabolites did not differ between PCOS and controls; however,
25(OH)D3correlated with embryo fertilization rates in PCOS patients alone (p=0.03).
For all subjects, 3epi25(OH)D3correlated with fertilization rate (p<0.04) and negatively
with HOMA-IR (p<0.02); 25(OH)D2correlated with cleavage rate, G3D3 and blastocyst
(p<0.05; p<0.009; p<0.002, respectively). 24,25(OH)2D3correlated with AMH,
antral follicle count, eggs retrieved and top quality embryos (G3D3) (p<0.03; p<
0.003; p<0.009; p<0.002, respectively), and negatively with HOMA-IR (p<0.01).
1,25(OH)2D3did not correlate with any of the metabolic or embryo parameters. In slim
PCOS, 25(OH)D3correlated with increased fertilization rates in PCOS, but other vitamin
D parameters did not differ to matched controls.
Conclusion: 3epi25(OH)D3, 25(OH)D2, and 24,25(OH)2D3, but not 1,25(OH)2D3, were
associated with embryo parameters suggesting that vitamin D metabolites other than
1,25(OH)2D3are important in fertility.
Keywords: vitamin D, vitamin D epimers, vitamin D metabolites, fertilization rates, PCOS
7
Cunningham et al. Vitamin D and Embryo Development in PCOS
INTRODUCTION
Polycystic ovarian syndrome (PCOS) is one of the most
common endocrine disorders amongst women of reproductive
age affecting 9–21% of the female population and is the main
cause of anovulatory infertility (1). It is associated with clinical
and biochemical hyperandrogenism, and insulin resistance (IR)
in PCOS is associated with obesity, type 2 diabetes, and
hypercholestrolemia (2). Vitamin D levels are low in 67–85% of
women with PCOS (3), which are suggested to exacerbate IR
and the free androgen index (FAI) in PCOS (4,5). IR itself is
both independent of and exacerbated by obesity and is present
in 65–80% of women with PCOS (6) and may be improved by
vitamin D replacement (7). In a recent meta-analysis, it was
shown that in weight matched PCOS women, vitamin D was
negatively predicted by weight hip ratio, glucose and LH (8).
Vitamin D deficiency has become the most common
nutritional deficiency throughout the world (9). Studies of sub-
fertile women have demonstrated that vitamin D deficiency
is present in between 58 and 91% of cases (9–12). Obesity
can exacerbate vitamin D deficiency, as a result of decreased
bioavailability from cutaneous and dietary sources because of
deposition in the body fat compartments (13).
Vitamin D3 (cholecalciferol) is endogenously produced or
taken as a dietary supplement, whist vitamin D2 (ergocalciferol)
is derived from the diet (primarily from mushrooms and fungi),
though both are hydroxylated to 25(OH)D3or 25(OH)D2
by multiple 25-hydroxylases (14,15) (Figure 1). 25(OH)D is
transported to the kidney and converted to either the active 1,25-
dihydroxyvitamin D (1,25(OH)2D3) by 1 alpha hydroxylase, or
to 24,25-dihydroxyvitamin D (24,25(OH)2D3), that is also active,
by the 24 alpha hydroxylase, in the renal tubular and other
cells widely in the body (Figure 1) (16). It has been recently
reported that extrarenal tissues may also convert 25(OH)D
to 1,25(OH)2D (17). 1,25(OH)2D binds to the vitamin D
receptor (VDR) subsequently heterodimerizes with the retinoid
X receptor for its action that may be effected in several hours (16);
however, a more rapid action has been reported with binding
membrane VDR or through the 1,25D3-membrane-associated,
rapid response steroid-binding protein receptor with activation
of protein kinases A and C (18). Vitamin D receptors have
been located within structures of the female reproductive tract,
including the ovary and endometrium (19,20).
Vitamin D2is derived from the diet as ergocalciferol
that has lower binding efficacy to VDR resulting in greater
serum clearance, limiting the formation of 25(OH)D2, though
1,25(OH)2D2has a high an affinity for VDR as 1,25(OH)2D3
(14,16). In the United States and other countries vitamin D2 is
available both as a supplement and as a pharmaceutical to treat
vitamin D deficiency.
3- epimerase isomerizes the C-3 hydroxy group of the
natural vitamin D from the αto the βorientation leading to
3epi25(OH)D3(14,21) that may be measured inadvertently
whilst measuring 25(OH)D3(22). 3epi25(OH)D3is thought to
be less potent physiologically as 25(OH)D3, and 1,25(OH)2-3-
epi-D3has less affinity to VDR thus less biologically active;
however, the 3-epimer may be as potent as 1,25(OH)2D3in other
circumstances such as PTH suppression (14,23) however, data is
sparse on the biological potency of the C3 epimers.
Two IVF cohort studies have suggested that clinical pregnancy
rates were significantly lower in women who were vitamin
D deficient (24,25), but no differences in the embryological
data have been associated with 25(OH)D3levels (25). However,
it remains unknown if there is a relationship of baseline
1,25(0H)2D3, 25(OH)D3, 25(OH)D2, 24,25(0H)2D3, or its
epimer 3epi25(OH)D3to fertilization in non-obese PCOS
subjects when age and weight are matched to control subjects,
and therefore this study was undertaken.
MATERIALS AND METHODS
This prospective cohort study was performed within the Hull IVF
Unit, UK following approval by the Yorkshire and The Humber
NRES ethical committee, UK and all gave their written informed
consent. The PCOS subjects were recruited using the revised 2003
criteria (26), namely any 2 out of 3 criteria were met; menstrual
disturbance (oligo or amenorrhoea), clinical and/or biochemical
signs of androgenism and polycystic ovaries on ultrasound, with
the exclusion of other conditions. All women were on folic
acid 400 mcg daily but no other medication. Exclusion criteria
were patients with diabetes, renal or liver insufficiency, acute or
chronic infections, systemic inflammatory diseases, age <20, age
>45, known Immunological disease.
Sample Collection
A fasting blood sample was taken in the luteal phase of the
cycle before commencing IVF treatment. The bloods were
centrifuged at 3,500 g for 15 min and placed into aliquots and
frozen at −80◦C until analysis. The bloods were analyzed
for FSH (Architect analyser, Abbott laboratories, Maidenhead,
United Kingdom), SHBG, insulin (DPC Immulite 200 analyser,
Euro/DPC, Llanberis United Kingdom), and plasma glucose
(Synchron LX20 analyser, Beckman-Coulter, High Wycombe,
United Kingdom). Free androgen index (FAI) was calculated by
dividing the total testosterone by SHBG, and then multiplying
by one hundred. Insulin resistance (IR) was calculated using
the homeostasis model assessment (HOMA-IR). Serum vitamin
D levels and testosterone were quantified using isotope-
dilution liquid chromatography tandem mass spectrometry (LC-
MS/MS). Vitamin D metabolites (1,25(OH)2D3), 25(OH)D2,
25(OH)D3, 24,25(OH)2D3 and 3epi25(OH)D3 and three labeled
internal standards (d6-25(OH)D3, d6-1,25(OH)2D3 and d6-3-
epi-25(OH)D3) were simultaneously extracted from 250 µL
serum using supportive liquid-liquid extraction and Diels-Alder
derivatization prior to LC-MS/MS analysis. Chromatographic
separations were achieved using Hypersil Gold C18 column
(150 ×2.1 mm; 1.9 µ) at flow rate 0.2 ml/min, operated in
Electrospray Ionization (ESI) positive mode and analyzed by
multiple reaction monitoring (MRM) method. The limit of
quantification (LOQ) for 1,25(OH)2D were 10 pg/mL, 3-epi-
25(OH)D, and 24,25(OH)2D were 50 pg/mL while 25(OH)D3
and 25(OH)D2 were 0.5 and 0.25 ng/mL, respectively. All
methods employed were performed in accordance with the
relevant guidelines and regulations.
Frontiers in Endocrinology | www.frontiersin.org 2January 2019 | Volume 10 | Article 13
8
Cunningham et al. Vitamin D and Embryo Development in PCOS
FIGURE 1 | 7-dehydrocholesterol in the skin is converted to previtamin D3 and then is thermally isomerize to vitamin D3. Transport of vitamin D3from the skin to the
liver is via the Vitamin D binding protein (DBP) transports 25 hydroxy vitamin D (25(OH)D3) to the kidney. 25(OH)D3/DBP is filtered by the glomeruli and 25(OH)D3is
taken up into the tubular cells, following DBP binding to megalin, a transmembrane protein. 25(OH)D3undergoes a second hydroxylation step by the
1-alpha-hydroxylase Cyp27B1, converting to the active 1α,25 (OH)2D3, whilst 24 hydroxylase Cyp27A1 converts to 24,25(OH)2D3. Keratinocytes contribute to the
3-epimerase activity converting 25(OH)D3to 3-epi-25(OH)D3, and 1α,25 (OH)2D3to 3-epi-1α,25 (OH)2D3, but the exact sites of activity remain unknown.
3epi25(OH)D3is equally converted by Cyp27A1 and Cyp27B1 as 25(OH)D3. *Vitamin D2derived from yeasts and fungi (mushrooms) is converted to 25(OH)D2in the
liver and to 1,25(OH)2D2and 24,25(OH)2D2in the kidney (adapted from 14, 16, and 21). It is unclear if the 3-epimers may be back converted.
All patients underwent a standard IVF antagonist protocol.
The patients commenced their rFSH stimulation on day 2 of their
menstrual cycle using either Merional (Pharmasure) or Gonal-F
(Merck Serono). A GnRH antagonist (Cetrotide: Merck Serono)
was used to prevent a premature LH surge.
The patients underwent ultrasound scans from day 7 to
observe the ovarian response to stimulation and were repeated
every 48 h. The scans were used to measure the diameters of
the follicles thus observing response and follicle numbers. Final
maturation was triggered when two or more leading follicles were
≥18 mm using human chorionic gonadotrophin [hCG, Pregnyl
(Merck Sharp and Dohme)].
Transcervical embryo transfer was performed and embryos
were classified using standard criteria (27) at the cleavage stage
(day 2–3 after egg collection) and for blastocyst stage (day 5–6
after egg collection). Top Quality embryos on Day 3 as per Alpha
Consensus (28). Embryo transfers were performed on either day
3 or ideally at day 5 (blastocyst) to give the best chance for
implantation as this timing is similar compared to natural cycle
embryos moving into the uterus.
Data Analysis and Statistics
Statistical analysis was performed using SPSS (v22, Chicago,
Illinios). Descriptive data is presented as mean ±SD for
continuous data and n (%) for categorical data. t-tests or
Mann Whitney tests were used to compare means/medians
where appropriate, and associations used Pearson’s correlation or
Spearman’s correlation as appropriate. A p<0.05 was considered
to indicate statistical significance. There was no comparative
study on which to base a formal power calculation; therefore,
power and sample size for a pilot study was performed (29);
therefore, to account for a minimum of 20 degrees-of-freedom
to estimate effect size and variability a minimum of 25 patients
per group were required to allow covariate adjustment.
RESULTS
Baseline characteristics of the 59 patients are shown in Table 1
where is can be seen that patients were non-obese, age, and
weight matched. There were significant differences in ovarian
reserve parameters antral follicle count (AFC) and anti-Mullerian
Hormone, (AMH), and androgen status between the groups,
however there was no significant difference in fasting insulin,
HOMA-IR or the vitamin D metabolites (Table 1).
There was a correlation between the levels of 25(OH)D3,
and embryo fertilization rates in PCOS patients (r=0.44; p
=0.03) that were not seen in the control group. However,
between the PCOS and control groups there were no differences
for any of the metabolic or embryo parameters for 25(OH)D2,
24R,25(OH)2D3, 1,25(OH)2D3, or 3epi25(OH)D3. When all of
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Cunningham et al. Vitamin D and Embryo Development in PCOS
TABLE 1 | Mean demographics and biochemical data.
Control (n=30) PCOS (n=29) p-value
Mean data (±S.D.) Mean data (±S.D.)
Age 32.6 ±4.7 30.9 ±4.8 0.14
BMI 25.5 ±3.6 26.0 ±3.8 0.56
Menarche 13.0 ±2.0 13.0 ±1.1 0.99
Anovulatory 5 25 0.0001***
Duration of subfertility 3.9 ±1.8 3.4 ±1.6 0.84
Total antral follicle count 17.2 ±6.8 38.4 ±17.8 0.0001***
Fasting insulin (mIU/ml) 7.68 ±4.0 8.13 ±4.7 0.69
Fasting glucose (mmol/L) 4.81 ±0.4 4.62 ±0.4 0.06
HOMA-IR 1.71 ±1.0 1.72 ±1.0 0.97
SHBG 110.9 ±82.4 63.9 ±49.8 0.01*
Testosterone (mmol/L) 0.8 ±0.4 1.4 ±0.8 0.0004***
Free androgen index 1.35 ±0.6 4.21 ±2.9 0.0001***
25-hydroxyvitamin D3
(ng/mL)
46.2 ±23.5 54.0 ±27.4 0.24
25-hydroxyvitamin D2
(ng/ml)
0.5 ±0.3 0.6 ±0.5 0.73
1,25-dihydroxyvitamin D3 0.03 ±0.02 0.04 ±0.2 0.63
24R,25-dihydroxyvitamin
D3
0.8 ±0.5 1.3 ±0.6 0.003**
3-epi-25-hydroxyvitamin
D3
0.4 ±0.4 0.7 ±1.2 0.89
*p<0.01, **p<0.001, ***p<0.0001.
the subjects were combined there was a correlation between the
levels of 25(OH)D2and cleavage rate (r=0.31; p=0.05), G3D3
(r=0.40; p=0.009) and blastocyst (r=0.40; p=0.022);
there was a correlation between 3epi25(OH)D3and fertility rate
(r=0.33; p<0.04) and a negative correlation with HOMA-IR
(r= −0.33; p<0.02); 24R,25(0H)2D3correlated with AMH
(r=0.1; p=0.03) antral follicle count (r=0.2; p=0.003),
eggs retrieved (r=0.14; p=0.009) and G3D3 (r=0.22; p=
0.002), and negatively with HOMA-IR (r= −0.07; p<0.01).
There was no correlation of the active 1,25(0H)2D3with any of
the metabolic or embryo parameters.
There was a correlation between the levels of 25(OH)D3with
both 24R,25(OH)2D3and 3epi25(OH)D3(r=0.91, p<0.001; r
=0.35, p<0.015, respectively.
As a cohort, 25-hydroxyvitamin D levels were low did not
differ between the controls and the PCOS group. The Endocrine
Society defines vitamin D deficiency, insufficiency and replete
as (≤20 ng/mL, 20–30 ng/mL and ≥30 ng/mL, respectively (30)
that was reflected in controls and PCOS as, deficient, 51 vs. 41%;
insufficient, 33 vs. 35%; deficient, 10 vs. 24%.
IVF cycle characteristics are represented in Table 2 showing
that the PCOS group had significantly greater numbers of follicles
aspirated and eggs retrieved compared to the controls, and the
mean fertilization and cleavage rates were significantly higher for
the PCOS group, though embryos quality did not differ.
There was a significantly negative correlation between SHBG
and 25-hydroxyvitamin D3 in the PCOS subjects, however after
adjusting for BMI, SHBG was not significantly associated with
25-hydroxyvitamin D3.
TABLE 2 | Mean outcome data for stimulated ovarian cycle for Control and PCOS
groups. G3D3: Top Quality embryos on Day 3 as per Alpha Consensus (28).
Control (N=30) PCOS (N=28) p-value
Mean (±S.D.) Mean (±S.D.)
Endometrium at
oocyte retrieval
10.31 ±1.78 10.72 ±2.06 0.42
Follicles aspirated 11.47 ±5.11 15.96 ±5.30 0.002**
Eggs retrieved 8.47 ±5.08 11.29 ±5.02 0.04*
Fertilization 4.82 ±2.65 8.43 ±3.87 0.0003***
Cleavage 4.68 ±2.72 7.26 ±4.40 0.01*
G3D3 3.00 ±2.29 4.17 ±3.47 0.16
Blastocyst 1.46 ±1.77 2.91 ±3.01 0.05
PDT 11 10 0.86
Clinical pregnancy 10 7 0.24
*p<0.01, **p<0.001, ***p<0.0001.
DISCUSSION
This study has shown that 25(OH)D3was associated with
higher fertility rates in PCOS compared to non-obese, age, and
weight matched control subjects, but that this was not seen
for the other vitamin D metabolites. This was surprising given
that there was no difference in the 25(OH)D3levels between
the PCOS and control group; however, it is recognized that
ova in PCOS may be at a less mature stage compared to
normal and therefore there is a possibility that they may be
more 25-hydroxyvitamin D responsive to allow those ovum
within the stimulated follicles to reach a more mature stage
prior to ovum retrieval, resulting in a greater capability to
achieve fertilization. PCOS women typically produced more
poor quality oocytes, with lower fertilization, cleavage and
implantation rates (31,32). The impaired oocyte maturation
and resultant embryonic developmental competence in PCOS
women is possibly due to the abnormal endocrine/paracrine
functions and the environment within the follicle at the time of
folliculogenesis (33,34). No differences in the embryological data
for 25(OH)D3were found, in accord with others (25). It may
have been speculated that the active 1,25(OH)2D3may have a
greater influence on fertility at higher levels, but this was not seen
in this study, with no correlation with fertilization or embryo
data. Vitamin D is involved in the regulation of AMH and FSH
gene expression (31,35), and high dose 25(OH)D3has been
shown to increase serum AMH levels in vitamin D insufficiency
(36). In this study only the metabolite 24R,25(OH)2D3correlated
with AMH and antral follicle count; 24R,25(OH)2D3is an active
metabolite [It can be converted to 1,24,25-trihydroxyvitamin D3
through the C24 oxidation pathway (37)] as it has been shown
to induce non-genomic signaling pathways and suppresses Apo
A-1 in hep G cells (38), may have a physiological role in the
growth plate formation (14), therefore a direct effect on the
ovary cannot be excluded. However, 24,25 dihydroxyvitamin D
is associated with blood levels of 25-hydroxyvitamin D and given
that both were very significantly associated it is unlikely that
24R,25(OH)2D3was having a unique biological effect and indeed
was dependent on serum 25(OH)D3levels.
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Cunningham et al. Vitamin D and Embryo Development in PCOS
There was a positive association for an increase in overall
fertility rate with the 3epi25(OH)D3that was not seen for the
other vitamin D metabolites. Little is known about the epimers
of vitamin D, with the assumption that they are biologically less
potent (21,23), but whilst that is likely for bone metabolism it
may not be the case for ovarian effects. Epimers are compounds
that have identical structure (and therefore identical molecular
weight) with the exception of a stereochemical difference at one
site. Other than the measurement of 3epi25(OH)D3to enable
the more accurate determination of 25(OH)D3, in children where
they have been shown to be higher, there has been little research
done on the C3 epimers (39) in order to know the significance
of this observation on fertility, but it is of interest to note
that serum lipids were discrepant for 25(OH)D epimeric forms
suggesting a differential effect (40). However, 3epi25(OH)D3is
associated with blood levels of 25-hydroxyvitamin D and given
that both were very significantly associated it is unlikely that
3epi25(OH)D3was having a unique biological effect and indeed
was dependent on serum 25(OH)D3levels.
Whole group analysis for 25(OH)D2, but not 25(OH)D3was
positively associated with increased cleavage rate, G3D3 and
blastocyst, though overall embryo quality did not differ. Whilst
vitamin D3 supplements are better than vitamin D2 to raise
vitamin D levels (41,42), their biological effects may not be
the same in different systems (43), and their differential effect
in the ovary needs to be clarified. The role of vitamin D in
fertilization remains controversial and the specific roles of 25-
hydroxyvitamin D levels largely unknown. Observational studies
have reported vitamin D levels within serum and follicular fluid
to be highly correlated and that those with higher serum and
follicular fluid levels of vitamin D had significantly higher clinical
pregnancy rates (12). Other studies have found no correlation
between serum and follicular fluid levels of vitamin D and
IVF outcomes (10,35,44). Conversely, two cohort studies
comparing serum vitamin D levels and pregnancy rates in women
undergoing fresh IVF showed that clinical pregnancy rates were
significantly lower in women who were vitamin D deficient
(24,25). Given the controversy, the implication of this data is
that in slim PCOS women undergoing IVF that their vitamin D
status should be determined, vitamin D replacement undertaken
for those deficient prior to IVF may be of benefit, or at least do
not harm, until future clarification becomes available.
These data suggest that vitamin D deficiency may not be a
homogeneous entity but rather may depend on the different
vitamin D metabolites present giving resultants effects, and
therefore may account for the heterogeneity and controversy
surrounding vitamin D deficiency effects and the response to
replacement (42,45). Whilst the effects of vitamin D and its
metabolites on bone and calcium metabolism are well-known,
vitamin D metabolite effects on other systems may not be
directly equipotent for each metabolite or comparable. For
example 1,25(OH)2-3-epi-D3may be biologically less active
than 1,25(OH)2D3, for increasing calcium, but may have
greater PTH suppression (39). However, the kidneys produce
1,25-dihydroxyvitamin D for regulating calcium and bone
metabolism (46). Therefore, measuring a blood level of 1,25-
dihydroxyvitamin D may not reflect its other biologic functions,
but rather local production of 1,25-dihydroxyvitamin D may
have its major benefit (47). Our observation that higher blood
levels of 25-hydroxyvitamin D are related to the outcome
measures could also be due to the higher substrate availability
for the local production of 1,25-dihydroxyvitamin D rather than
25-hydroxyvitamin D having a direct effect.
When weight and aged matched then there was no difference
in vitamin D metabolite levels between PCOS and normal
women. These results differ from previous studies that found
Vitamin D deficiency to be more common in PCOS subjects
(48); however, in this study the PCOS patients were specifically
non-obese and it is well-recognized that vitamin D levels fall
in obesity that may account for this observation (6). Negative
correlations with HOMA-IR were seen for both 24R,25(OH)2D3,
and 3epi25(OH)D3, but no association was seen for either
25(OH)D2or 25(OH)D3, suggesting that an association with
insulin resistance may depend on the metabolites present. There
were no correlations between testosterone or oocyte quality in the
PCOS patients in this study These observations are in accord with
some studies, though not with others (4,49); however, in those
reported studies patients were not intentionally weight matched.
High dose 25(OH)D3replacement was not associated with an
improvement in insulin sensitivity in PCOS subjects (50).
This study was specifically designed to look at the relationship
of vitamin D levels with fertilization rates and therefore was not
powered to look at pregnancy rates that would require a much
larger sample population. Furthermore, the overall sample size
is small and further work is needed, particularly to determine if
these findings are true for the differing PCOS phenotypes within
the Rotterdam criteria. However, the strengths of this study were
that the patients were age and weight matched from the same
ethnic background and that all of the vitamin D metabolites were
measured by state of the art methods.
In conclusion, non-obese age and weight matched PCOS
women showed that 25(OH)D3was associated with the
fertilization rate, compared to controls; however, vitamin D
metabolites were associated with embryology parameters and
HOMA-IR, suggesting a possible relationship between differing
vitamin D metabolites, oocyte maturation and insulin sensitivity
in non-obese PCOS patients.
AUTHOR CONTRIBUTIONS
TC was involved in the study design, acquisition of data, analysis
and interpretation of data, and paper drafting. HM, NA, and AL
were involved in vitamin D analysis and drafting the manuscript.
VA and SD were involved in analysis and interpretation of data,
and paper drafting. SA, EK, SM, and TS were involved in the
study design, supervision, paper drafting, and contributed to the
interpretation of the data. All authors read and approved the final
manuscript.
FUNDING
The publication of this article was funded by the Qatar National
Library.
ACKNOWLEDGMENTS
This work was part of an MD thesis and its publishing is in line
with the University of Hull policy, and can be accessed online
https://hydra.hull.ac.uk/resources/hull:14051 online (51).
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Cunningham et al. Vitamin D and Embryo Development in PCOS
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
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Copyright © 2019 Cunningham, Allgar, Dargham, Kilpatrick, Sathyapalan,
Maguiness, Mokhtar Rudin, Abdul Ghani, Latiff and Atkin. This is an open-access
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13
REVIEW
published: 19 February 2019
doi: 10.3389/fendo.2019.00079
Frontiers in Endocrinology | www.frontiersin.org 1February 2019 | Volume 10 | Article 79
Edited by:
Daniel David Bikle,
University of California, San Francisco,
United States
Reviewed by:
Roger Bouillon,
Katholieke Universiteit Leuven,
Belgium
Jan Josef Stepan,
Charles University, Czechia
*Correspondence:
Rolf Jorde
rolf.jorde@unn.no
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 21 December 2018
Accepted: 30 January 2019
Published: 19 February 2019
Citation:
Jorde R (2019) The Role of Vitamin D
Binding Protein, Total and Free
25-Hydroxyvitamin D in Diabetes.
Front. Endocrinol. 10:79.
doi: 10.3389/fendo.2019.00079
The Role of Vitamin D Binding
Protein, Total and Free
25-Hydroxyvitamin D in Diabetes
Rolf Jorde 1,2
*
1Tromsø Endocrine Research Group, Department of Clinical Medicine, UiT The Arctic University of Norway, Tromsø, Norway,
2Division of Internal Medicine, University Hospital of North Norway, Tromsø, Norway
Vitamin D is important for bone health, but may also have extra-skeletal effects. Vitamin D
and its binding protein DBP have immunological effects and may therefore be important
in the development of type 1 diabetes (T1DM), and low serum levels of 25-hydroxyvitamin
D (25(OH)D) are associated with later development of type 2 diabetes (T2DM). However,
it has so far been difficult to convincingly show an effect of vitamin D supplementation
on prevention or treatment of diabetes. The serum level of 25(OH)D has traditionally
been used as a marker of a subject’s vitamin D status. This measurement includes both
25(OH)D bound to DBP and albumin as well as the free from of 25(OH)D. However,
according to the free hormone hypothesis, the free form is the biologically active.
Previously the free form of 25(OH)D had to be calculated based on measurements
of 25(OH)D, DBP, and albumin, but recently a method for direct measurement of free
25(OH)D has become commercially available. This is important in clinical conditions
where the amount of DBP is affected, and has caused a renewed interest in which vitamin
D metabolite to measure in clinical situations. In the present review the relations between
DBP, total and free 25(OH)D in T1DM and T2DM are described.
Keywords: diabetes, free vitamin D, vitamin D binding protein (DBP), single nucelotide polymorphisms, 25- hydroxy
vitamin D
INTRODUCTION
Vitamin D is produced in the skin upon UV-B exposure and is obtained through the diet where
fatty fish is the main source. Regardless of how it is obtained, vitamin D has to be hydroxylated
first in the liver to 25-hydroxyvitamin D (25(OH)D) and then in the kidneys to the active form
1,25-dihydroxyvitamin D (1,25(OH)2D) (1). These hydroxylations may also occur in peripheral
tissues (2).
In the circulation the major part of vitamin D, 25(OH)D and 1,25(OH)2D are bound to the
vitamin D binding protein (DBP), and to a lesser extent also to albumin. Only a small fraction
circulates in the free form (3). To exert their action, the vitamin D metabolites have to cross the cell
membrane into the cell [and for vitamin D and 25(OH)D also to be hydroxylated], where the active
form 1,25(OH)2D connects to the nuclear vitamin D receptor (VDR) (1).
14
Jorde DBP, Total and Free 25(OH)D and Diabetes
The endocytic receptors megalin and cubulin are present in
the renal tubuli and parathyroid cells (4), and at least in the
kidney enable transportation of the DBP-vitamin D complexes
into the cells (5). In other (and perhaps most) cell types, the
vitamin D metabolites have to pass the cell membranes in their
free un-bound form by passive diffusion (6).
The serum concentrations of vitamin D and 25(OH)D are
>100 times that of 1,25(OH)2D, and the DBP binding coefficients
as well as the potential for passive diffusion through cell
membranes differ between these vitamin D metabolites (6).
Accordingly, it is difficult to say which vitamin D metabolite,
or vitamin D metabolite-DBP complex is quantitatively the
most important for VDR activation and the one that should be
measured for evaluation of a subject’s vitamin D status (6,7).
For this purpose, one has traditionally measured the serum
25(OH)D level, since this metabolite is abundant, easy to
measure, and has a long half-life and therefore stable levels.
Furthermore, the hydroxylation from vitamin D to 25(OH)D is
substrate driven and the serum 25(OH)D level correlates strongly
with sun exposure and vitamin D intake and also correlates with
known vitamin D effects, like the suppression of the parathyroid
hormone (PTH) secretion (1).
The serum 25(OH)D that is measured is the total 25(OH)D,
which includes the DBP and albumin bound 25(OH)D as well
as the free form. Since the major part of 25(OH)D is bound
to DBP, the concentration of total 25(OH)D will depend on
the serum DBP concentration. The DBP concentration is fairly
stable throughout life, but increases with pregnancy and estrogen
supplementation. DBP is synthesized in the liver and accordingly
the serum DBP concentration is reduced in liver failure as well
as in malnutrition (8,9). Loss of proteins in the urine (like in
some subjects with diabetes) may also cause low serum DBP
levels (10,11). Thus, in situations with high serum DBP levels
like pregnancy, an even larger portion of the total 25(OH)D in
plasma is bound to DBP and accordingly the free form is reduced.
Conversely, in patients with liver cirrhosis where the serum level
of DBP is low, the free fraction is increased. Although there
is a strong correlation between total and free 25(OH)D (12),
measurement of total 25(OH)D may therefore not always reflect
the free form.
According to the free hormone hypothesis, it is the free form
of the hormone, which easily diffuses through cell membranes,
that is the biologically active, and the one to be measured (13).
This is exemplified for thyroid hormones, where the serum
concentration of tree thyroxine is regulated in a negative feedback
manner by the secretion of thyroid stimulating hormone (TSH).
In this system, changes in the concentration of thyroid hormone
binding globulin (TBG) will be compensated by increased or
decreased secretion of TSH keeping the free concentration of
thyroxine stable (14). This demonstrates the utility of the free
hormone concept for thyroid hormones.
This concept does not necessarily apply to the vitamin
D system where the active hormone 1,25(OH)2D can be
transported into (at least some) cells in a DBP-complex,
and also have its activating hydroxylations intracellularly.
Furthermore, 25(OH)D is in essence a pro-hormone not
regulated by negative feed-back control. Changes in DBP will
not induce changes in the hydroxylation of vitamin D to
25(OH)D since this is a substrate driven process. Increased
serum 25(OH)D concentrations may be accompanied by an
increased level of FGF-23, increased CYP24A1 expression and 24-
hydroxylase activity, and accelerated degradation of 25(OH)D to
24,25(OH)2D (15). However, this mechanism must for 25(OH)D
be of minor importance since the increase in free 25(OH)D
and total 25(OH)D after vitamin D supplementation is, at least
until serum 25(OH)D levels of approximately 150 nmol/L, quite
linear (12). Therefore, whether the total or the free form of
25(OH)D is the best vitamin D parameter cannot be decided on
theoretical grounds only, but has to be tested in clinical situations
as well (16,17).
There are many single nucleotide polymorphisms (SNPs) in
the DBP gene (GC gene, globulin–complex gene). Combinations
of two of these (rs7041 and rs4588) result in three polymorphic
alleles and six major phenotypes. These phenotypes may have
different binding affinities for the vitamin D metabolites (18)
and the serum 25(OH)D levels do differ between subjects with
different DBP phenotypes (12). The distribution of the six
variants also differs between races (19).
In addition to the skeleton vitamin D deficiency has
been associated with a number of diseases, like mortality,
cancer, immunological diseases, cardio-vascular diseases,
and diabetes (20). Most of these relations are based on
observational studies only, where 25(OH)D has been measured
in old serum samples and subsequent diseases recorded.
For these studies, measurement of total serum 25(OH)D
has been employed, whereas there has been little focus on
DBP [where the major part of the circulating 25(OH)D
is bound] or the free form which potentially may be the
most important.
The serum level of free 25(OH)D has traditionally been
calculated based on measurements of total 25(OH)D, DBP,
and albumin concentrations (21–23). However, measurement
of DBP depends on type of antibody employed (monoclonal
or polyclonal) (19), and it has usually been assumed that the
vitamin D binding-coefficient for each of the six prevalent
DBP phenotypes are equal. The validity of the free 25(OH)D
calculations have therefore been questioned (24). Lately, kits for
direct measurement free 25(OH)D has become commercially
available which has caused a renewed interest in the relation
between free serum 25(OH)D, as well as DBP, and disease states
(25). However, further validation and standardization of this
assay is still needed in subjects with major illnesses or with
abnormal DBP or protein concentrations (16).
In the present review these relations will be summarized for
the metabolic disorders type 1 and type 2 diabetes (T1DM and
T2DM), presented separately.
T2DM
Serum 25(OH)D and T2DM
There are many reasons for why vitamin D could influence
the development of T2DM. Thus, the vitamin D activating
hydroxylases and the VDR are found in the pancreatic beta-
cells (26,27), 1,25(OH)2D may induce insulin secretion (28), and
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Jorde DBP, Total and Free 25(OH)D and Diabetes
vitamin D may have an anti-inflammatory effect that may prevent
insulin resistance (29).
In line with this, there are a number of observational studies
on the relation between serum 25(OH)D concentration and
incident diabetes, and practically all confirm an association
(30). Thus, in a study by Afzal et al. on 31,040 subjects with
measurement of serum 25(OH)D followed for up to 34 years,
participants who had a 20 nmol/L reduction in 25(OH)D had a
16% increased risk of T2DM (31). Similarly, Ye et al. combined
22 studies in a meta-analysis that included 8,492 cases and 89,698
controls and found a 21% increased risk of T2DM per 25 nmol/L
lower 25(OH)D concentration (32).
However, for vitamin D there is a strong possibility of revers
causation and other methods than observational studies are
needed for confirmation, as recently reviewed by Angelotti and
Pittas (30).
There are a few RCTs with vitamin D specifically designed
for prevention of T2DM in subjects at risk. Thus, Davidson
et al. included 109 subjects with prediabetes and randomized
them to high dose vitamin D (mean weekly dose 88,865 IU)
vs. placebo. However, no significant effects on insulin secretion,
insulin sensitivity or development of diabetes were found after
1 year (33). Similarly, in a study from Tromsø, Norway,
Jorde et al. randomized 511 subjects with reduced glucose
tolerance to 20,000 IU vitamin D per week vs. placebo for
a maximum of 5 years, but found no difference between the
groups in development of T2DM (34). However, both studies
were underpowered for detection of minor effects. And finally,
the effect of giving vitamin D to subjects with established
T2DM do at best show a marginal effect on HbA1c with a
reduction of 0.32% in HbA1c as compared with placebo according
to a review by Lee et al. that included nine trial with 3,324
participants (35).
Another approach to the vitamin D—T2DM question is
the Mendelian randomization. Several SNPs are associated
with serum 25(OH)D level; SNPs in the DHCR7 gene
related to vitamin D synthesis, the CYP2R1 gene related
to 25-hydroxylation, and the CYP24A1 gene related to 24-
hydroxylation and degradation (36). When these SNPs are
combined to a genetic score, the highest vs. the lowest
scores result in 5–20% difference in serum 25(OH)D levels.
However, in the most recent and largest meta-analysis including
five studies with 28,144 cases and 76,344 non-cases, no
significant association with T2DM was found, neither for
the individual SNPs tested, nor when combined to a genetic
score (32).
There are, however, many shortcomings of the Mendelian
randomization approach. So far it only predicts differences in
serum 25(OH)D concentration and not the free fraction, and the
alleles tested only explain a small part of the variance in serum
25(OH)D level.
One may therefore conclude that although a low serum
25(OH)D level do predict development of T2DM, this is most
likely due to confounding or reverse causality, although minor
effects cannot be excluded. Hopefully the ongoing D2d study that
has included 2,423 participants with prediabets randomized to
4000 IU vitamin D daily vs. placebo may settle this question (37).
Free 25(OH)D and T2DM
There are several reports where the directly measured free
fraction of 25(OH)D has been compared with total 25(OH)D
regarding biological effects of vitamin D. Thus, Johnsen et al.
found a better correlation for free than for total 25(OH)D
regarding bone density (24), whereas that was not found in study
by Michaelsson et al. (38). For PTH similar relations have been
found for free and total 25(OH)D in most studies (24,39–41),
whereas Lopez-Molina et al. in healthy children found better
correlation with markers of phosphocalcic metabolism for free
than for total 25(OH)D (42). Shieh et al. found in the early phase
(first 4 weeks) of vitamin D treatment the free 25(OH)D, but not
the total 25(OH)D, to be associated with a decrease in serum PTH
(43). In inflammatory diseases the results are also mixed with
free 25(OH)D correlating better to disease activity in ulcerative
colitis (44), whereas total 25(OH)D correlates best to activity in
systemic lupus erythematosus (45). For markers of inflammation
(IL-6) in older men free and total 25(OH)D appear to correlate
equally (46). And finally and most important, in a study by Yu
et al. the free but not total 25(OH)D was associated with risk
of mortality in patients with coronary artery disease (47). The
study included 1,387 patients followed for a median time of
6.7 years, during which period 205 patients died. The all-cause
mortality was 64% higher in the lowest free 25(OH)D quartile
vs. the highest free 25(OH)D quartile, whereas the corresponding
analysis using 25(OH)D did not show a significant difference or
trend across the quartiles.
So far, there are no studies where the free 25(OH)D has been
compared with total 25(OH)D as predictor for development of
T2DM. However, there is a publication by Lee et al. that included
1,189 non-diabetic subjects where the free and total form of
25(OH)D were measured and related to acute insulin response
and glucose disposition index based on intravenous glucose
tolerance tests (48). Both free and total 25(OH)D were positively
associated with these measures, but after adjustment for BMI,
only free 25(OH)D was significant related to insulin secretion.
Based on the above papers, one cannot conclude that
measurements of free vs. total serum 25(OH)D has any advantage
regarding vitamin D responses. This is also difficult to decide,
as comparisons of P-values and correlation coefficients give
indications only.
DBP and T2DM
In addition to being the carrier protein for vitamin D and its
metabolites, DBP has a number of other effects. It acts as a
carrier for free fatty acids (49), it binds actin and may prevent
actin polymerization during tissue damage (50,51), may act as a
macrophage activator and play a part in the inflammation process
by influencing the T-cell response (52). These immunological
effects may differ between the phenotypes (53), and the level of
DBP as well as the different DBP phenotypes might therefore at
least theoretically affect the development of not only T1DM (see
later) but also T2DM.
However, in a case-cohort study design with 958 cases and
3,489 controls Jorde et al. found no association between DBP
phenotypes (based on genotyping of rs4588 and rs7041) and
incident T2DM (54). Furthermore, there were no relations
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Jorde DBP, Total and Free 25(OH)D and Diabetes
between the DBP phenotypes and lipids and blood pressure, but
a slight relation to hip circumference.
Prior to our study Wang et al. made a meta-analysis on
DBP SNPs and T2DM that included six studies (three Caucasian
and three Asian cohorts) with 1,191 cases and 882 controls. No
overall association between the DBP SNPs rs4588 and rs7041 and
T2DM was found. However, when analyzing the Asian cohorts
separately, there were significant associations with T2DM for
both rs7041 and rs4588 (55).
Also after the meta-analysis by Wang et al., Ye at al.
meta-analyzed the DBP SNP rs4588 regarding T2DM in
European cohorts including 28,144 cases and 76,344 controls.
A strong relation between rs4588 and serum 25(OH)D was
found, but not with T2DM (OR 1.00 (CI, 0.97 −1.03)
(32). Accordingly, at least in Caucasians there appears to
be no relation between DBP phenotypes and development
of diabetes.
To the author’s knowledge, there are no longitudinal studies
regarding serum levels of DBP and T2DM. However, there is
one cross-sectional study by Leong et al. on 2,122 adult subjects
that included 201 with diabetes (56). The effect estimate per 50
mg/L DBP increase was 0.79 (95% CI 0.65–0.96) for diabetes,
and there was a marginal relation between higher DBP and lower
fasting blood glucose levels. However, as a cross-sectional study
it could not examine the impact of biological variability of DBP
over time.
T1DM
Serum 25(OH)D and T1DM
The 1α-hydroxylase, necessary for activation of vitamin D, is
expressed in immune cells like the B- and T-cells and the
antigen presenting cells (2). These cells may therefore synthesize
active vitamin D locally. Vitamin D has immune-modulatory
effects (57), and since T1DM is an autoimmune disorder, a
role for vitamin D in pathogenesis as well as treatment thus
possible (58).
However, in a study by Thorsen et al. using a case-cohort
design that included 459 children with T1DM and a control
group of 1,561, no association between maternal serum 25(OH)D
levels sampled repeatedly during pregnancy and subsequent
T1DM in the offsprings was found (59). Furthermore, in two
large Danish populations, one case-cohort study with 912 cases
and 2,866 controls followed for a maximum of 31 years and
a case-control study with 527 matched pairs followed for a
maximum of 23 years, Jacobsen et al. found no relation between
neonatal vitamin D status and later risk of T1DM (60). On the
other hand, there might be a link between intake of vitamin D in
childhood and development of T1DM as reported by Hyppönen
et al. in a birth-cohort study with 12,055 pregnant women in
northern Finland (61). This was also the conclusion in a meta-
analysis by Dong et al. from 2013 that included eight studies
(six case-control and two cohort studies) with vitamin D intake
during early life where the pooled OR for T1DM was 0.71 (95%
CI, 0.51–0.98) (62).
Furthermore, the serum levels of 25(OH)D are lower in
subjects with newly diagnosed T1DM (63) as well as later in the
course of disease compared to similarly aged subjects (64). There
may also be a beneficial effect by vitamin D supplementation
in newly diagnosed T1DM. This was reviewed by Gregoriou
et al. (65) who found a positive effects on the daily insulin dose,
fasting, and stimulated C-peptide response by vitamin D in two
studies. However, only 67 patients were randomized and the
effect was marginal.
To the author’s knowledge there are no studies reporting free
25(OH)D levels in T1DM.
DBP and T1DM
Since the immunological effects of DBP may differ between the
DBP phenotypes (53), relations between the DBP SNPs rs4588
and rs7041 and T1DM are of interest. This was reviewed by
Penna-Martinez and Badenhoop who found that in the majority
of the studies there was no relation between these SNPs and
T1DM (66). As an example, Cooper et al. who included 720
cases and 2,610 controls and used a Mendelian randomization
approach, found no relation between rs4588 and T1DM (67),
whereas in the two studies that did find an association with rs7041
the total number of cases was only 154 (68,69).
There are a few cross-sectional reports on serum DBP levels
in patients with T1DM. In a study by Blanton et al. that
included 203 subjects with T1DM and 153 controls, the serum
DBP levels were ∼10% lower in the T1DM patients (70). A
similar result was found by Thraikill et al. but they could for
a large part ascribe this to increased urinary loss of DBP in
the urine (11). Low serum DBP levels have also been described
in diabetic BB rats together with low serum 1,25(OH)2D
levels accompanied with reduced duodenal calcium absorption,
indicating the possible physiological importance of urinary DBP
loss (71).
CONCLUSIONS
For preventing or treating diabetes, the majority of clinical
studies do not indicate a major role for vitamin D
supplementation, with a possible exception for T1DM in
children. As for many other presumed extra-skeletal effects
of vitamin D, the effect on glucose metabolism must be small
(if present at all) and accordingly difficult to demonstrate. In
most of the vitamin D RCTs the results are also hampered by
the inclusion of subjects who are not truly vitamin D deficient
(72). However, since such subjects (and in particular young
children) need vitamin D for bone health, there are many ethical
problems in including vitamin D deficient subjects in long lasting
RCTs. The “perfect” vitamin D RCT will therefore probably not
be performed.
However, regarding vitamin D and health, the two crucial
questions are how much vitamin D we need for skeletal
health (which everyone agrees is vitamin D dependent),
and if supplementation above that will give any additional
health benefits.
So far, there are too few studies on the relative importance
of measuring total or free 25(OH)D in diabetes and glucose
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Jorde DBP, Total and Free 25(OH)D and Diabetes
metabolism, and too few studies on the importance of DBP
concentration on development and progression of diabetes, to
draw firm conclusion. However, since it is difficult to show
an effect of vitamin D supplementation regarding diabetes, it
follows that finding the right form or metabolite of vitamin D
to measure (7), may for diabetes simply be a search for another
biomarker (73). In disease states with clearly altered DBP levels,
like pregnancy and liver cirrhosis, the situation obviously is
different (9).
AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and
approved it for publication.
ACKNOWLEDGMENTS
UiT The Arctic University of Norway is gratefully acknowledged
for their support.
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Conflict of Interest Statement: The author declares that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Jorde. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) and the
copyright owner(s) are credited and that the original publication in this journal
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Frontiers in Endocrinology | www.frontiersin.org 7February 2019 | Volume 10 | Article 79
20
ORIGINAL RESEARCH
published: 27 March 2019
doi: 10.3389/fendo.2019.00171
Frontiers in Endocrinology | www.frontiersin.org 1March 2019 | Volume 10 | Article 171
Edited by:
Jonathan H. Tobias,
University of Bristol, United Kingdom
Reviewed by:
Jan Josef Stepan,
Charles University, Czechia
Michaël R. Laurent,
University Hospitals Leuven, Belgium
*Correspondence:
Xiangbing Wang
wangx9@rwjms.rutgers.edu
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 07 January 2019
Accepted: 01 March 2019
Published: 27 March 2019
Citation:
Wang X, Sheng Z, Meng L, Su C,
Trooskin S and Shapses SA (2019)
25-Hydroxyvitamin D and Vitamin D
Binding Protein Levels in Patients With
Primary Hyperparathyroidism Before
and After Parathyroidectomy.
Front. Endocrinol. 10:171.
doi: 10.3389/fendo.2019.00171
25-Hydroxyvitamin D and Vitamin D
Binding Protein Levels in Patients
With Primary Hyperparathyroidism
Before and After Parathyroidectomy
Xiangbing Wang 1
*, Zhifeng Sheng 2, Lingqiong Meng 3, Chi Su 1, Stanley Trooskin4and
Sue A. Shapses 3
1Divisions of Endocrinology, Metabolism, and Nutrition, Departments of Medicine and Surgery, Rutgers-Robert Wood
Johnson Medical School, New Brunswick, NJ, United States, 2Institution of Metabolism and Endocrinology, The Second
Xiangya Hospital, Central South University, Changsha, China, 3Department of Nutritional Sciences, Rutgers University, New
Brunswick, NJ, United States, 4Divisions of General Surgery, Departments of Medicine and Surgery, Rutgers-Robert Wood
Johnson Medical School, New Brunswick, NJ, United States
Objective: To evaluate vitamin D binding protein and free 25-hydroxyvitamin D [25(OH)D]
levels in healthy controls compared to primary hyperparathyroidism (PHPT) patients, and
to examine PHPT before and after surgery.
Methods: Seventy-five PHPT patients and 75 healthy age, gender, and body mass
index (BMI) -matched control subjects were examined. In addition, 25 PHPT patients
underwent parathyroidectomy and had a 3-month follow up visit. Levels of total and free
25(OH)D, DBP, and intact parathyroid hormone (iPTH) were determined before and 3
months after surgery.
Results: There was no significant difference in age and BMI between PHPT patients and
controls. Levels of 25(OH)D and DBP were lower in PHPT patients compared to controls
(p<0.01). There was no significant difference in calculated free and bioavailable 25(OH)D
levels between PHPT patients and controls. Calcium and iPTH levels decreased to normal
but DBP and DBP-bound-25(OH)D increased (P<0.001) after parathyroidectomy.
Levels of DBP were inversely correlated with iPTH (r= −0.406, P<0.001) and calcium
levels (r= −0.423, P<0.001).
Conclusion: Serum DBP levels were lower in patients with PHPT and
parathyroidectomy restored DBP levels. We suggest that lower DBP levels is one
of contributing mechanisms of low total 25(OH)D in PTHP patients and the total
25(OH)D levels might not reflect true vitamin D status in PHPT patients.
Keywords: vitamin D binding protein, vitamin D deficiency, parathyroid hormone, calcium metabolism,
hyperparathyroidism, parathyroidectomy
INTRODUCTION
Total 25(OH)D level has been recognized as an optimal indicator of vitamin D nutrition status,
and lower 25(OH)D concentration is usually considered as vitamin D deficiency or insufficiency
in clinical practice. Low total 25(OH)D concentration, which is common in PHPT patients, is
associated with the severity of the disease and high parathyroid adenoma weight (1–3). Low
21
Wang et al. DBP and 25OHD in PHPT Patients
25(OH)D levels also exist in many chronic conditions such as
end-stage liver disease and nephrotic syndrome, and in critical
illness where intact parathyroid hormone levels are not elevated
(4,5). The majority of circulating 25(OH)D tightly bound to
DBP, with a smaller amount (10–15%) bound to albumin. Less
than 1% of circulating vitamin D metabolites exists in a free,
unbound form (5). The variations in the 25(OH)D levels in these
conditions result from variations in the binding of 25(OH)D to
DBP (4).
Previous studies showed that DBP are lower in PHPT patients
compared with age, BMI, and gender matched (6–8) or genetic
background matched controlled subjects (7). It is unclear how
DBP is regulated in and if the elevated iPTH plays a role in PHPT,
or if DBP simply a biomarker of circulating 25(OH)D. Since the
majority (>85%) of circulating 25(OH)D is bound to DBP, we
suggest that decreased DBP might be one of mechanisms of low
total 25(OH)D levels in PHPT patient (8). The causes of lower
DBP concentration in the serum of PHPT patients remained
unknown. We hypothesize that the elevated iPTH or calcium
levels inhibit DBP production in the liver of PHPT patient. In
our current study, we investigated the effects of lower calcium
and iPTH levels by parathyroidectomy on DPB in PHPT patients.
We also compared levels of 25(OH)D, DBP, and calculated free
and bioavailable 25(OH)D in patients with PHPT with normal
controls. The aim of this study is to investigate the effects of
parathyroidectomy on DBP and DBP-bound 25(OH)D levels in
PHPT patients.
METHODS
Study Subjects
Seventy-five PHPT patients (61 Caucasians, eight African
American, four Asians, and two Hispanic Americans) were
seen at the Endocrinology and General Surgery clinics of
Robert Wood Johnson University Hospital from January 2010 to
December 2016 at prior to treatment. Most of the patients were
relatively asymptomatic with less severe PHPT profile (9). The
inclusion criteria were: (1) a serum calcium level >10.6 mg/dL
(8.6–10.4 mg/dl) and intact PTH (iPTH) >66 pg/mL (15–65
pg/mL), (2) age 20–80 years, and (3) 24-h urine calcium >100 mg
(100–300 mg/24 h) with fraction excretion of calcium >0.01.
The exclusion criteria were: (1) hormone replacement therapy
or contraceptive pills, (2) hepatic dysfunction, or (3) renal
dysfunction, and (4) BMI >40 (kg/m2). Seventy-five age, gender,
and BMI-matched healthy volunteers (62 Caucasians, eight
African Americans, four Asians, and one Hispanic American)
(10) from the community were included as controls after a
multistep screening process and did not take contraceptive
pills. The healthy controls took 400 IU vitamin D supplement.
Supplemental vitamin D intake in patients before surgery is
not known. Twenty-five PHPT patients (seven males and 18
females) underwent parathyroidectomy (PTX) monitored by
intro-operative iPHT levels and were examined at 3 months
during their follow-up visit after surgery. All minimally invasive
PTX were done by one surgeon and all patients were advised to
take 0.25 mcg calcitriol for 1–2 weeks and 1,000–2,000 IU vitamin
D for 1–3 months after PTX to prevent hypocalcemia as standard
post-operative clinical care (11). All subjects and patients signed
an informed consent and the use of human subjects in this study
was approved by the IRB at Rutgers University.
Sample Collections and Assays
Venous blood samples were collected from patients and
controlled subjects after a 12-h overnight fast. Twenty-five PHPT
patients had parathyroid surgery and finished 3 months’ post-
surgery follow up visit. Serum was separated and stored at −70 C
for measurement of 25(OH)D and DBP levels. Intact-PTH,
serum calcium, and albumin were determined by commercial
laboratories. The laboratory uses both internal and external
standards, and also participated in the international Vitamin
D External Quality Assessment Scheme to ensure the quality
and accuracy of the 25(OH)D analysis and serum 25(OH)D
levels (radioimmunoassay; DiaSorin) CV <12.5%). DBP levels
in serum were determined using a commercial polyclonal ELISA
kit (ALPCO, Salem, NH). The intra- and inter-assay coefficients
of variation are 5.0 and 12.7%, respectively. Free, bioavailable,
albumin-bound and DBP-bound 25(OH)D concentrations were
calculated using equations adapted from Bikle et al. (12).
Statistical Analyses
Results are expressed as mean ±SD. Shapiro-Wilk test was used
to check normality. Two-tailed Student’s t-test and Wilcoxon
Rank Sum test were used to compare values between groups with
normally and non-normally distribution, respectively. Changes
before and after parathyroidectomy were compared with a paired
Student’s t-test. Correlation coefficients and linear regression
were used to assess relationships between variables. A P<0.05
was defined as the level of significance. Statistical analysis was
performed with SAS v9.4.
TABLE 1 | Subject characteristics and serum concentrations.
Variable Control PHPT P-value
n=75 n=75
Age (years) 58.0 ±8.1 59.3 ±12.3 0.314
BMI (kg/m2) 29.9 ±2.1 30.6 ±4.8 0.373
Calcium (mg/dL) 9.4 ±0.5 11.1 ±0.6 <0.001
iPTH (pg/mL) 37.9 ±17.3 140.4 ±70.5 <0.001
25(OH)D (ng/mL) 28.3 ±5.4 23.6 ±8.3 <0.001
DBP (mg/dL) 42.1 ±7.0 35.2 ±7.9 <0.001
Albumin (g/dL) 4.5 ±0.2 4.3 ±0.3 <0.001
DBP-bound
25(OH)D (ng/mL)
26.4 ±5.1 21.8 ±7.7 <0.001
Albumin-bound
25(OH)D (ng/mL)
1.9 ±0.5 1.8 ±0.8 0.082
Bioavailable
25(OH)D (ng/mL)
1.9 ±0.5 1.8 ±0.8 0.083
Free 25(OH)D
(pg/mL)
4.7 ±1.1 4.7 ±2.0 0.573
BMI, body mass index; iPTH, intact parathyroid hormone; 25(OH)D, 25-hydroxyvitamin
D; DBP
, vitamin D binding protein. Data shows as mean ±standard deviation.
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Wang et al. DBP and 25OHD in PHPT Patients
RESULTS
Seventy-five PHPT patient (23 men, 11 premenopausal women,
and 41 postmenopausal women) and 75 control subjects (19 men,
11 premenopausal women, and 45 postmenopausal women) were
included in this study. The mean concentrations of calcium,
albumin, 25(OH)D, iPTH, and DBP determined in the serum
samples from control subjects and PHPT patients are shown
in (Table 1). Both total 25(OH)D and DBP levels were about
17% lower in PHPT patients compared to control subjects
(P<0.001). There was no significant difference in albumin-
bound 25(OH)D, but DBP-bound 25(OH)D was also 17%
lower in PTHP patients compared to control subjects (P<
0.001). In addition, albumin levels were significantly lower in
PHPT patients than in control subjects (p<0.001). There
were no significant differences between bioavailable and free
25(OH)D between healthy controls and PHPT patients (Table 1).
Comparison of individual 25(OH), DBP, free 25(OH)D, and
bioavailable 25(OH) D between healthy controls and PHPT
patients were showed in Figure 1.
FIGURE 1 | (A-D) Comparison of serum levels of DBP and 25 (OH) D between PHPT patients and control subjects. DBP, vitamin D binding protein; 25OHD,
25-hydroxyvitamin D. ***P<0.001.
TABLE 2 | Spearman correlation coefficients between DBP and other variables (n=150).
Age BMI Calcium iPTH 25(OH)D Albumin Free 25(OH)D Bioavailable
25(OH)D
DBP −0.210a−0.192a−0.423c−0.406c0.253b0.139a−0.344c−0.295c
Age 0.113 0.076 0.055 0.132 −0.074 0.301c0.278c
BMI 0.038 0.074 −0.272c−0.141 −0.121 −0.145
Calcium 0.751c−0.292c−0.218b−0.057 −0.114
iPTH −0.418c−0.312c−0.153 −0.233b
25(OH)D 0.225b0.767c0.786c
Albumin 0.092 0.284c
Free
25(OH)D
0.972c
BMI, body mass index; iPTH, intact parathyroid hormone; 25(OH)D, 25-hydroxyvitamin D; DBP, vitamin D binding protein. Bold means significan. aP<0.05; bP<0.01; cP<0.001.
Frontiers in Endocrinology | www.frontiersin.org 3March 2019 | Volume 10 | Article 171
23
Wang et al. DBP and 25OHD in PHPT Patients
Levels of DPB (n=150) were positively correlated with total
25(OH)D (r=0.253, P<0.01) but inversely correlated with
iPTH (r= −0.406, P<0.001) and calcium (r= −0.423, P<
0.001; Table 2). Levels of iPTH inversely correlated with total
25(OH)D (r= −0.418, P<0.001) and bioavailable 25(OH)D (r
= −0.233, P<0.01; Table 2).
In PHPT patients who underwent parathyroidectomy, serum
iPTH, and calcium decreased to normal but DBP levels increased
by 15% (P<0.01, Table 3). Serum total 25(OH) D were increased
by 43 % (p<0.001) but DBP-bound 25(OH)D also increased
by 43% (P=0.001). As a result, there was an attenuated rise
in bioavailable (23%, P=0.024) and free 25(OH)D (21%, P
=0.021, Table 3). Comparison of individual 25(OH), DBP, free
25(OH)D, and bioavailable 25(OH) D before and after PTX was
showed in Figure 2. Multiple regression showed that none of
the variables (Ca, PTH or 25(OH)D) predicted the change in
DBP after parathyroidectomy (not shown). In addition, there
were no predictors for the rise in 25(OH)D due to surgery.
Multiple Regression indicated that only the change in albumin
predicted change in bioavailable 25(OH)D (p=0.027), but not
free 25(OH)D (p=0.122) after parathyroidectomy.
DISCUSSION
The results of our current study demonstrate that PHPT patients
have lower serum levels of DBP and total serum 25(OH)D
consistent with our previous study (8) and study by Battista et al.
(7). In addition, our data confirmed our previous studies that
the calculated free or bioavailable 25(OH)D remained unchanged
compared with normal control subjects (8). We also showed
that PTX increases DBP and DBP-bound 25(OH)D levels. Thus,
based on our findings and because supplemental vitamin D raises
25(OH)D, but not DBP (13–15), we suggest that the increased
DBP level after PTX might be due to the decreased iPTH or
lowered calcium levels after surgery. This supports the hypothesis
that DBP is not simply a biomarker of 25(OH)D. Our current
TABLE 3 | PHPT profile changes before and after parathyroidectomy.
Before After P-value
n=25 n=25
BMI (kg/ m2) 31.0 ±5.6 29.0 ±4.3 0.127
Calcium (mg/ dl) 11.0 ±0.6 9.7 ±0.4 <0.001
iPTH (pg/ ml) 121.5 ±40.5 44.7 ±8.2 <0.001
25(OH)D (ng/ ml) 26.4 ±7.8 37.7 ±12.1 <0.001
DBP (mg/ dl) 38.9 ±9.8 44.7 ±8.2 0.001
Albumin (g/ dl) 4.3 ±0.9 4.5 ±0.2 0.046
DBP-bound 25(OH)D
(ng/ mL)
24.6 ±7.3 35.4 ±11.1 <0.001
Albumin-bound
25(OH)D (ng/ mL)
2.0 ±0.5 2.36 ±0.75 0.024
Bioavailable 25(OH)D
(ng/mL)
1.94 ±0.94 2.36 ±0.74 0.024
Free25(OH)D (pg/mL) 4.89 ±2.05 5.90 ±1.9 0.021
BMI, body mass index; DBP, vitamin D binding protein; iPTH, intact parathyroid hormone;
25(OH)D, 25-hydroxyvitamin D. Data are means ±standard deviation (Paired t-test).
results also support the concept that the lower DBP levels in
PHPT compared to healthy matched controls may be one of
the factors contributing to the low total 25(OH)D levels in
PHPT patients. Another possible factor leading to the low total
25(OH)D levels in PHPT patients include the conversion to 1,25
or 24,25 (OH)2D due to elevated iPTH or FGF-23 (6,16).
Aloia et al. found that black Americans have lower levels of
total 25(OH)D but the free 25(OH)D remains relative unchanged
by direct measurement of free 25(OH)D (17). Pre-menopausal
women have higher serum DBP, estradiol, and 25(OH)D levels
than postmenopausal women (18). The calculated free 25OHD
was also lower in postmenopausal women than that of control
subjects, but to a much lesser degree than total 25OHD (18).
In a recent study, Pilz et al. found that women taking estrogen
containing contraceptive measures have higher total 25(OH)D
but unaltered free 25(OH)D levels by direct measurement (19).
The results suggest that total 25(OH)D levels might not be
an accurate marker of bioactive vitamin D status in at least
a few situations, including black Americans or women taking
hormonal contraceptive pill or other clinical situations (5,20)
Bioavailable 25(OH)D may be a better measure of vitamin D
status with respect to bone mineral density (BMD) and mineral
metabolism, as has been shown in nephrotic syndrome patients
(21). Lai et al. found that cirrhosis patients with low albumin had
lower DBP, total 25(OH)D, and free 25(OH)D levels and suggest
that total 25(OH)D is not accurate marker for vitamin D status
in these patients (22). Yu et al. reported that it is bioavailable
and free 25(OH)D levels, not total 25(OH), associated with the
risk of mortality in Chinese patients with coronary artery disease
(23). Our results suggest that the total 25(OH)D levels in PHPT
patients may not be a good indicator of vitamin D status before or
after surgery since there is a much lower rise in both bioavailable
and free 25(OH)D concentrations.
The appropriate management of asymptomatic PHPT still
require more evidence from clinical studies (1,24) despite the
guidelines for PHPT have been revised recently (25). There are
controversies about vitamin D supplementation in the PTHP
patient with low 25(OH) levels. Marcocci et al. reviewed three
studies; two demonstrated that vitamin D supplementation had
no significant influence on serum and urinary calcium levels, and
one study showed no clinical benefit, while in the third study of
27 PHPT patients, 12 patients developed either increased serum
calcium levels or increased urine calcium excretion (24). Our data
show here that there are no significant differences in free and
bioavailable 25(OH)D levels between PHPT patients and control
subjects. Given the pre-existing high serum Ca in PHPT patients,
we suggest that clinicians should be aware of this treating PHPT
patients with vitamin D, especially when using a loading dose of
vitamin D supplementation (26).
The DBP concentration is relatively stable throughout life
but is altered by gender, menopausal status (10), and genetic
backgrounds (27–29). In the current study, we matched PHPT
patients and control subjects for these factors. The underlying
mechanism for lower DBP concentration in PHPT patients,
at least in part, may be explained by the higher iPTH levels
inhibition of liver-derived DBP in PHPT patient, a finding
that has been reported previously (8) and PTH/PTH-related
Frontiers in Endocrinology | www.frontiersin.org 4March 2019 | Volume 10 | Article 171
24
Wang et al. DBP and 25OHD in PHPT Patients
FIGURE 2 | (A-D) Comparison of serum levels of DBP and 25(OH)D before and after parathyroidectomy. DBP, vitamin D binding protein; 25OHD, 25-hydroxyvitamin
D. *P<0.05; **P<0.01; ***P<0.001.
protein receptor is highly expressed in the liver (30). Conditions
such as malnutrition and liver failure might affect DBP,
albumin, and other liver-specific protein status (22). Serum DBP
concentrations were inversely correlated with iPTH and calcium
levels and DBP increased after decreasing iPTH and calcium
by parathyroid surgery. It is also possible that a reduced DPB
is a compensatory mechanism in PHPT to ensure that under
conditions of low total 25(OH)D, there is adequate circulating
free or bioavailable 25(OH)D. Also, the mechanism regulating
the rise in DBP after parathyroidectomy remains unclear, but
it is suggested that studying this population may help to better
understand the binding protein and its regulation of normal
vitamin D metabolism.
The limitations of the study are the relatively small sample
size, including only 25 PHPT patients had 3 months’ post-
surgery data. Total 25OHD levels were measured by RIA, and
not by mass spectrometry which is considered more accurate,
but we used internal and external controls to increase accuracy.
Another limitation is that this study does not include serum
phosphate or FGF-23 levels and the study design cannot confirm
a mechanism of low total 25(OH)D or DBP in PHPT. All
patients took calcitriol for 1–2 weeks after surgery and advised
to take a vitamin D supplement, as standard post-operative
clinical care (11) to prevent risk of low serum calcium levels.
As a result, this may be another reason for the increase in
serum total 25(OH)D at 3 months after parathyroidectomy.
Moreover, calculated free 25(OH)D utilize equations that use
average binding coefficients for DBP and albumin may not be as
accurate as direct measurements (27).
In conclusion, total 25(OH)D and DBP levels are lower in
PHPT patients but calculated free 25(OH) remained relatively
unchanged. Parathyroidectomy increased DBP and DBP-bound
25(OH)D levels. Further research is required to investigate
whether free 25OHD is the better marker of vitamin D status in
the PHPT patient.
AUTHOR CONTRIBUTIONS
ZS, LM, and CS contributed to recruiting patients, data collection
and analysis, and manuscript preparation. ST contributed to
Parathyroidectomy and manuscript preparation. SS contributed
to experimental design, recruiting control subjects, data analysis
and manuscript preparation. XW contributed to experimental
design, data analysis and manuscript preparation.
ACKNOWLEDGMENTS
We thank Dr. Wei Sun MD., Ph.D. for assisted in DBP ELISA
assay; Aseel Al-Dayyeni MD, and Aliza Rubin, RN for help
recruiting PHPT patients, Professor Huizgou Fan for assisted in
the preparation of this manuscript.
Frontiers in Endocrinology | www.frontiersin.org 5March 2019 | Volume 10 | Article 171
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Wang et al. DBP and 25OHD in PHPT Patients
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Wang, Sheng, Meng, Su, Trooskin and Shapses. This is an open-
access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms.
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26
REVIEW
published: 28 May 2019
doi: 10.3389/fendo.2019.00317
Frontiers in Endocrinology | www.frontiersin.org 1May 2019 | Volume 10 | Article 317
Edited by:
Giacomina Brunetti,
University of Bari Aldo Moro, Italy
Reviewed by:
Ian R. Reid,
The University of Auckland,
New Zealand
Jan Josef Stepan,
Charles University, Czechia
*Correspondence:
Daniel David Bikle
daniel.bikle@ucsf.edu
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 25 March 2019
Accepted: 01 May 2019
Published: 28 May 2019
Citation:
Bikle DD and Schwartz J (2019)
Vitamin D Binding Protein, Total and
Free Vitamin D Levels in Different
Physiological and Pathophysiological
Conditions. Front. Endocrinol. 10:317.
doi: 10.3389/fendo.2019.00317
Vitamin D Binding Protein, Total and
Free Vitamin D Levels in Different
Physiological and Pathophysiological
Conditions
Daniel David Bikle 1,2
*and Janice Schwartz 1
1Department of Medicine, University of California, San Francisco, San Francisco, CA, United States, 2Endocrine Research
Unit, San Francisco Veterans Affairs Medical Center, San Francisco, CA, United States
This review focuses on the biologic importance of the vitamin D binding protein (DBP)
with emphasis on its regulation of total and free vitamin D metabolite levels in various
clinical conditions. Nearly all DBP is produced in the liver, where its regulation is
influenced by estrogen, glucocorticoids and inflammatory cytokines but not by vitamin
D itself. DBP is the most polymorphic protein known, and different DBP alleles can
have substantial impact on its biologic functions. The three most common alleles—Gc1f,
Gc1s, Gc2—differ in their affinity with the vitamin D metabolites and have been variably
associated with a number of clinical conditions. Although DBP has a number of biologic
functions independent of vitamin D, its major biologic function is that of regulating
circulating free and total levels of vitamin D metabolites. 25 hydroxyvitamin D (25(OH)D)
is the best studied form of vitamin D as it provides the best measure of vitamin D status.
In a normal non-pregnant individual, approximately 0.03% of 25(OH)D is free; 85% is
bound to DBP, 15% is bound to albumin. The free hormone hypothesis postulates that
only free 25(OH)D can enter cells. This hypothesis is supported by the observation
that mice lacking DBP, and therefore with essentially undetectable 25(OH)D levels,
do not show signs of vitamin D deficiency unless put on a vitamin D deficient diet.
Similar observations have recently been described in a family with a DBP mutation.
This hypothesis also applies to other protein bound lipophilic hormones including
glucocorticoids, sex steroids, and thyroid hormone. However, tissues expressing the
megalin/cubilin complex, such as the kidney, have the capability of taking up 25(OH)D still
bound to DBP, but most tissues rely on the free level. Attempts to calculate the free level
using affinity constants generated in a normal individual along with measurement of DBP
and total 25(OH)D have not accurately reflected directly measured free levels in a number
of clinical conditions. In this review, we examine the impact of different clinical conditions
as well as different DBP alleles on the relationship between total and free 25(OH)D, using
only data in which the free 25(OH)D level was directly measured. The major conclusion
is that a number of clinical conditions alter this relationship, raising the question whether
measuring just total 25(OH)D might be misleading regarding the assessment of vitamin
D status, and such assessment might be improved by measuring free 25(OH)D instead
of or in addition to total 25(OH)D.
Keywords: vitamin D binding protein, vitamin D, free 25(OH)D, free hormone hypothesis, megalin, polymorphisms,
liver cirrhosis, pregnancy
27
Bikle and Schwartz DBP and Free 25(OH)D
INTRODUCTION
Vitamin D enters the body either from its production in the
skin or absorption from the intestine. In either case, vitamin
D must be transported to tissues such as the liver where it
is metabolized to its major circulating form, 25(OH)D, by a
variety of enzymes with 25-hydroxylase activity, the major one
being CYP2R1. 25(OH)D is then transported to tissues such as
the kidney where it gets further metabolized to its biologically
active metabolite 1,25 dihydroxyvitamin D (1,25(OH)2D) by the
mitochondrial based CYP27B1. CYP24A1, found in most tissues,
is the major enzyme catabolizing 1,25(OH)2D, thus controlling
its impact on a cell specific basis. Vitamin D binding protein
(DBP) is the key transport protein which, along with albumin,
binds over 99% of the circulating vitamin D metabolites. For most
cells it is the unbound 25(OH)D that enters cells (free hormone
hypothesis), but at least in some cells such as in the kidney, and
likely in the parathyroid gland and placenta, DBP participates in
the transport of the 25(OH)D into the cell via a megalin/cubilin
complex. Although our focus will be on the transport function of
DBP and how that relates to the total and free vitamin D levels
in different physiologic and pathophysiologic conditions, DBP
has a number of functions independent of its role as a vitamin
D transport protein. These functions will be briefly reviewed as
they do contribute to the role DBP plays in health and sickness
independent of its role in vitamin D transport. DBP is a highly
polymorphic protein with at least 120 isoforms distinguished by
electrophoresis. Of these, three major isoforms have received the
most interest—Gc1f, Gc1s, and Gc2. Their structural differences
affect DBP function in ways that have an impact on a number of
clinical conditions that will be reviewed.
VITAMIN D BINDING PROTEIN
Genomic Regulation
The human DBP gene is located on chromosome 4q12-q13. It
is 35 kb in length and comprised of 13 exons encoding 474
amino acids including a 16 amino acid leader sequence, which
is cleaved before release. Numerous tissues express DBP, but the
liver is the major source (1). The expression of DBP is increased
by estrogen (2) as appreciated with the rise in DBP during
pregnancy (3,4) and with oral contraceptive administration (5).
However, the exact mechanism for this induction is not clear
as a response element for the estrogen receptor in the DBP
promoter has not been identified. Androgens, on the other hand,
do not appear to affect DBP expression (2). Dexamethasone and
certain cytokines such as IL-6 also increase DBP production,
whereas TGFβis inhibitory (6). As for estrogen, the mechanism
underlying such regulation is unclear. However, these cytokines
and glucocorticoids are likely to play a role in the increase
in DBP production following trauma (after an initial decrease
in levels due to actin clearance, see below) (7) and acute
liver failure (8), which we will discuss subsequently. Primary
hyperparathyroidism, on the other hand, is associated with
a reduction in DBP levels, likely contributing to the lower
25(OH)D levels in these patients as the free 25(OH)D is not
reduced (9). Vitamin D itself or any of its metabolites do not
regulate DBP production (10).
Structure and Polymorphisms
The mature human DBP is approximately 58 kD in size, although
differences in glycosylation of the protein for different alleles
alter the actual size. DBP is the most polymorphic gene known.
Before the appreciation of its role as a carrier of the vitamin
D metabolites these polymorphisms in DBP were used by
population geneticists to track different populations, referring
to the protein as Gc globulin. Over 120 variants have been
described based on electrophoretic properties (11) as noted above
with 1,242 polymorphisms currently listed in the NCBI database
(12). Of these variants, the Gc1f and Gc1s (rs7041 locus) and
Gc2 (rs4588 locus) are the most common (Figure 1). Gc1f and
Gc1s involve two polymorphisms, one at aa 432 (416 in the
mature DBP) and one at 436 (420 in the mature DBP). The
1f allele encodes the sequence of aa between 432 and 436 as
DATPT, the 1s allele encodes the sequence EATPT. This subtle
difference in charge makes Gcf run faster (fast) than the Gcs
(slow) during electrophoresis. The Gc2 allele encodes DATPK
which runs slower still. Glycosylation further distinguishes the
Gc1 variants from the Gc2 variant. The threonine (T) in Gc1
binds N-acetylgalactosamine to which galactose and sialic acid
bind in tandem. The lysine (K) in comparable position in Gc2
is not glycosylated (13,14). This affects the conversion of DBP
to DBP-MAF (macrophage activating factor), which involves a
partial deglycosylation removing the galactose and sialic acid by
the sequential action of sialidase and β-galactosidase by T and
B cells (15). The significance of this for the biologic function is
described below.
DBP is comprised of 3 structurally similar domains. The first
domain is the binding site for the vitamin D metabolites (aa 35–
49). Fatty acid binding utilizes a single high affinity site for both
palmitic acid and arachidonic acid, but only arachidonic acid
competes with 25(OH)D for binding (16,17). The actin binding
site is located at aa 373–403, spanning parts of domains 2 and
3, but part of domain 1 is also involved (18,19). The C5a/C5a
des Arg binding site is located at aa 130–149 (20). DBP serves
as a cochemotactic factor for C51/C5a des Arg in its regulation
of neutrophil functions (21). Membrane binding sites have been
identified in aa 150–172 and 379–402 (22).
Biologic Function
Binding to and Transport of Vitamin D Metabolites
DBP was discovered by Hirschfeld in 1959 (23), and originally
called group specific component (Gc-globulin), but it was not
until 1975 that its function as a vitamin D transport protein
was appreciated (24). In normal individuals, ∼85% of circulating
vitamin D metabolites are bound to DBP. Albumin binds
∼15% of these metabolites and does so with much lower
affinity. Approximately 0.4% of total 1,25(OH)2D3and 0.03%
of total 25OHD3are free in serum from normal non-pregnant
individuals. The affinity of DBP for the vitamin D2metabolites
is somewhat less than that for the vitamin D3metabolites (25).
The designation of “bioavailable” vitamin D metabolite is the sum
of the free vitamin D metabolite and that bound to albumin,
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Bikle and Schwartz DBP and Free 25(OH)D
FIGURE 1 | The major DBP alleles. The amino acid differences between the three major DBP alleles are depicted. These differences affect not only their
electrophoretic properties but also their glycosylation pattern. In particular Gc2 is not glycosylated, which prevents it from forming the DBP-macrophage activating
factor (DBP-MAF). Other biologic differences are discussed in the text.
thus measuring around 15% in normal individuals [review in
(26)]. However, the degree to which the albumin fraction is
truly bioavailable is not clear (27). The free hormone hypothesis
postulates that only the non-bound fraction (the free fraction)
of hormones that otherwise circulate in blood bound to their
carrier proteins is able to enter cells and exert their biologic
effects. However, at least for some tissues, a transport system
has been identified that takes up the 25(OH)D (and presumably
other vitamin D metabolites) attached to DBP. That system
involves megalin/cubilin.
The role of megalin for vitamin D metabolism was discovered
by Nykjaer et al. (28), who found extensive loss of DBP in
the megalin knockout mouse and 25(OH)D in its urine. These
mice have very poor survival rates. More recently, a kidney
specific knockout of megalin was developed with a good survival
rate, enabling longer term studies that demonstrated reduced
circulating levels of the vitamin D metabolites, hypocalcemia,
and osteomalacia (29). Cubilin, together with megalin, forms
part of the complex facilitating this transport mechanism [review
in (30)]. Other tissues express the megalin/cubilin complex
including the parathyroid gland and placenta, but its role
outside the kidney has received little interest (30). Moreover,
activated monocytes may be able to accumulate DBP by a
megalin independent process, although this too needs further
study (31,32).
The physiologic role of DBP is well-illustrated in the DBP
knockout mouse. In these mice the vitamin D metabolites are
presumably all free and/or bioavailable as albumin levels are
normal. Unlike the megalin knockout mice, mice lacking DBP
do not show evidence of vitamin D deficiency unless placed
on a vitamin D deficient diet despite having very low levels of
serum 25(OH)D and 1,25(OH)2D and increased loss of these
metabolites in the urine (33). Tissue levels of 1,25(OH)2D were
normal in the DBP knockout mice, and markers of vitamin
D function such as expression of intestinal TRPV6, calbindin
9k, PMCA1b, and renal TRPV5 were maintained. Moreover,
injection of 1,25(OH)2D into these DBP knockouts showed a
more rapid increase in the expression of Cyp24A1, TRPV5,
and TRPV6 than in DBP intact controls (34). However, on
a vitamin D deficient diet they quickly developed vitamin D
deficiency. More recently, a family has been described to have
a mutation in the DBP gene deleting it from the homozygous
patient and decreasing its concentration to 50% of normal
in a heterozygous sibling (35). The homozygous patient had
nearly undetectable levels of total 25(OH)D, although the free
concentration measured directly was comparable to that of
the normal sibling, as was that of the heterozygote sibling.
Parathyroid hormone, calcium, and phosphate were all normal.
Thus, DBP does not appear necessary for getting the vitamin D
metabolites into cells, supporting the free hormone hypothesis,
but DBP clearly serves as a critical reservoir for the vitamin
D metabolites, reducing the risk of vitamin D deficiency when
intake or epidermal production is limited.
The DBP alleles have been reported to differ in their affinity
to 25(OH)D. Gc1f was initially reported as having the highest
affinity and Gc2 the lowest among the common alleles (36),
but results from other laboratories have not confirmed these
differences, and the results from later studies themselves are
inconsistent (37,38). In one such study evaluating the half life of
25(OH)D in serum, subjects homozygous for the Gc1f allele were
found to have the shortest half life indicating a reduced affinity
(39). On the other hand, serum containing the Gc1f variant of
DBP reduced the ability of 25(OH)D and 1,25(OH)2D to induce
cathelicidin in monocytes more than that of serum with the Gc2
allele, suggesting the opposite order of affinity (31). Schwartz
et al. (40) recently reported that DBP haplotype had significant
effects on total 25(OH)D, free 25(OH)D, and DBP levels. The
lowest total and free levels of 25(OH)D were seen with the Gc
2/2 haplotype which also tends to have the lowest DBP levels.
Other studies have also found lower total 25(OH)D levels in
subjects with the Gc2 allele (41–45). The reason the Gc2 allele
is associated with lower DBP levels is unknown. DBP haplotype
also affected percent free 25(OH)D. The lowest free percentage
was seen with the 1s/1s haplotype and the highest one with the
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Bikle and Schwartz DBP and Free 25(OH)D
1f/1f haplotype, suggesting that in this survey the Gc1s allele
had a higher affinity for 25(OH)D than the Gc1f allele, with
the Gc2 allele in between. Furthermore, the different Gc alleles
affect the response to vitamin D supplementation. Individuals
with the Gc2 variant have been shown to respond to vitamin
D supplementation with a more robust increase in 25(OH)D
(46). Moreover, within the Gc2 polymorphic region (rs4588),
individuals in an Iranian population with an AA genotype within
this polymorphic region showed a greater increase in 25(OH)D
levels following vitamin D supplementation than those with
the GG genotype did (47). Similar results were found with a
different polymorphism at rs2282679 in Caucasian women (48).
Rs2282679, an intronic polymorphism in the DBP gene that
does not alter DBP structure, was previously shown in GWAS
studies to be associated with lower 25(OH)D and DBP levels in
several different populations (49–51). The clinical significance
of these allelic differences is unclear. Differences in these alleles
were not found to contribute to a difference in fracture rate
in a large study including African Americans and Caucasians
(52) or other calcemic and cardiometabolic diseases in the
Canadian Multicentre Osteoporosis Study (50). However, as
reviewed by Malik et al. (13) and Speeckaert et al. (53), a large
number of chronic diseases including type 1 and 2 diabetes
(54–56), osteoporosis (57–59), chronic obstructive lung disease
(60), endometriosis (61), inflammatory bowel disease (62),
some cancers (63–66) [although see (66–68)], and tuberculosis
(69) have been associated with DBP variants. Other SNPs at
rs4588 have been associated with susceptibility to the metabolic
syndrome (70). At the Gc1 locus (rs7041) the G allele is associated
with increased susceptibility to hepatitis C viral infection (71).
Karras et al. (72) has summarized a number of studies showing
the impact of DBP and DBP polymorphisms on various outcomes
of pregnancy. These studies demonstrate the recent interest in
the impact of polymorphisms on DBP function, but it remains
to be seen whether these initial results will be generalized across
different populations.
Actin Scavenging
A major function of DBP that has received considerably less
interest than that of vitamin D metabolite binding is its role
in actin scavenging. Following trauma (7), sepsis (73–75), liver
trauma (8,76,77), acute lung injury (78), preeclampsia (79),
surgery (80,81), and burn injuries (82), large amounts of
actin are released from the damaged cells forming polymerized
filamentous F-actin that, in combination with coagulation factor
Va, can lead to disseminated intravascular coagulation and
multiorgan failure unless cleared (83). The actin scavenging
system consists of gelsolin and DBP. Gelsolin depolymerizes F-
actin to G (globular) actin. DBP, with its high affinity for G-actin
(Kd =10 nM), prevents the repolymerization and clears it from
the blood (84,85). No clear difference among the major DBP
variants has been observed regarding binding to G-actin (53).
The DBP-actin complexes are rapidly cleared (half life in blood
approximately 30 min) (81), primarily by the liver, lungs and
spleen. These tissues have receptors for the DBP-actin complexes
(86). The acute conditions result in a fall in DBP levels, potentially
decreasing the bioavailability of the vitamin D metabolites (8,87,
88), with a rise in the DBP-actin complexes (7,73,77,78). The
ability of the organism to respond to the insult by increasing DBP
production is correlated to survival (7,8,89), and has led to the
consideration of the use of DBP therapeutically (90,91).
Neutrophil Recruitment and Migration With
Complement 5a (C5a) Binding
Neutrophil activation during inflammation increases their
binding sites for DBP (92), and DBP binding to these sites
facilitates C5a induced chemotaxis (21) as well as other
chemoattractants such as CXCL1 during inflammation (93).The
interaction with C5a involves residues 130–149 of DBP, a region
which is common to all major DBP alleles (20), and no difference
in these alleles has been found with respect to their promotion of
C5a mediated chemotaxis (21). Binding of 1,25(OH)2D but not
25(OH)D blocks the promotion by DBP of C5a activity (94).
Fatty Acid Binding
DBP binds fatty acids but with lower affinity (Ka =105-106M−1)
than albumin and via a single binding site (16,95). Most of the
fatty acids binding to DBP are mono-unsaturated or saturated,
with only 5% poly-unsaturated. However, only poly-unsaturated
fatty acids such as arachidonic acid and linoleic acid compete
with vitamin D metabolites for DBP binding (17,96). This
suggests that the different fatty acids alter the configuration
of DBP affecting the binding of the vitamin D metabolites
rather than directly competing with the vitamin D metabolites
for their binding site. The role of DBP in fatty acid transport
appears limited.
Formation of the DBP-Macrophage Activating Factor
(DBP-MAF) and its Functions
As described above, DBP-MAF is formed from certain alleles
(Gc1s and 1f) of DBP following deglycoslyation during
inflammatory processes (97). These deglycosylation steps are
required for the role of DBP in macrophage activation (15), but
further removal of the N-acetyl-galactosamine (NaGal) reduces
this activity (98). DBP-MAF is able to activate osteoclasts (99)
independent of its 25(OH)D binding function, and it has been
shown to stimulate bone resorption in the osteopetrosis (OP)
and the incisor absent (IA) rat (100). DBP-MAF has also shown
efficacy in a number of tumor models (101–103). Removal of
NaGal by α-NaGalase blocks DBP-MAF formation contributing
to the loss of immunosuppression in cancer patients (104). α-
NaGalase is produced in the liver, and appears to be directly
related to tumor burden (105). Preparations of DBP-MAF may
have therapeutic potential (14).
FREE HORMONE HYPOTHESIS
As previously noted, the free hormone hypothesis postulates that
only the non-bound fraction (the free fraction) of hormones
that otherwise circulates in blood bound to their carrier proteins
is able to enter cells and exert their biologic effects (Figure 2).
Examples include the vitamin D metabolites, which we are
discussing in this review, sex steroids, cortisol, and thyroid
hormone. These are lipophilic hormones assumed to cross
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Bikle and Schwartz DBP and Free 25(OH)D
FIGURE 2 | The Free Vitamin D hypothesis. As noted in the text, vitamin D (OH) metabolites are bound to D Binding Protein (DBP) and to a lesser extent albumin in
the circulation. These cross the cell membrane as the free (unbound) metabolite in most tissue. However, In the kidney, parathyroid gland, and placenta, the
megalin/cubilin complex can transport bound D (OH) metabolites into cells.
the plasma membrane by diffusion and not by an active
transport mechanism. One of the earliest clinical examples
leading to the formulation of the free hormone hypothesis
came from observations by Recant and Riggs (106) that patients
with protein losing nephropathy developed quite low levels of
thyroid hormone (PBI) along with increased urinary losses but
without evidence of hypothyroidism. Subsequent studies have
established the free hormone hypothesis for the thyroid and
steroid hormones (107,108), and measurements of the free
concentrations of thyroid hormone, estrogen, and testosterone
are standard practice. As will be discussed subsequently, this is
likely to become the case for free 25(OH)D. As noted earlier,
mice lacking DBP lost substantial amounts of the vitamin
D metabolites in the urine with marked reductions in their
circulating levels of 25(OH) D, but they did not develop evidence
of rickets until put on a low vitamin D diet. Such results indicate
the importance of the free fraction of 25(OH)D for biologic
functions and the role of DBP as a circulating reservoir (33).
To address the clinical relevance of the free hormone
hypothesis for vitamin D metabolites, a method to measure
the free concentration needed to be developed. This was
originally performed by centrifugal ultrafiltration to directly
determine the free levels of 25(OH)D and 1,25(OH)2D (109,
110) in various clinical situations. However, this method is
labor intensive and has recently been replaced at least for
free 25(OH)D by a two-step ELISA that directly measures
free 25(OH)D (Future Diagnostics Solutions B.V., Wijchen,
Netherlands) using monoclonal antibodies from DIAsource
Immunoassays (Louvain-la-Neuve, Belgium). The antibody in
the current assay does not recognize 25(OH)D2as well as
25(OH)D3(77% of the 25(OH)D3value), so underestimates
the free 25(OH)D2. However, under most situations where
the predominant vitamin D metabolite is 25(OH)D3, the data
compare quite well to those obtained from similar populations
using the centrifugal ultrafiltration assay (111,112). The initial
studies with the centrifugal ultrafiltration method established
affinity constants for DBP and albumin binding to 25(OH)D and
1,25(OH)2D in a healthy young adult (DD Bikle) and may not
be generalizable to a broad range of individuals from different
ethnic backgrounds or in different clinical conditions. However,
prior to the development of a high throughput ELISA assay to
measure the free concentration directly, these affinity constants
proved useful in calculating the free concentrations (113,114)
from measurements of DBP, albumin and the total vitamin D
metabolite of interest according to the formula:
free vitamin D metabolite =total vitamin D metabolite
1+Kaalb∗albumin+(KaDBP∗DBP)
As noted previously, the affinity of 25(OH)D for albumin is
much less that than for DBP, leading some to consider albumin-
bound 25(OHD) to be essentially “free” or “available” and define
“bioavailable 25(OH)D” as free 25(OH)D plus albumin-bound
25(OH)D. Given that the albumin bound 25(OH)D (15%) is
considerably higher than the free level (0.03%), this would imply
that approximately 500 times as much 25(OH)D is available to
cells than if only the free fractions were available. There is little
evidence to support albumin bound 25(OH)D as being readily
available to cells.
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Bikle and Schwartz DBP and Free 25(OH)D
In sera from normal healthy younger individuals, the
calculated values of free 25(OH)D and 1,25(OH)2D using
DBP measured with polyclonal antibodies correlate reasonably
well with the directly measured free levels using centrifugal
ultrafiltration for both metabolites or the ELISA assay for
25(OH)D. However, when applied to clinical populations with
altered DBP levels either during physiologic (e.g., pregnancy)
or pathologic (eg. liver disease) conditions, the calculated values
no longer are consistent with those measured directly by either
centrifugal ultrafiltration or the newly developed ELISA (115).
Part of this is due to the disparity between assays for both
the vitamin D metabolite (e.g., 25(OH)D) and DBP, each of
which have generally relied on immunoassays. However, mass
spectroscopy is becoming the gold standard for measurement
of the vitamin D metabolites (116,117) and is being developed
for the measurement of DBP and its various isoforms as well
(42,118). The adoption of mass spectroscopy should reduce the
variation in these measurements from different laboratories. But
a major problem in attempting to calculate the free fraction of
vitamin D metabolites is the assumption that all DBP alleles
have the same affinity for the vitamin D metabolites, and that
this is invariant under varying clinical conditions. As noted
previously, the rank order of affinity of the different alleles for the
vitamin D metabolites remains controversial, but differences have
been found. Regardless, these potential differences in measured
affinity do not begin to explain the large differences between the
calculated and directly measured free metabolite levels in various
disease states (40). Although there are statistically significant
correlations between calculated and directly measured free
25(OH)D, the relationship accounts for only 13% of the variation.
Calculated free 25(OH)D concentrations are consistently higher
than directly measured concentrations in a variety of studies,
such as those performed during the third trimester of pregnancy
and in patients with liver disease or cystic fibrosis (115,119–
122). These studies suggest changes in the affinity of 25(OH)D
to DBP independent of allelic variations in at least some of these
clinical conditions.
CLINICAL STUDIES
Healthy Populations
Determinations of free 25(OH)D concentrations in healthy
populations show highly significant correlations with total
25(OH)D concentrations whether measured directly or
indirectly. Assays to directly measure free 25(OH)D are not
currently available for use in clinical care but have been used
in research investigations. As noted above, calculated 25(OH)D
values are usually higher than when measured directly, which
is based on multiple unsubstantiated assumptions such that
results obtained with the two methods can differ markedly in
different clinical conditions. For these reasons only results from
studies with directly measured free 25(OH)D will be discussed.
When measured with the direct immunoassay, free 25(OH)D
levels have been reported to be between 0.02 and 0.09% of
total 25(OH)D concentrations and generally range from 0.5
to 8.1 pg/mL in 95% of healthy adults (Figure 3). However,
clinical conditions that alter either DBP, the affinity of DBP for
FIGURE 3 | Distribution of free 25(OH)D in Adults and Selected Patient
Groups. Distribution of directly-measured free 25(OH)D in normal adults (in
green), pregnant women (pink), cirrhotics (orange), and nursing home
residents (gray). Distributions are shifted leftward toward lower free 25(OH)D
concentrations in pregnant women in the 2nd and 3rd trimesters concordant
with increased DBP while decreased synthetic function and DBP in cirrhotics
shifts free 25(OH) concentrations to the right toward higher levels. The
mechanism for higher free 25(OH) concentrations in Nursing home residents is
likely related to D supplementation, somewhat lower, albumin, and the
pro-inflammatory state of frailty. Figure generated form data in
Schwartz et al. (40).
25(OH)D metabolites or albumin, or disposition of vitamin
D, may alter free 25(OH)D concentrations or relationships
between free and total 25(OH)D concentrations. In this regard,
a number of medications, hormones, and smoking have been
shown to affect DBP levels (123). Thus, as shown in Figure 3, the
free concentration of 25(OH)D varies among different clinical
conditions. DBP haplotypes have also been hypothesized to
alter the affinity between total 25(OH)D and free 25(OH)D,
although, as shown in Figure 4, the variation in percent
free 25(OH)D levels is less affected by DBP haplotype than
clinical condition.
Free 25(OH) D in Conditions That Alter DBP
Pregnancy
As pregnancy progresses there are time dependent changes
in DBP with almost two-fold increases between the second
and third trimesters. Despite these marked DBP changes,
mean free 25(OH)D may be the same as or only slightly lower
than in non-pregnant women but with less variability than
in other groups (40,124). The slope of the free 25(OH)D
vs. total 25(OH) D relationship, however, is significantly
less steep than in healthy individuals. The same conclusion
was drawn from earlier studies with measurements of free
1,25(OH)2D (109). These results suggest that the affinity
of DBP for vitamin D metabolites is decreased during
pregnancy, perhaps compensating for increased DBP
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Bikle and Schwartz DBP and Free 25(OH)D
FIGURE 4 | Percent free 25(OH)D in adults by clinical condition or DBP Haplotype. Percent free 25(OH)D concentrations for selected clinical groups on the left panel
(community outpatients, NH=nursing home patients, cirrhotics, pregnant women, prediabetics, and normal individuals) and by DBP haplotype on the right . Boxplots
show 10th, 25th, median, 75th, and 90th percentile values. Individual points represent values above the 90th and below the 10th percentiles. Both clinical subgroup
and DBP genotype significantly effect percentage free 25(OH)D. Between group comparisons for clinical conditions were significant for all but healthy persons
compared with pregnant women or outpatients, or for pregnant women compared with outpatients. For DBP haplotypes, smaller but significant differences were
detected between the 1s/1s haplotype and the 1s/1f,1f/2, 1f/1f, and 1s/2 haplotypes and between the 1s/2 and 1f/2 and 1f/1f haplotypes and between the 1s/1f and
1f/1f haplotypes. Data are reproduced with permission from Schwartz et al. (40).
concentrations and the needs of both the mother and fetus
for calcium.
Liver Disease
Liver diseases that are associated with impaired protein synthetic
function such as cirrhosis and acute liver failure result in
reductions in DBP and albumin. In addition, the relationship
between free 25(OH)D and total 25(OH)D is significantly steeper
in patients with cirrhosis than in healthy people indicating altered
affinity of DBP for 25(OH)D (40) (Figure 4). The net result
is that directly measured free 25(OH)D is higher and shows
greater variability in patients with cirrhosis compared to healthy
individuals and stable outpatients with other chronic conditions
(40,110,115) despite lower total 25(OH) D concentrations.
Results regarding the effects of cirrhosis or acute liver failure
on the relationship of total to free 25(OH)D are consistent,
creating a strong argument for assessment of free 25(OH)D to
assess vitamin D status in the presence of liver pathology as total
25(OH)D measurements may be misleading.
Renal Disease
Nephrotic syndrome, acute renal failure, acute tubular necrosis,
or chronic kidney disease associated with renal tubular necrosis
may have decreased transport capacity for DBP from the
glomerular filtrate into the renal tubules. Heavy proteinuria
can lead to loss of DBP as well as 25(OH)D in the urine as
the maximal transport capacity of the megalin/cubulin system
is saturated. Reports in the literature have not included direct
measurement of free 25(OH)D in these conditions, but a small
study of nephrotics showed lower total and free 1,25(OH)2D
compared to people with normal renal function (125).
Clinical Conditions Not Associated With
Altered DBP Levels
Obesity
High BMIs are associated with reductions in total and free
25(OH)D but not DBP or elimination of half-life measurements
of 25(OH)D (126). The underlying mechanism for these changes
is unknown but may be related to the pro-inflammatory state
and circulating cytokines present in obesity, although increased
volume of distribution (into fat) has also been invoked.
DBP Haplotypes
Investigations using direct measurements of free 25(OH)D have
detected statistically significant but not marked differences in free
25(OH)D concentrations between healthy individuals with the
six common DBP haplotypes (Figure 4). This is in contrast to the
marked differences between haplotypes reported with calculated
free 25(OH)D levels (122,127). As noted previously with directly
measured free 25(OH)D, the lowest free 25(OH)D is seen with
the Gc 2/2 haplotype and the highest levels with the 1s alleles. Per
cent free was highest with the 1f/1f haplotype in our studies (40)
(see Figure 4).
Nursing Home Subjects
In a vitamin D dose titration study (128) of nursing home
residents, who are older, have more chronic co-morbidities, and
receive more medications than younger people or community-
dwelling elderly, free 25(OH)D levels rose along with increases
in total 25(OH)D. The per cent free was higher than in younger
adults. Relationships between free and total 25(OH)D were also
steeper than those of normal subjects or younger outpatients
suggesting altered affinity of 25(OH)D to DBP in this group.
Slightly lower albumin concentrations may have also had a
small contribution. Inflammation and/or elevated cytokines that
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Bikle and Schwartz DBP and Free 25(OH)D
accompany very old age or multiple morbidities may have
also contributed to altered affinity of 25(OH)D to DBP in this
group (129).
Associations With Markers of Vitamin D Biologic
Function
PTH is generally found to be negatively correlated with
free 25(OH)D as well as total 25(OH)D. Reports variably
conclude that one or the other shows a slightly more
significant relationship, but neither explains more than a small
amount of the variability in the relationship. Moreover, if
the megalin/cubilin complex is operative in the parathyroid
gland as it is in the kidney, PTH levels may not be able to
distinguish between free and total 25(OH)D with respect to
biologic action. However, further insight into the impact of free
vs. total 25(OH)D on PTH levels may be gained from several
recent studies showing that with high dose D supplementation,
changes in iPTH were significantly related to changes in directly
measured free 25(OH)D but not to changes in total 25(OH)D
(128,130,131), suggesting that free 25(OH)D might be a better
marker of the biologically available fraction at higher total
25(OH)D concentrations or when 25(OH)D is changing. Data on
relationships between directly measured free 25(OH)D and bone
density or markers of bone turnover are inconsistent.
Other Conditions
There are limited data on the effect of oral contraceptives or
hormone replacement therapy with estrogen, but free 25(OH)D
levels and relationships between total and free 25(OH)D do not
appear to be significantly influenced by the use of these agents
at currently prescribed dosages and routes of administration.
Similarly, stable medical conditions such as hypertension,
prediabetes, diabetes, osteoporosis, or mild renal disease do
not appear to significantly alter relationships between free and
total 25(OH)D.
Summary of Clinical Studies
The impact of clinical conditions on free 25(OH)D is that the
absolute level, the percent free 25(OH)D and the relationship
between free and total 25(OH)D concentrations, differ in
pregnant women, 336 people with cirrhosis, and elderly people
with multiple morbidities compared to normals or community-
dwelling outpatients. These relationships are affected to a much
smaller extent by BMI in all groups. It is key that while DBP
haplotype variation is associated with differences in per cent free
25(OH)D, the DBP haplotype effects are far smaller in magnitude
than those of pregnancy, cirrhosis, or very old nursing home
residents with multiple chronic conditions. Thus, total 25(OH)D
measurements may be misleading in persons with altered total-
to-free relationships, although for other clinical conditions
the relationship between total and free 25(OH)D may be
less affected.
CONTRIBUTION TO THE FIELD
25(OH)D measurements in the blood currently provide the
standard assessment of vitamin D status. Nearly all 25(OH)D
circulates as the bound form, with the vitamin D binding protein
(DBP) accounting for approximately 85% of the binding, with
albumin accounting for most of the rest. However, it is the very
small percentage that is not protein bound (0.03% in normal
individuals) that is able to cross the membrane of most cells.
Conditions that alter levels of DBP or its binding to 25(OH)D
alter the relationship between free and total levels. If the free
concentration provides a more accurate assessment of vitamin D
status, measuring only total 25(OH)D levels may be misleading
in situations where the relationship between total and free
25(OH)D levels is altered as in liver disease and pregnancy or in
individuals with different DBP alleles. This review examines the
impact of different DBP alleles and clinical conditions that do the
relationship between free and total 25(OH)D levels, concluding
that in a number of clinical situations measuring the free level
may provide a better index of vitamin D status than total levels in
such situations.
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication.
FUNDING
Grant support provided by: NIH AR 055924 (DB), VA
I01BX003814 (DB).
ACKNOWLEDGMENTS
We appreciate the provision of data by our coauthors in our
publication Schwartz et al. (40), that is included in this review.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Bikle and Schwartz. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
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distribution or reproduction is permitted which does not comply with these terms.
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38
REVIEW
published: 10 July 2019
doi: 10.3389/fendo.2019.00470
Frontiers in Endocrinology | www.frontiersin.org 1July 2019 | Volume 10 | Article 470
Edited by:
Zhongjian Xie,
Central South University, China
Reviewed by:
Giovanni Lombardi,
Istituto Ortopedico Galeazzi
(IRCCS), Italy
Stefano Pagano,
University of Perugia, Italy
*Correspondence:
Richard R. Kew
richard.kew@stonybrookmedicine.edu
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 30 April 2019
Accepted: 28 June 2019
Published: 10 July 2019
Citation:
Kew RR (2019) The Vitamin D Binding
Protein and Inflammatory Injury: A
Mediator or Sentinel of Tissue
Damage? Front. Endocrinol. 10:470.
doi: 10.3389/fendo.2019.00470
The Vitamin D Binding Protein and
Inflammatory Injury: A Mediator or
Sentinel of Tissue Damage?
Richard R. Kew*
Department of Pathology, Stony Brook Cancer Center, Renaissance School of Medicine, Stony Brook University,
Stony Brook, NY, United States
Neutrophils are the most abundant type of white blood cell in most mammals including
humans. The primary role of these cells is host defense against microbes and clearance of
tissue debris in order to facilitate wound healing and tissue regeneration. The recruitment
of neutrophils from blood into tissues is a key step in this process and is mediated by
numerous different chemoattractants. The neutrophil migratory response is essential for
host defense and survival, but excessive tissue accumulation of neutrophils is observed in
many inflammatory disorders and strongly correlates with disease pathology. The vitamin
D binding protein (DBP) is a circulating multifunctional plasma protein that can significantly
enhance the chemotactic activity of neutrophil chemoattractants both in vitro and in vivo.
Recent in vivo studies using DBP deficient mice showed that DBP plays a larger and
more central role during inflammation since it induces selective recruitment of neutrophils,
and this cofactor function is not restricted to C5a, as prior in vitro studies indicated,
but can enhance chemotaxis to many chemoattractants. DBP also is an extracellular
scavenger for actin released from damaged/dead cells and formation of DBP-actin
complexes is an immediate host response to tissue injury. Recent in vitro evidence
indicates that DBP bound to G-actin, and not free DBP, functions as an indirect but
essential cofactor for neutrophil migration. DBP-actin complexes always will be formed
regardless of what initiated an inflammation, since release of actin from damaged cells
is a common feature in all types of injury and DBP is abundant and ubiquitous in all
extracellular fluids. Indeed, these complexes have been detected in blood and tissue
fluids from both humans and experimental animals following various forms of injury.
The published data strongly supports the premise that DBP-actin complexes are the
functional neutrophil chemotactic cofactor that enhances neutrophil chemotaxis in vitro
and augments neutrophilic inflammation in vivo. This review will assess the fundamental
role of DBP in neutrophilic inflammation and injury.
Keywords: inflammation, neutrophil accumulation, chemotactic factor, vitamin D binding protein (DBP), tissue
injury
39
Kew DBP and Inflammatory Injury
HISTORICAL CONTEXT AND BRIEF
BACKGROUND
The vitamin D binding protein (DBP, also abbreviated VDBP)
was initially named the group-specific component of serum
(abbreviated as Gc-globulin). Jan Hirschfeld is credited with the
first publication in 1959 describing the migration pattern of an
unknown serum protein using agarose gel electrophoresis and
rabbit anti-human serum (i.e., immunoelectrophoresis) (1). This
report described qualitative differences in the electrophoretic
migration of α2-globulin proteins in serum obtained from
10 normal healthy blood donors (1). These immunoreactive
proteins would display a consistent migration pattern (either
fast, intermediate, or slow) that was specific to an individual
donor, and thus was named the group-specific component of
serum (1). This paper, together with two subsequent papers by
Hirschfeld published the following year (2,3), initiated this six
decade long investigation into the structure and function of DBP.
A major milestone for DBP research occurred in 1975 when
Diager et. al. reported that the serum group-specific component
binds vitamin D, and consequently, the authors proposed to
name the protein the vitamin D binding α-globulin (4), later
shortened to the vitamin D binding protein. A few years later
several reports described what initially was thought to be a
larger cellular form of DBP (5–7) but subsequently was shown
unequivocally to be plasma DBP binding to G-actin monomers in
a 1:1 molar complex (8). Thus, by 1980, DBP was known to have
two distinct binding functions: vitamin D and G-actin. Structural
studies would later confirm the vitamin D sterol and G-actin
binding pockets in DBP (9,10). Beginning in the mid-1980’s
and continuing into the early 1990’s, numerous reports identified
DBP and plasma gelsolin as part of the actin scavenging system
in blood. These proteins work in tandem to clear extracellular
actin released from dead or damaged cells for removal from
the circulation, largely in the liver (11). Furthermore, several
studies demonstrated that serious injury (burns, traumatic injury,
acetaminophen-induced hepatotoxicity) generate large amounts
of extracellular actin that consumes DBP and reduces its plasma
concentration (12–15). Low levels of plasma DBP directly
correlate with poor overall survival in these studies (12–15).
Although the actin binding properties of DBP have been clearly
established and shown to be physiologically relevant, this role
largely has been overshadowed by the vitamin D sterol binding
function, particularly over the past 15 years with increasing
awareness of the essential role of vitamin D in health and disease.
Besides, the vitamin D binding protein is not the best choice
of name to promote its actin binding function! There are many
facets of this multifunctional plasma protein, several of which are
discussed in other reviews of this series. The topic herein will
focus primarily on the actin binding capacity of DBP, how this
function plays a role in chemotaxis enhancement of neutrophils
and may act as a possible mediator of inflammatory tissue injury.
DBP-ACTIN COMPLEXES AND TISSUE
INJURY
Tissue injury often causes cell damage and death leading to
the release of intracellular contents into extracellular fluids.
Many intracellular molecules (HMGB1, mitochondrial DNA,
ATP, etc.) have different roles in the extracellular environment
and function as “alarmins” (also known as danger-associated
molecular patterns or DAMPs) to signal the immune system
to the presence of tissue injury (16). Although numerous
alarmins have been described and their role in tissue injury
well-characterized, it is quite surprising that very little attention
has been paid to actin, the major cytoskeletal protein that
is the most abundant intracellular protein in any organism.
Actin essentially exists in two states inside a cell: monomeric
globular actin (G-actin), or G-actin polymerized into filaments
(F-actin) (17). These two forms are in a constant state of
polymerization and depolymerization that is highly regulated by
several intracellular actin binding proteins, and this dynamic
process is very prevalent in motile cells such as leukocytes
(18). During tissue injury large quantities of actin can be
released into extracellular fluids where the protein escapes
normal intracellular regulatory mechanisms and the protein
spontaneously will form F-actin filaments. In animal models,
extracellular F-actin filaments have been shown to alter the
coagulation and fibrinolytic systems leading to occlusion and
damage of the microcirculation (particularly in the lung) in a
manner similar to how fibrin damages the microvasculature in
disseminated intravascular coagulation (15,19). Accordingly, an
effective extracellular scavenging system has evolved to dispose
of actin released from dead or damaged cells (15). The system
is composed of two plasma proteins that have complementary
functions: gelsolin, which severs F-actin filaments into G-actin
monomers, and DBP that binds G-actin in a high affinity (Kd
of 10−9M) 1:1 molar complex for transport and eventual
clearance primarily in the liver. This process consumes DBP and
a decreased plasma concentration of actin-free DBP has been
shown to be an effective but indirect marker of tissue injury in
cases of severe trauma in both humans and animal models (15).
Although there has been interest in the potential therapeutic role
of gelsolin as an extracellular actin scavenger to treat several
conditions, less attention has been focused on its partner DBP,
despite the fact that over the past 30 years numerous studies
(>400 listed on PubMed) have reported that DBP is associated
with various acute and chronic diseases in several different organs
[reviewed in (20,21)]. There is no apparent common connection
in these various studies, but a very broad view of all these reports
would suggest that DBP has a fundamental role during tissue
injury, perhaps due to its actin scavenging function.
The vitamin D binding protein is an abundant plasma
protein that is part of the albumin gene family and shares
considerable amino acid homology and structural similarity
with these proteins (11). Several groups have published the
crystal structure of DBP that revealed an α-helix triple domain
arrangement (characteristic of the albumin family) that forms a
broad U-shaped or saddle-shaped molecule (9,22,23). The N-
terminal (domain I) and the C-terminal domains (III) form the
front and back of the saddle and domain II the seat (10,22,23).
This shape is designed to perfectly fit a molecule of G-actin,
and this has been confirmed by crystal structure analysis of
DBP bound to G-actin (10). Vitamin D sterol binding pocket
in domain I is distinct from the actin binding domain, and a
molecule of DBP can bind both ligands simultaneously without
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Kew DBP and Inflammatory Injury
apparent alteration in binding affinity or protein function (24).
Although DBP mRNA expression has been reported in many
different tissues, plasma DBP is synthesized by hepatocytes in
the liver and circulates in blood with a range of 5–9 µM (300–
500 µg/ml) (11). It has a relatively rapid turnover in plasma with
a half-life of about 2 days (compared to 22 days for albumin)
(25). In contrast to albumin whose plasma levels decrease during
inflammation, DBP levels in blood are stable or rise slightly
during the acute phase response of inflammation (26). Moreover,
DBP is ubiquitous in vivo and significant quantities (0.1–1 µM
range) have been detected in all extracellular fluid compartments
(cerebrospinal, bronchoalveolar, synovial, etc.) (25,26). Although
DBP is the primary transport molecule for vitamin D sterols in
the blood and extracellular fluids, its concentration in plasma
is much higher than vitamin D. Normally only 1–2% of the
total circulating DBP pool has vitamin D bound and this
percentage never rises above 5% (27). This is in contrast with
other transport proteins in blood where about 50% of the total
protein pool is bound with ligand (25). This fact prompted
many to speculate that DBP must have other essential functions,
perhaps related to its ability to scavenge G-actin (28). Indeed,
significant tissue injury can result in a large percentage of
circulating DBP complexed with actin (12–15). Actin-induced
depletion of plasma DBP to levels below 3.5 µM (200 µg/ml)
have been shown to be an effective but indirect marker of tissue
injury that correlates with poor prognosis in cases of sepsis,
multiple trauma and acetaminophen-induced liver failure (12–
15). Clinical outcome and decreased plasma levels of DBP have a
statistical correlation similar to the other outcome metrics such
as the APACHE II score (sepsis), Kings College criteria (liver
failure) and the TRISS-like method (multiple trauma) (13–15).
The vitamin D binding protein and actin are abundant
and ubiquitous proteins of the intra- and extracellular
compartments, their expression is stable during inflammation
and cell injury causes immediate complex formation. Thus,
global transcriptome analysis of mRNAs increased or decreased
during inflammatory injury most likely would not identify
actin or DBP (29). For these reasons we believe that the
potential role of DBP-actin complexes during inflammation has
been overlooked. Varying levels of DBP-actin complexes are
continually formed in vivo as a result of minor tissue trauma,
menstrual cycles, infections, surgery, etc. Hence, low levels of
circulating DBP-actin would be a routine physiological process
and should be transient and inconsequential. On the other hand,
prolonged generation and/or high concentrations of DBP-actin
in extracellular fluids potentially could act as a danger signal
(alarmin) of ongoing and significant tissue injury. Although
previous research has focused on actin-free DBP in plasma,
the role of DBP-actin complexes in tissue injury has not been
determined, most likely because it has been assumed that these
complexes are inactive by-products of cell damage that are
rapidly cleared from the circulation. Interestingly, a previous
study examining fulminant hepatic necrosis (FHN) in humans
showed that 72% of plasma DBP was complexed with actin in
patients who died of the disease whereas only 22% of total DBP
was bound to actin in FHN survivors (12). However, while this
data was very compelling, the authors of this study focused on
actin-free DBP, not the level of DBP-actin complexes. Although
DBP-actin complexes generally have been viewed as benign
by-products of cell injury, recent studies from our lab (both in
vitro and in vivo) have shown that these complexes may serve
as an alarmin and possess a cytokine-like function. Thus, the
accumulated evidence has shown that DBP, via its actin binding
function, likely plays a role in both the mediation and resolution
of tissue injury.
CELL-ASSOCIATED DBP
The vitamin D binding protein interacts with many different
cell types to achieve its diverse functions (delivery of vitamin D
sterols, binding and/or clearance of G-actin, chemotactic cofactor
function, macrophage activating-factor), and it appears that the
protein must first bind to its target cell surface in order to mediate
these effects. Numerous studies have reported DBP on the
surface of most cell types including B-lymphocytes (30–32), T-
lymphocytes (33), testicular cells (34), placental cytotrophoblasts
(35,36), pancreatic acinar cells (37), monocytes (38), neutrophils
(39), and renal proximal tubule cells (40). It is clear that cell-
associated DBP is not a novel cellular form but rather circulating
DBP bound to the cell surface (41). Although binding to the
plasma membrane is the first step in the interaction of DBP
with cells, it is unlikely that a cell surface binding site for
DBP would be a specific high affinity (Kd≤10−8M) receptor.
DBP is abundant and ubiquitous in all fluid compartments
and circulating blood leukocytes are bathed in 5–9 µM DBP
(11). Therefore, a relatively low affinity binding site with a Kd
significantly above the plasma concentration of DBP would seem
most logical, otherwise leukocytes in blood could act as a “sink”
for DBP and diminish the circulating pool. Several studies have
shown that DBP binds with low affinity to megalin, also known
as LDL receptor related protein 2 (LRP2) (42–44). Megalin is a
large scavenger receptor that is primarily expressed on the surface
of epithelial cells in the kidney (proximal tubules), endocrine
glands, and reproductive organs (45). Megalin on renal proximal
tubules binds and captures DBP (both free and bound to vitamin
D) in glomerular filtrate for re-uptake into the circulation (45).
DBP also binds specifically, but with low avidity, to chondroitin
sulfate glycosaminoglycans (CS-GSGs), and more specifically to
the CS-GAG group on CD44, which is widely expressed on
most leukocytes (46,47). However, several papers have reported
that DBP bound to the surface of cells cannot be removed
by numerous high salt and/or detergent washes, suggesting a
relatively tight binding site (30,33,38).
Our lab has extensively investigated the binding of DBP to
human neutrophils and myeloid cell lines (U937, HL-60) since
cell binding is the critical first step in the process of DBP
functioning as a chemotactic cofactor. DBP does not follow the
kinetics of saturable binding to a single high-affinity receptor,
but instead displays a multiphasic, time-dependent interaction
with neutrophils over the course of 60 min at 37◦C: weak
binding (5–20 min), strong binding (20–50 min), shedding into
the extracellular fluid (>45 min) (46). There is very little (if any)
binding of radiolabeled DBP to cells incubated in an ice bath
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Kew DBP and Inflammatory Injury
(1◦C), but at 37◦C three distinct phases of DBP binding are
observed, indicating that a dynamic process is required to express
this binding site. In addition, if neutrophils are first stimulated
with chemotactic factors or calcium ionophores at 37◦C prior to
incubation with labeled DBP, the delay in tight binding phase
is essentially eliminated. The initial weak binding of DBP to
neutrophils probably is mediated by low avidity molecules such
as CD44 or other chondroitin sulfate proteoglycans upregulated
from internal stores in neutrophil cytoplasmic granules (46,48).
These weak binding molecules may serve to capture DBP from
extracellular fluids and loosely tether it to the cell surface.
The biochemical characterization of the tight binding site
for DBP on the plasma membrane of neutrophils, and
mechanisms of the subsequent protease-mediated shedding,
were very extensive (46,48–50) and showed that: (a) DBP
bound to the plasma membrane can only be dissociated
using harsh denaturing conditions; (b) solubilization of cells
revealed that the bound DBP partitions to the detergent
insoluble fraction, which contains the actin cytoskeleton; (c)
confocal immunofluorescence microscopy revealed that DBP
and actin co-localize on the surface of neutrophils; (d)
immunoprecipitation of DBP bound to (or shed from) cells,
followed by mass spectrometry analysis showed that the major
binding partners were actin, CD44 and annexin A2. Annexin
A2 is known as a molecular facilitator since it binds multiple
diverse molecules (including CD44 and actin) to assemble plasma
membrane complexes in a phospholipid and calcium-dependent
manner (51). Interestingly, two reports published more than a
dozen years before our studies on neutrophil cell surface DBP
binding site, described that DBP was tightly bound to actin on
the surface of human B lymphocytes (30) and monocytes (38).
The third phase, DBP shedding from the cell surface, was
shown to be due to the action of the enzyme elastase on the
DBP binding site, since DBP is not modified (cleaved) during
this process (49). Elastase is a physiologically important protease
with broad substrate specificity, its expression is restricted mainly
to neutrophils and abundant quantities of this protease are
stored in the azurophil (primary) granules in the cytoplasm
of both immature and mature neutrophils (52). Inhibition of
elastase will inhibit shedding and cause DBP to accumulate on
the plasma membrane, and also will inhibit the chemotactic
cofactor function of DBP (49). Previous reports have described
proteolytically active elastase on the cell surface of neutrophils
in vitro (53,54). Interestingly, both DBP binding, and shedding
of the binding site, are constitutive processes and occur in the
absence of an inflammatory or chemotactic stimulus, however
these stimuli accelerate the process (46,49). Moreover, as will be
discussed below, DBP binding to cell surface actin on neutrophils
and subsequent elastase-mediated shedding from the plasma
membrane, may be a key step in understanding its function as
a chemotactic cofactor.
In addition to DBP uptake from fluids, we also have previously
reported that human neutrophils contain an intracellular store
of DBP in specific (secondary) granules (39), and this finding
was verified in a follow-up study investigating the DBP binding
site in neutrophils (46). Neutrophil specific granules contain a
diverse array of molecules including several binding proteins
such as haptoglobin and vitamin B12 binding protein (55). It is
not clear why these cells have DBP stored in intracellular granules
formed during myelocyte stage of neutrophil development in
the bone marrow (56). Perhaps cells utilize this store of DBP
during chemotaxis and phagocytosis when there is dynamic
rearrangement of cell structures and release of granule contents
(56). The amount of DBP was calculated to be 3 ng/106
neutrophils, a rather small quantity but considering that an
inflammatory exudate may contain billions of neutrophils, the
amount of DBP that neutrophils potentially could release in an
inflammatory lesion may not be insignificant.
NEUTROPHILS, INFLAMMATORY INJURY,
AND THE CHEMOTACTIC COFACTOR
FUNCTION OF DBP
Neutrophils are the primary “rapid response” cells of the innate
immune system that are essential for host defense (57,58).
Individuals with a marked reduction in circulating neutrophils
(neutropenia), either due to a rare congenital abnormality
or more commonly a consequence of cancer chemotherapy,
are highly susceptible to severe and often life-threatening
bacterial and fungal infections. The importance of maintaining
adequate numbers of circulating neutrophils is highlighted by
the fact that ∼60% of the total bone marrow output of all
blood cells is dedicated just to produce neutrophils (59). It is
estimated that the average healthy person has a steady-state
production of 100 billion neutrophils per day under homeostatic
conditions, whereas cell production increases significantly and
rapidly during an infection, a process known as emergency
granulopoiesis (60,61). However, it has been well-known
for more than a century that excessive tissue accumulation
of neutrophils is observed in many inflammatory disorders
(neutrophilic inflammation). Cytotoxic products from activated
neutrophils mediate significant tissue injury and are linked to
the pathogenesis of numerous acute and chronic diseases (57–
59). The destructive potential wrought by excessive numbers of
responding neutrophils is highlighted by the fact that this cell
type can liquify and obliterate tissue, i.e., a cavity resulting from
an abscess (62). There has been remarkable progress over the
past 5–8 years in understanding various aspects of neutrophil
biology, from their birth in the bone marrow to their death at
sites of inflammation and everything in between. Perhaps most
interesting is that neutrophils possess considerable plasticity and
are far more adaptable to their environment than previously
thought. Furthermore, neutrophils can display several disparate
functions that contradict their long-time moniker as “masters of
tissue destruction.” Many excellent reviews on various aspects of
neutrophil biology have been published recently (63–66).
Migration of neutrophils from the bloodstream into various
tissues is a critical stage during inflammation triggered by both
infectious and non-infectious stimuli (63). Although much is
known about the chemoattractants and their receptors that
initiate and direct the neutrophil migratory response, little
is known about factors in physiological fluids that regulate
tissue recruitment of these cells. More than 35 years ago
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42
Kew DBP and Inflammatory Injury
several investigators had demonstrated that normal human
serum possesses a heat stable chemotactic enhancing activity for
complement activation peptide C5a (67–69). A 60 kDa protein
(called co-chemotaxin) was partially purified from human serum
and shown to be capable of enhancing the neutrophil chemotactic
activity of C5a and its stable degradation product C5a des
Arg (70). Previously, we were the first of several groups to
identify that DBP was the serum co-chemotaxin for C5a (71,
72), and several other groups subsequently confirmed our
initial observations that DBP can augment the chemotactic
activity of C5a/C5a des Arg (73–79). These studies all reported
that DBP does not possess chemotactic activity but requires
chemoattractants to exert its cofactor activity. Other studies
also reported that DBP is not able to augment other C5a-
mediated functions in neutrophils such as oxidant generation
and degranulation (release of cytoplasmic granule contents)
(71,79). But most curious was the observation that DBP
could not enhance the chemotactic activity of other major
neutrophil chemoattractants: formylated peptides, CXCL8 (IL-
8), leukotriene B4or platelet activating factor (71,72), leading to
the general consensus that the chemotactic enhancing properties
of DBP appeared to be restricted to C5a/C5a des Arg. All of
these reports used an in vitro chemotaxis assay, employing
either blind-well or Boyden chambers, to measure an increase
in neutrophil migration when DBP was added. Although this
assay is very sensitive and quantitative, it has limitations that
resulted in the mistaken conclusion that DBP was specific
for complement peptide C5a. C5a is a very potent neutrophil
chemotactic factor and on a molar basis is 10–50 times more
potent than other major chemoattractants when tested in vitro
(71,80). Therefore, the chemotactic enhancing activity of DBP
during the short incubation (30 min) filter-based assays was
particularly noticeable when C5a was used as the stimulus.
However, utilizing a different in vitro assay (under agarose), that
requires a longer incubation period (4 h), revealed that DBP
could enhance neutrophil movement to other chemoattractants
as well (81). Moreover, in vivo studies using the DBP null mice
(discussed below) have shown that the chemotactic cofactor
activity of DBP is not specific for C5a as previously thought
but can augment the chemotactic activity of perhaps many
leukocyte chemoattractants. These recent in vitro and in vivo
studies described below have helped to better define how DBP
functions to enhance neutrophil chemotaxis that may contribute
to neutrophilic inflammation.
LESSONS FROM THE DBP NULL MOUSE
The initial reports describing the chemotactic cofactor function
of DBP were received enthusiastically at that time in the
inflammation research community (71,72). However, that high
level of interest faded with the subsequent failure to describe
a clear mechanism of chemotaxis enhancement, and to provide
confirmation that DBP enhances neutrophil chemotaxis in
vivo. Previous attempts to define a mechanism using in vitro
approaches were not successful. For example, our lab performed
numerous chemical cross-linking and co-immunoprecipitation
experiments that demonstrated clearly DBP does not physically-
associate with the C5a receptor (50). In addition, we reported
that and DBP does not bind to C5a, and cell binding of C5a and
DBP are independent events (82). Other groups have shown that
DBP does not alter the number of neutrophil C5a receptors or
the receptor Kdfor C5a, thereby discounting another obvious
explanation for its co-chemotactic effect (79,83). The question
of physiological relevance and whether DBP enhances neutrophil
recruitment to tissues in vivo needed to wait more than 10 years
until a DBP null mouse was generated.
The vitamin D binding protein has been very well-studied in
multiple different populations worldwide for over 50 years, but
no natural homozygous deficiency of DBP had been reported in
humans, or to the best of our knowledge, any mammal. There was
no clear reason why a DBP-deficiency was never identified but
it was widely speculated that a DBP deletion may be embryonic
lethal. Nevertheless, Nancy Cooke’s lab at the University of
Pennsylvania produced the first mice that were homozygous
null for DBP (84). These mice were healthy, of similar size
and appearance as wild-type mice, and the DBP null males and
females were fertile and produced normal sized litters (84). This
phenotype was somewhat surprising at the time given the fact
that a natural DBP deficiency had not been observed. Analysis
of blood chemistry values revealed that DBP null mice had
essentially the same levels of serum calcium, phosphorus, PTH,
and alkaline phosphatase as DBP sufficient wild type mice when
fed a standard vitamin D replete mouse chow diet (84). However,
the serum of DBP null mice had >95% reduction in both 25(OH)
and 1,25 (OH)2vitamin D (84). Moreover, when placed on a
vitamin D deficient diet, the DBP null mice quickly developed
secondary hyperparathyroidism and bone mineralization defects
such as osteoid thickening that were not seen in DBP sufficient
wild-type mice (84). The initial DBP null strain was backcrossed
onto a C57BL/6J background for 10 generations and was used
in further studies which showed that the lack of circulating
DBP does not alter the tissue distribution, uptake, activation or
biological potency of vitamin D (85). Thus, DBP does not alter
bioavailable vitamin D but appears to function as a circulating
reservoir of 25(OH) vitamin D.
Since no case of homozygous deficiency in humans has been
reported, a long-standing unresolved question has been: can DBP
null mice function as a murine model for DBP deficiency in
humans? Surprisingly, during the preparation of this review,
the first report of a DBP deficient human was published
(86). The individual is a 58 year-old female who presented
with long-standing and progressive ankylosing spondylitis and
severe vitamin D deficiency that did not respond to vitamin
D supplementation. Laboratory results showed a deletion of
the DBP (GC) gene and corresponding absence of circulating
DBP. Although this individual had a profound deficiency in
both 25(OH) and 1,25 (OH)2vitamin D, her blood calcium
levels were normal (86). The bone abnormalities and blood
chemistry values of this DBP deficient woman were similar to
those observed in DBP null mice fed a vitamin D-deficient diet
(84,86). In vitro studies using dermal fibroblasts from this DBP
null patient showed that DBP does not alter cellular uptake
of bioactive vitamin D and expression of the responsive gene
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43
Kew DBP and Inflammatory Injury
CYP24A1, a very similar result also was previously reported
using cells from DBP null mice (85). However, no information
was provided about this patient’s response to infections or
tissue injury. This report provides some degree of validation
that DBP null mice can act as a model for DBP deficiency
in humans.
DBP NULL MICE AND INFLAMMATORY
INJURY MODELS
Our in vivo studies of inflammatory injury utilized the DBP
null mouse strain developed by Nancy Cooke’s lab (84,87). This
DBP null strain was re-derived the by in vitro fertilization using
DBP−/−sperm and a wild-type C57BL/6J female (DBP+/+)
to produce DBP+/−hemizygotes which were cross-bred to
generate the DBP null (−/−) and wild type (+/+) mouse
colonies (81). These DBP−/−and DBP+/+mice have been
used in three injury models: acute lung injury, multiphasic
(acute, chronic, fibrotic) lung injury, and acute muscle injury
(81,88). Each model clearly showed that DBP null mice
always have less inflammation, and most noticeably, significantly
fewer neutrophils at the site of injury. The markedly reduced
neutrophilic inflammation observed with DBP null mice is
very consistent among the injury models and reproducible
between experiments, but the underlying cellular and molecular
mechanisms that are responsible for generating this phenotype
is only partially understood. A proposed tentative model is
described in a separate section below.
The first study to examine the in vivo role of DBP in
inflammatory injury used a model of acute complement-
dependent alveolitis, induced by either immune complexes or
purified mouse C5a (81). In both alveolitis models, DBP null
mice had significantly reduced (∼50%) neutrophil recruitment
to the lungs compared to their wild-type DBP+/+counterparts,
and lung histology showed significantly less inflammation in the
null mice (81). Another important observation is that addition of
exogenous DBP to the lungs of DBP null mice completely rescued
their neutrophil recruitment defect. The same study also showed
that bronchoalveolar lavage (BAL) fluid from wild-type mice had
extensive DBP-actin complexes (∼75% of total DBP) 4h after
induction of alveolitis. Although as predicted there were no DBP-
actin complexes in DBP null mice, there was detectable actin in
the BAL fluid from these animals, indicating actin release from
damaged cells (81). These results indicate that DBP null mice
have impaired neutrophil recruitment due to lack of DBP and not
a cellular defect since the total number, receptor expression and
chemotaxis of circulating DBP null neutrophils are essentially
identical to cells from their wild-type DBP+/+counterparts.
A second study investigated if a systemic DBP deficiency
could attenuate multiphasic lung injury and tissue remodeling
induced by bleomycin (81). Wild type and DBP null mice
received bleomycin by oropharyngeal aspiration; lung injury was
evaluated after 7, 16, or 21 days. DBP null mice all survived to
day 21 and did not display overt signs of morbidity whereas all
wild-type mice died between day 13 and 16 and showed clear
signs of respiratory distress. Bronchoalveolar lavage (BAL) fluid
from wild-type mice had extensive DBP-actin complexes (60–
75% of total DBP) whereas DBP null mice had no complexes but
had evidence of free actin. Analysis of BAL fluid on days 7 and
16 post-treatment showed that both mouse strains had similar
numbers of lung macrophages and lymphocytes, but DBP null
animals had significantly fewer lung neutrophils. Histological
analysis of the lungs on day 16 showed that DBP null mice
had a 50% decrease in fibrosis and collagen deposition as
compared wild-type animals. This study demonstrated that a
systemic deficiency in DBP provides significant protection from
bleomycin-induced inflammation and fibrosis in mice.
The third in vivo study utilized an acute muscle injury model
induced by injection of 50% glycerol into the thigh muscle (88).
All animals survived the procedure, but intramuscular glycerol
injection showed lysis of skeletal myocytes, and inflammatory
cell infiltrates in both strains of mice. The muscle inflammatory
cell infiltrate in DBP null mice had remarkably few neutrophils
as compared to wild-type mice. The neutrophil chemoattractant
CXCL1 was significantly reduced in muscle tissue from DBP null
mice. Plasma obtained 48 h after glycerol injection revealed that
DBP null mice had significantly lower levels of systemic cytokines
IL-6, CCL2, CXCL1, and G-CSF. Multiplex analysis of 36
cytokines indicated that DBP null mice had a less inflammatory
and more pro-reparative cytokine profile than their wild-type
DBP+/+counterparts (88).
These in vivo studies comparing null mice to wild-types
showed that DBP may have a central role during inflammation
since it induces selective recruitment of neutrophils, and the DBP
cofactor function is not restricted to C5a as prior in vitro studies
indicated, so the physiological implications are much broader.
So how does DBP contribute to inflammation by enhancing
neutrophil recruitment? The accumulating in vitro and in vivo
evidence appears to suggest that DBP-actin complexes may act
as an alarmin and trigger pro-inflammatory functions. DBP
is the major extracellular scavenger for actin released from
damaged/dead cells and formation of DBP-actin complexes is an
immediate host response to tissue injury. All of the in vivo injury
models discussed above had evidence of DBP-actin complexes
only in wild-type mice, and DBP repletion in the acute alveolitis
model reversed the neutrophil recruitment defect in the DBP
null mice. The currently accepted actin scavenger hypothesis
states that injury-induced depletion of plasma DBP will diminish
the actin binding capacity in blood and extracellular fluids,
leading to formation of actin filaments that obstruct flow and
damage small blood vessels (15). Furthermore, if the current actin
scavenger hypothesis is correct, it follows that DBP null mice
should succumb rapidly to an intravenous bolus of actin which
would obstruct the pulmonary vasculature. However, contrary
to what would be predicted by the actin-free DBP hypothesis,
we observed that DBP null mice do not succumb to a bolus
of purified actin injected intravenously (89). In fact, DBP null
mice were largely resistant to the lung inflammation and injury
observed in wild-type mice, and the null mice appeared utilize
plasma gelsolin to clear the actin bolus. Wild-type mice also
had a large percentage of their total plasma DBP pool (78%)
bound to actin 1.5 h after i.v. injection. Moreover, in vitro studies
showed that purified DBP-actin complexes added to cultured
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Kew DBP and Inflammatory Injury
endothelial cells caused direct cell damage (at 4 h) or death (at
24 h), providing clear evidence that DBP-actin complexes have
a direct detrimental effect on cells (89). Thus, results from DBP
null mice provide strong evidence to refute the prevailing actin
scavenger hypothesis and suggest that the inverse hypothesis
may be valid, i.e., an increase in DBP-actin complexes, and
not a reduction in actin-free DBP, correlates with inflammation
and injury.
A POSSIBLE ROLE FOR VITAMIN D IN
INFLAMMATORY INJURY?
Vitamin D is known to regulate numerous genes that are involved
in the immune and inflammatory response. In vitro and in vivo
studies have shown that active vitamin D produces a tolerogenic,
anti-inflammatory, and reparative phenotype as evidenced by
immune cell activation status and cytokine profiles (90,91). DBP
null mice have almost no detectable serum vitamin D but actually
are vitamin D sufficient when fed a vitamin D replete chow
diet (84,87). Although plasma DBP does not alter the tissue
availability of vitamin D, the effect on immune cells in the blood
is not known. The lack of plasma DBP in mice may permit greater
delivery of bioactive vitamin D to immune cells during their
transit in the blood, potentially altering their transcriptomes to
dampen inflammation and limit tissue damage. Superimposed on
this possible scenario of transcriptional regulation by vitamin D
is a lack of DBP-actin complexes during tissue injury. Perhaps
both mechanisms together cause DBP null mice to have less
neutrophilic inflammation and resultant tissue damage following
injury. However, these possibilities remain to be investigated.
Finally, it is interesting to note that we previously reported that
bioactive vitamin D (1,25 dihydroxy-vitamin D3) bound to DBP
at physiologically relevant concentrations of 10 and 100 pM,
completely abolished the DBP chemotactic cofactor function of
human neutrophils in vitro, but had no effect on chemotaxis
to optimal concentrations of four different chemoattractants
(80). In contrast, 25-hydroxy-vitamin D3bound to DBP had no
effect on the chemotactic cofactor function (80), thus providing
evidence of a direct inhibitory effect of bioactive vitamin D on the
DBP chemotactic cofactor function for neutrophils.
PROPOSED MODEL OF DBP-ACTIN
COMPLEXES AND INFLAMMATORY
INJURY
Recent evidence indicates that DBP bound to G-actin, and not
free DBP, functions as an indirect but essential cofactor for
neutrophil migration, thus, providing a possible mechanism to
explain how DBP functions to enhance neutrophil migration in
vitro and recruitment to sites of inflammation in vivo (81,88,
89). However, it is not clear how DBP-actin complexes enhance
neutrophil recruitment and inflammation. It is interesting to
speculate that DBP-actin complexes may augment a chemotactic
signal and cause release of other proinflammatory molecules
stored within neutrophils. Perhaps the most attractive candidate
in this scenario is calprotectin, a 24 kDa heterodimer composed
of S100A8 and S100A9 that can be rapidly released from
neutrophils (92). Abundant quantities of S100A8 and S100A9
are stored in the cytosol of neutrophils, and upon cell activation
these molecules can be released into the extracellular space
as active heterodimers or heterotetramers (92). S100A8/A9
has multiple proinflammatory functions and has been shown
to mediate both neutrophil bone marrow development and
facilitate chemotaxis of mature circulating cells, most likely by
binding to toll-like receptor 4 (TLR4) on the plasma membrane
(92). The proposed mechanism involving DBP-actin complexes
has clear physiological relevance since DBP is abundant and
ubiquitous in all fluid compartments, and release of G-actin
from damaged/dead cells is a consistent feature in all types
of inflammatory injury. Moreover, we have previously reported
that DBP binds to actin on the neutrophil plasma membrane
followed by elastase-mediated shedding of these complexes,
perhaps as microvesicles, into the extracellular fluids (46,48,49).
We propose that DBP-actin complexes can bind (or re-bind
following shedding) to a neutrophil surface receptor that triggers
S100A8/A9 release. In turn, S100A8/A9 binds in an autocrine
or paracrine manner to neutrophil TLR4 inducing a signal that
synergizes with the chemoattractant receptor signal to enhance
migration. Furthermore, it is well-known that TLR4 ligation and
signaling synergizes with signals from chemoattractant receptors
to enhance leukocyte chemotaxis both in vitro and in vivo
(93,94).
This provisional model may explain how a deficiency of
DBP results in significantly decreased neutrophilic inflammation.
However, there are several “unknowns” with this model that need
to be investigated. First, the putative receptor for binding DBP-
actin complexes has not been identified, but other studies have
shown that the receptor Clec9A binds F-actin and is involved
in sensing damaged cells (95–97). It is not known if the Clec9A
receptor also binds G-actin complexed with DBP or if even
if this receptor is expressed on neutrophils. Second, it is not
known if the putative DBP-actin receptor signals release of
S100A8/A9 when it is ligated with complexes. Third, a question
remains do all DBP-actin complexes function the same or are
there modifications that differentiate between inflammatory and
benign complexes. Finally, do other cell types besides neutrophils
and endothelial cells also respond to DBP-actin complexes,
particularly hepatocytes and Kupffer cells in the liver (the
primary site of DBP-actin clearance).
FUTURE DIRECTIONS
Many questions about the functions of DBP remain to be
answered. However, new investigative tools and experimental
approaches will be needed to decipher these functions. For
example, new mouse models with tissue specific inducible
expression of DBP, or a mouse model constructed with
selective deletions in functional regions within DBP (vitamin
D, actin, cell binding regions) by utilizing CRISPER-Cas9
gene editing technology. Another essential tool currently
needed is an antibody that only recognizes a neoepitope on
DBP-actin complexes, and not DBP or G-actin monomers.
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Kew DBP and Inflammatory Injury
This antibody then could be used to develop an ELISA
that specifically detects only DBP-actin complexes in
biological fluids. In closing, research into the biological
functions of DBP has progressed far since Hirschfeld’s initial
description 60 years ago, but perhaps in the near future
new experimental approaches using advanced technologies
(single cell transcriptomics, mass cytometry, advanced
microscopy, and in vivo imaging, etc.) and bioinformatic
analysis may reveal what DBP actually has been doing
all along.
AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and
has approved it for publication.
ACKNOWLEDGMENTS
We would like to acknowledge the long-term grant support
from the U.S. National Institutes of Health (R01GM063769) that
facilitated these investigations.
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03.009
Conflict of Interest Statement: The author declares that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Kew. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) and the
copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
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48
REVIEW
published: 24 October 2019
doi: 10.3389/fendo.2019.00718
Frontiers in Endocrinology | www.frontiersin.org 1October 2019 | Volume 10 | Article 718
Edited by:
Zhongjian Xie,
Central South University, China
Reviewed by:
Michael F. Holick,
Boston Medical Center, United States
Melissa Orlandin Premaor,
Federal University of Minas
Gerais, Brazil
*Correspondence:
Rene F. Chun
rchun@mednet.ucla.edu
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 26 April 2019
Accepted: 04 October 2019
Published: 24 October 2019
Citation:
Chun RF, Shieh A, Gottlieb C,
Yacoubian V, Wang J, Hewison M and
Adams JS (2019) Vitamin D Binding
Protein and the Biological Activity of
Vitamin D. Front. Endocrinol. 10:718.
doi: 10.3389/fendo.2019.00718
Vitamin D Binding Protein and the
Biological Activity of Vitamin D
Rene F. Chun 1
*, Albert Shieh 2, Carter Gottlieb 1, Vahe Yacoubian 1, Jeffrey Wang 1,
Martin Hewison 3and John S. Adams 1
1Department of Orthopaedic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States,
2Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States, 3Institute of
Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom
Vitamin D has a long-established role in bone health. In the last two decades, there
has been a dramatic resurgence in research interest in vitamin D due to studies that
have shown its possible benefits for non-skeletal health. Underpinning the renewed
interest in vitamin D was the identification of the vital role of intracrine or localized,
tissue-specific, conversion of inactive pro-hormone 25-hydroxyvitamin D [25(OH)D] to
active 1,25-dihydroxyvitamin D [1,25(OH)2D]. This intracrine mechanism is the likely
driving force behind vitamin D action resulting in positive effects on human health.
To fully capture the effect of this localized, tissue-specific conversion to 1,25(OH)2D,
adequate 25(OH)D would be required. As such, low serum concentrations of 25(OH)D
would compromise intracrine generation of 1,25(OH)2D within target tissues. Consistent
with this is the observation that all adverse human health consequences of vitamin D
deficiency are associated with a low serum 25(OH)D level and not with low 1,25(OH)2D
concentrations. Thus, clinical investigators have sought to define what concentration
of serum 25(OH)D constitutes adequate vitamin D status. However, since 25(OH)D is
transported in serum bound primarily to vitamin D binding protein (DBP) and secondarily
to albumin, is the total 25(OH)D (bound plus free) or the unbound free 25(OH)D the crucial
determinant of the non-classical actions of vitamin D? While DBP-bound-25(OH)D is
important for renal handling of 25(OH)D and endocrine synthesis of 1,25(OH)2D, how
does DBP impact extra-renal synthesis of 1,25(OH)2D and subsequent 1,25(OH)2D
actions? Are their pathophysiological contexts where total 25(OH)D and free 25(OH)D
would diverge in value as a marker of vitamin D status? This review aims to introduce and
discuss the concept of free 25(OH)D, the molecular biology and biochemistry of vitamin
D and DBP that provides the context for free 25(OH)D, and surveys in vitro, animal, and
human studies taking free 25(OH)D into consideration.
Keywords: vitamin D, free vitamin D, bone, immunology, DBP, CYP27B1, VDR
INTRODUCTION
The benefits of vitamin D for mineral homeostasis and bone health are well-established. Deficiency
of vitamin D, rickets in children and osteomalacia in adults, can be treated or prevented with oral
supplements of vitamin D. Despite promising pre-clinical observations and vitamin D-deficiency
association studies, the impact of vitamin D on other aspects of human health such as common
49
Chun et al. Free 25D
cancers, cardiovascular disease, type 2 diabetes obesity,
autoimmune disorders, and infectious disease remains
controversial (1–5). Randomized, controlled supplementation
trials are required to better define the extra-skeletal roles
of vitamin D. However, these trials are complicated by two
unanswered questions: (1) what is the best marker of vitamin
D status and (2) what constitutes a level that is sufficient to
promote the health benefits of vitamin D?
ENDOCRINE VITAMIN D METABOLISM
AND ACTION
The name “vitamin D” in this review refers to a collection
of secosterol molecules detectable in the serum of vertebrates
(left panel, Figure 1). Briefly, cholecalciferol or vitamin D3
(vitamin D) results from the ultraviolet B (UVB; 290–315 nm)-
mediated photolytic-conversion of 7-dehydrocholesterol (DHC)
in skin (6–8). Vitamin D can also be obtained from (i)
food, principally from fortified dairy and juice products, (ii)
consumption of fresh caught fish (e.g., salmon) (9), and (iii) oral
vitamin D supplements. Vitamin D2 (ergocalciferol) is naturally
found in fungi (e.g., mushrooms) and sometimes used in food
fortification and supplementation regimes. Regardless vitamin
D2 proceeds through the same modifications as described for
vitamin D3 below.
Once in the general circulation vitamin D is bound to its
serum carrier, vitamin D binding protein (DBP) and to a lesser
extent albumin (10,11), and is subject to a first hydroxylation
step by vitamin D substrate-dependent 25-hydroxylase [CYP2R1
(12) and possibly a yet unidentified hydroxylase(s) (13)] in
the liver resulting in 25-hydroxyvitamin D (25(OH)D). Very
little 25(OH)D (∼5%) is secreted in to the bile (14). Rather,
the bulk of 25(OH)D re-enters the circulation, once again
bound to either DBP or albumin for endocrine transport to
target tissues. DBP- and albumin-bound 25(OH)D in urine
is reclaimed by tubular epithelial cells in the kidney (15).
Here internalized 25(OH)D is freed from its carrier protein(s),
becoming substrate for (i) the low capacity 25-hydroxyvitamin
D-1-alpha-hydroxylase (CYP27B1) and production of the
active, hormonal form of vitamin D, 1,25-dihydroxyvitamin D
(1,25(OH)2D) or (ii) the high calpacity 25-dihydroxyvitamin D-
24 hyxdoxylase (CYP24A1) to form the largely non-biologically
active metabolites 24,25-dihydroxyvitamin D (24,25(OH)2D)
and 1,24,25-trihydroxyvitamin D, respectively (16). The various
hydoxylated forms gain access to the general circulation
bound to DBP or albumin. Rheostatic endocrine control
over the reciprocal production of 1- and 24-hydroxylated
vitamin D metabolites is exerted by parathyroid hormone and
FGF23. Parathyroid hormone increases activity of CYP27B1-
hydroxylase (17,18), decreases product output by CYP24A1
(19,20); thus, increases the “activation” quotient of product
1,25(OH)2D:substrate 25(OH)D in the serum. On the other
hand, FGF-23 blunts CYP27B1 activity (21) and promotes
CYP24A1 activity; thus, decreases the 1,25(OH)2D:25(OH)D
activation quotient and increases the 24,25(OH)2D:25(OH)D
inactivation quotient (22,23).
As noted above, 1,25(OH)2D can be chaperoned in an
endocrine mode to potential target tissues that employ serum
bound, extracellular 1,25(OH)2D as a specific ligand for
transactivation of the vitamin D receptor (VDR) in the target cell
driving 1,25(OH)2D-VDR directed differential gene expression
(24). A major caveat in the concept of direct endocrine action
of 1,25(OH)2Dis the fact all adverse physiological consequences
of vitamin D deficiency in humans are associated with a low
serum 25(OH)D, not a low 1,25(OH)2D level [Table 1; (25)].
In fact, in the basal state, before treatment to raise 25D levels,
subjects with low serum 25D often have serum 1,25D levels
that are relatively elevated as a consequence of compensatory
secondary hyperparathyroidism. In this instance the increase in
the host’s circulating concentration of PTH drives an increase in
renal 1,25D production. This suggests that 25D deficiency in the
serum is a cause for “endocrine resistance” to circulating levels
of the 1,25D hormone at the level of the gut. After vitamin D
restoration treatment with return of 25D balance to normal and
resolution of secondary hyperparathyroidism, there is an increase
in intestinal calcium absorption even though there is a relative
decrease, or no change in the circulating serum 1,25D level. This
suggests that there may be local conversion of 25D to 1,25D in the
gut outside of the serum compartment that is driving intestinal
calcium absorption and/or that only measuring the total amount
of vitamin D metabolite(s) in the serum, may be an inadequate
biomarker of response to restoration of 25D levels in the blood
to normal.
LOCAL VITAMIN D METABOLISM AND
ACTION
In non-renal tissues, the CYP27B1 converts 25(OH)D to
1,25(OH)2D for local usage in paracrine, autocrine, and
intracrine regulated activities (26). Perhaps the most relevant
physiological/pathophysiological example of these events (e.g.,
those confined to the local tissue microenvironment outside
of circulating serum compartment) are the human granuloma
forming diseases like sarcoidosis and tuberculosis (27). In
these disease states, cells of the innate immune response,
principally macrophages, express the same metabolic machinery
to synthesize 1,25(OH)2D intracellularly when presented with
a CYP27B1 activating signal and with sufficient 25(OH)D in
the extracellular space to serve as substrate for the CPY27B1.
When the extracellular concentration of 25(OH)D falls below the
equivalent of ∼20 ng·mL−1or 50 nM, the intracrine production
of 1,25(OH)2D via the CYP27B1-hydroxylase becomes limiting;
unlike the renal CYP27B1, the enzyme in the macrophage
is highly substrate-drive (28). Taking the human granuloma-
forming, macrophage dominant infectious disease tuberculosis
(TB) as an example, in the face of deficient extracellular
substrate 25(OH)D the macrophage CYP27B1 is unable to
generate enough active 1,25(OH)2D metabolite to effectively
ligand sufficient VDR in that cell to promote expression of
vitamin D-dependent antimicrobial genes (29,30). The end result
is failure of the macrophage to mount an effective autophagy-
related, vesicular killing response to ingested Mycobacterium
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FIGURE 1 | Vitamin D metabolism and action. Vitamin D metabolites (Left). 7-dehydrocholesterol (7-DHC) is photoconverted to vitamin D3by UV exposure of skin.
CYP2R1 in the liver hydroxylates vitamin D3to 25-hydroxyvitamin D3(25(OH)D). Another hydroxylation to 25(OH)D3by CYP27B1 occurs in the kidney but also in a
number of extra-renal tissues produces the active 1,25-dihydroxyvitamin D3(1,25(OH)2D3). 1,25(OH)2D3is the cognate ligand for the vitamin D receptor (VDR), a
nuclear transcription factor, that directs 1,25(OH)2D3regulated gene transcription. CYP24A1 is responsible for the hydroxylation that yields 24,25-dihydroxyvitamin D
(24,25(OH)2D3) and subsequent catabolism to non-biologically active metabolites. Intracrine mechanism in immune cells (Right). Vitamin D action in immune cells is
reliant upon the intracrine (local) production of active 1,25(OH)2D within the macrophage. Vitamin D status of the host as defined by serum, extracellular 25(OH)D
levels impact immune response, as 25(OH)D is the substrate for CYP27B1. A complex interplay between monokine signaling, that can both be responsive to and
stimulatory of 1,25(OH)2D synthesis, and regulatory signaling among innate and adaptive immune cells is shown.
TABLE 1 | Vitamin D and human health.
Adverse health outcomes significantly associated with low serum
25(OH)D level
Low bone density Obesity
Hip fractures Insulin resistance
Non-vertebral fractures Type 1 diabetes
Heart attack Type 2 diabetes
Hypertension Cancer
Stroke Preterm delivery
Neurocognitive dysfunction Pre-eclampsia
Proximal muscle weakness Inflammation/infection
Autoimmune diseases Multiple sclerosis
Numerous health conditions have been associated with low total serum levels of 25(OH)D
and are listed in the table. Conditions pertaining to bone health are indicated in blue. Table
is based on reviews by Rosen et al. (2) and Rosen and Taylor (4).
tuberculosis (M. tb) (31,32). In vitro this failure can be rescued
in a 25(OH)D concentration-dependent fashion by exchanging
vitamin D deficient human serum with vitamin D sufficient
serum (>30 ng·mL-1 or 75 nM); in other words, rescue of
the macrophage innate immune is achieved by conditioning
activated macrophages ex vivo in serum from the same host after
treatment of the host with vitamin D in vivo (30). Demonstrating
successful rescue from 25(OH)D deficiency in vivo in humans
exposed to or in the very early phases of infection with M. tb.
would support the value of 25(OH)D-driven innate immune
competence in prevention and use as adjunctive therapy early in
the course of this disease (33).
The local antimicrobial capacity of the macrophage is
subject to intracrine, autocrine, and paracrine feedforward
and feedback immune regulatory circuits. This regulatory
network is depicted schematically in the right panel of
Figure 1. Stimulation of the Toll-like receptor signaling pathway
by pathogen-associated molecular pattern (PAMP) molecules
induces expression of CYP27B1 and VDR as well as of monokines
(e.g., IL-15 and IL-1β). In an autocrine mode, these two
monokines act to amplify expression of the CYP27B1 and
1,25(OH)2D-VDR-directed generation of antimicrobial peptides
(34). In a paracrine fashion IL-1ß mobilizes and activates
cells of the adaptive immune response (35–37). Activation
of the Th1 subset of “helper” lymphocytes promotes: (1)
production of IFN-γ, the most potent known stimulator of
the macrophage CYP27B1-hydroxylase (38); and (2) induction
of expression of the VDR in adaptive immune response cells
(39,40). When IFN-γ-driven production of 1,25(OH)2D in the
macrophage is robust enough to allow escape of the active
vitamin D metabolite into the local, pericellular inflammatory
microenvironment, this 1,25(OH)2D is sufficient to drive VDR-
dependent gene expression in activated lymphocytes such as
inhibiting proliferation of those lymphocytes. As such, the
predominant paracrine action of 1,25(OH)2D in this setting is
to modify the adaptive immune response (41) and turn down
IFN-γand macrophage CYP27B1 gene expression, preventing a
potential overzealous adaptive (auto)immune response harmful
to the host. Therefore, 25(OH)D “sufficiency” in the serum of
the host appears to be paramount in providing the optimal IFN-
γ-mediated feedback control on 1,25(OH)2D synthesis by the
macrophage and appropriate antimicrobial response to ingested
microbes. For example, failure of this normal feedback control
in disseminated infection with M. tb. may result in escape of
1,25(OH)2D from the local immune microenvironment into
the serum tuberculosis, resulting in a form of endocrine-acting
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Chun et al. Free 25D
“1,25(OH)2D intoxication” and life-threatening hypercalcemia.
This form of extra-renal 1,25(OH)2D intoxication can occur
in certain granuloma-forming diseases such as tuberculosis
(where pathogen is known) and sarcoidosis (where pathogen
is unknown).
These scenarios indicate that the serum level of
bioavailable 25(OH)D to macrophages is a key determinant
of normal/abnormal physiological control of innate and
adaptive immunity in the host with low serum 25(OH)D
levels. Cell and molecular biology experiments have established
co-existent expression of the CYP27B1 and VDR in the same
cell (Table 2). This observation makes all of these cell-types
potential candidates for intracrine metabolism and action of
1,25(OH)2D generated from 25(OH)D available to that cell from
its extracellular microenvironment in vivo. Beyond tuberculosis
just discussed, many human diseases are associated with “low”
serum 25(OH)D levels compared to matched controls without
the disease (Table 1). In cardiovascular disease, the leading cause
of mortality in the US, mortality is inversely related to serum
25(OH)D <20 ng/ml (42,43), though this is disputed (44). All
of these disease states are associated with altered host immunity.
Perhaps this is the common link to adverse outcomes in this
otherwise diverse set of disorders.
DEFINING VITAMIN D STATUS
The total serum 25(OH)D level (the sum of 25(OH)D that
is bound to carrier proteins and is free in the circulation) is
the currently accepted marker of choice for defining vitamin
D status in any given individual. Its preferential use is based
on (i) analysis of the existing body of clinical research, (ii)
assay advances that confer easy and accurate measurements
and (iii) the fact that it is the most abundant and stable of
the various vitamin D metabolites in serum; 25(OH)D has a
relatively long half-life (15 days) compared to 1,25(OH)2D (15 h).
However, health organizations across the globe differ significantly
in their definition of what level of total circulating 25(OH)D
constitutes sufficiency and deficiency for a “normal” population
without evidence of active bone disease (e.g., osteoporosis,
TABLE 2 | Cell types that express both CYP27B1 and VDR.
Cells co-expressing a functional CYP27B1 and VDR
Macrophage Enterocyte
Dendritic cell Decidual stromal cell
Parathyroid cell Fetal trophoblast
Osteoblast Prostate epithelial cell
Osteoclast Vascular endothelial cell
Keratinocyte Pancreatic βcell
Mammary epithelial cell Renal tubular cell
In contrast to the endocrine action based on kidney production of 1,25(OH)2D that travels
through the general circulation to VDR-possessing target cells, a number of cell types
harbor both CYP27B1 and VDR making intracrine metabolism and action possible. The
red arrow indicates the macrophage, the cell type in which the intracrine metabolism of
25(OH)D to 1,25(OH)2D and 1,25(OH)2D-directed gene expression has been most clearly
demonstrated ex vivo.
hyperparathyroidism, etc.). In North America, the Institute of
Medicine has recommended the value of ≥20 ng/ml 25(OH)D
(50 nmol/L) for sufficiency (45), whereas the Endocrine Society
recommended for ≥30 ng/ml (75 nmol/L) (46). The Vitamin
D Council states that individuals should strive for levels above
40 ng/ml (100 nmol/L) (47). In contrast to this, the UK Science
Advisory Council on Nutrition defined vitamin D deficiency as
serum levels of 25(OH)D <10 ng/ml (25 nmol/L), but did not
recommend an optimal level for human health (48). Although the
10 ng/ml level is held by some other European nations, some have
recommended higher levels (49). Investigators in the vitamin D
field have highlighted the limitations of the total 25(OH)D as the
routine biomarker for vitamin D status in various commentary
and review articles (50,51) and discussed other potential markers
and the possible need of a “vitamin D panel” (52,53).
Human data indicates that the threshold for detection of
a relative increase in the serum iPTH at the individual and
population level occurs when the total serum 25(OH)D level
falls below ∼30 ng/ml (54,55). Thus, PTH offers a measurable
biological consequence of “low” 25(OH)D. However, PTH
levels are not exclusively controled by 25(OH)D as serum
concentration of ionized calcium is sensed at the parathyroid
gland by the calcium-sensing receptor (CaSR). When serum
calcium levels drop, CaSR signal transduction in the parathyroid
yields an increase in PTH production that then enters the general
circulation resulting in PTH’s endocrine effects.
Another reason for the uncertainty concerning the validity of
total 25(OH)D as an exclusive serum marker for vitamin D health
is due to the complex molecular biology and biochemistry of
25(OH)D-associated bioactivities. This is particularly relevant to
the intracrine, paracrine or endocrine conversion of 25(OH)D to
active 1,25(OH)2D, because (i) the local concentrations vitamin
D metabolites outside of the serum compartment cannot be
easily measured in vivo and (ii) the subsequent molecular actions
of 1,25(OH)2D in conjunction with its binding by the VDR
is dependent on diverse mechanisms beyond simple variations
in circulating 25(OH)D. These include: (1) the transport and
target tissue uptake of 25(OH)D; (2) the directed intracellular
transport of 25(OH)D to the inner mitochondrial to CYP27B1
for enzymatic conversion of 25(OH)D to 1,25(OH)2D; (3) export
if 1,25(OH)2D from the mitochondia and binding of 1,25(OH)2D
to VDR; and (4) competing catabolism of 1,25(OH)2D by
the enzyme CYP24A1 also located on the inner mitochondrail
membrane. In this review, we will briefly discuss the molecular
biology and biochemistry behind these processes, with particular
emphasis on the role of free 25(OH)D as a key determinant of
the downstream actions of 1,25(OH)2D, specifically in bone and
mineral health.
MOLECULAR BIOLOGY AND
BIOCHEMISTRY OF VITAMIN D ACTION
1,25(OH)2D is the active vitamin D molecule with 25(OH)D
being its immediate precursor (panel A, Figure 1). It is
1,25(OH)2D that drives vitamin D-regulated gene expression
in target cells. Under normal conditions, the level of serum
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1,25(OH)2D is tightly regulated within a narrow range (30–
60 pg/ml) at a level that is 1,000X less plentiful than its
25(OH)D precursor. In non-pregnant humans, 1,25(OH)2D
in the serum comes almost exclusively from expression of
CYP27B1 in the kidney. Circulating 1,25(OH)2D’s endocrine
actions are to regulate the serum level of calcium by optimizing
intestinal calcium absorption and/or calcium resorption from
the skeleton (56). Not surprisingly then, when kidney failure
occurs, there is (i) an accompanying decrease in renal CYP27B1
capacity, (ii) a fall in 1,25(OH)2D production and (iii) a
reduction in the serum calcium level. In the opposite case,
when a pathophysiological extra-renal source of CYP27B1
(e.g., the macrophage in granuloma-forming diseases; see
right panel, Figure 1 and Table 1) becomes dysregulated
and dominant, serum 1,25(OH)2D-driven hypercalcemia
and/or hypercalciuria [1,25(OH)2D intoxication] occurs
overriding the calcium-lowering actions of the CaSR in
the parathyroid gland and in the kidney (57). Only in
these two abnormal calcemic states is the serum level of
1,25(OH)2D of the host informative to the clinician in evaluating
a patient.
At the molecular level, 1,25(OH)2D binds VDR with the
highest affinity among all vitamin D metabolites regardless of
whether 1,25(OH)2D is coming from the serum outside the
target cell (endocrine mode), from the local microenvironment
outside of the serum compartment (paracrine mode), or from
the inside of the cell (intracrine mode) (58). In the 1,25(OH)2D-
liganded state the VDR preferentially forms heterodimers
with retinoic acid X receptor (RXR). In the 1,25(OH)2D-
occupied state the VDR and its unliganded RXR partner
heterodimer become transacting complexes binding to specific
cis-acting vitamin D response elements (VDREs) in the genome.
The heterodimer interacts with the transcriptional machinery
resulting in 1,25(OH)2D-regulated (positive or negative) gene
expression and corresponding bioactivities. Due to the three-
dimensional “looping” nature of DNA-protein interactions,
VDRE-like enhancer and repressor motifs can found at
considerable distance from the transcriptional start site of a
vitamin D regulated gene (59,60).
Owing to a rapidly growing skeleton with a high demand for
calcium and phosphate for skeletal mineralization and in the
face of normal VDR and CYP27B1 activity, children suffering
from low 25(OH)D levels and secondary decreases in optimal
intestinal absorption in dietary calcium and phosphate can
develop vitamin D deficient rickets (61). This is commonly
observed in subpopulations of impoverished, dark-skinned
children (i) who require up to 10 times more cutaneous
sunlight exposure that lightly pigmented children to make
the same amount of vitamin D in their skin and (ii) in whom
consumption of natural vitamin D-rich foods (e.g., fish) and
vitamin D supplemented foods is compromised. In nations of
lower income, children usually consume a diet rich in grains;
grains are a rich source of phytates known to chelate ingested
calcium further decreasing intestinal calcium absorption.
Vitamin D deficiency rickets can also be hastened in children
subjected to religious/cultural practices that (e.g., occlusive
garb) that effectively eliminate skin exposure to sunlight. In
vitamin D deficient rickets the serum calcium and phosphate
is low and PTH and 1,25(OH)2D usually elevated for the
subject’s age (62). A low 25(OH)D level (usually <10 ng/ml)
is the distinguishing marker for vitamin D deficient rickets,
distinguishing it from Human Vitamin D Resistant Rickets
(HVDRR; high 1,25(OH)2D) and Pseudo Vitamin D Deficient
Rickets (PDDR; low 1,25(OH)2D) (63). Appropriate dietary
supplementation with calcium and vitamin D to normalize the
serum 25(OH)D level (e.g., >30 ng/ml) alleviates nutritional
rickets (61,64).
Prevention of nutritional rickets in children and osteomalacia
in adults is the primary concern behind the recommendations
on vitamin D intake and serum 25(OH)D level attainment.
Various medical organizations have evaluated the existing body
of research data to support their positions and are exhaustively
reported elsewhere (45,48). However, two key studies are
emblematic of the basis for the determinations. In one line
of evidence, PTH is used as a biomarker of bone health. In
some studies, an inverse association between PTH (declining)
and 25(OH)D (increasing) has been observed (55,65,66). In
one study involving 1,536 post-menopausal women (55) an
inflection point where the decline in serum PTH levels off
was identified at a 25(OH)D level of 30 ng/ml, leading some
authorities in the vitamin D field to call for this to demarcate
the cutoff for vitamin D sufficiency (46). Another often quoted
study involved post-mortem (N=675) determination of
25(OH)D levels and comparisons to histomorphometric analysis
of transiliac crest biopsies (67). From this dataset, the IOM
(45) concluded that at a serum 25(OH)D level of 20 ng/ml and
above, 99% of the normal population (e.g., without a known bone
disease) should have no pathological accumulation of osteoid
(unmineralized portion of bone indicative of rickets in children
and osteomalacia in adults). Interestingly, the original authors
(67) using different criteria to analyze the same data concluded
that 30 ng/ml 25(OH)D as the recommended level for optimal
skeletal health.
TOTAL 25(OH)D OR FREE 25(OH)D?
The measured total 25(OH)D concentration in serum is present
at nearly 1,000-fold higher levels compared to 1,25(OH)2D in
serum and easily and accurately measured from a small amount
of sample (25–50 µl) (68). As such, total 25(OH)D has become
the de facto biomarker of the state of vitamin D deficiency
or sufficiency of the host. However, the majority (>99%) of
serum 25(OH)D is bound (Figure 1, left box) to carrier proteins
(∼85% to vitamin D binding protein [DBP]; ∼15% to albumin).
The affinity of DBP for 25(OH)D is ∼1,000-fold greater than
that of albumin for 25(OH)D (11). Cells that express the cell
surface receptor proteins megalin and cubulin can internalize
the DBP-bound-25(OH)D complex into an endolysosome with
ultimate release of 25(OH)D from DBP into the cell interior
for further metabolism and/or catabolism of 25(OH)D. This
mechanism of endocytosis, intracellular release of 25(OH)D from
acid-hydrolyzed DBP has been most clearly demonstrated at the
luminal membrane of the tubular epithelial cells in the kidney
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(15). Here internalized 25(OH)D is the substrate used by the
CYP27B1 to form 1,25(OH)2D for endocrine distribution. In
cells not expressing megalin, 25(OH)D entry into its target cell
is proposed to be accomplished by diffusion of the unbound,
free 25(OH)D across the lipid bilayer of the plasma membrane
to the cell interior (15). Thus, the biologically relevant substrate
for some cells is DBP-bound 25(OH)D (essentially equal to
total 25(OH)D) and for others it is the free 25(OH)D or a
related metric of bioavailable 25(OH)D (sum of free 25(OH)D
and albumin-bound-25(OH)D).
Though the serum protein carriers of vitamin D metabolites
are well-characterized, it is now clear that vitamin D metabolites
also reside in non-serum locales such as body fat and inside cells,
suggesting that specific vitamin D binding proteins other than
DBP and albumin may also play a role in vitamin D biology.
Notably intracellular vitamin D binding proteins (IDBPs) were
identified as a result of investigations into apparent vitamin D
resistance in New World Primates [NWP; reviewed in Adams
et al. (69,70)]. The high serum levels of steroid hormones
in general and vitamin D in particular, relative to Old World
Primates (OWP), was shown to be associated with a form
of target tissue insensitivity to 1,25(OH)2D (71). The first
indications of a functional role for IDBPs was the observed
diminished ability of NWP cells to effectively upregulate VDR-
target genes, like the CYP24A1, despite having comparable
amounts of VDR (72,73) and VDR functionality (74). Using
cell extracts from NWP and OWP in VDR-1,25(OH)2D
radiolabel binding assays, a protein in NWP cell extracts was
observed to prevent VDR-1,25(OH)2D binding when mixed
with OWP extracts. This inhibition of binding was abolished
after trypsin digestion or heat denaturation (75). Upon further
characterization, IDBP was found to (i) bind 25(OH)D as well as
other steroid hormones (76) and (ii) be part of the heat shock
protein 70 family (77,78). With cDNA constructs for IDBP
obtained, transient and stable overexpression in vitro in tissue
culture studies revealed that IDBP could increase 1,25(OH)2D
synthesis, possibly by chaperoned delivery of 25(OH)D to the
CYP27B1 (79). The ATPase domain of IDBP was essential to this
function (80,81), with the BCL2-associated athanogene 1 (BAG1)
serving as an IDBP co-chaperone (82).
Measurement of the free, unbound form of 25(OH)D in
serum is challenging, because its levels are low (4–8 pg/ml
range) and has historically required radioactive tracers of
25(OH)D with equilibrium or centrifugal dialysis methods
that are cumbersome and impractical for use in clinical
laboratory testing services (10,11). Free and bioavailable
25(OH)D levels in the serum can be mathematically calculated
using equations that incorporate the binding affinity of DBP
for 25(OH)D and the concentration of DBP in the serum
(11,83). This approach of calculating the free, biologically active
fraction of 25(OH)D has also been taken with testosterone
(84) and thyroid hormone (85). Unfortunately, for free
25(OH)D, it was discovered that one commonly used DBP
ELISA kit used to calculate for free 25(OH)D had differential
sensitivities to the common phenotypic variants of DBP
(discussed later in this review) that resulted in inaccurate
measurement of DBP concentrations in some samples
TABLE 3 | Hormone binding proteins found in human serum.
Binding protein Metabolites Percent free
DBP
4.5–5.5 µMa
Total 25(OH)D
25–75 nMa
Free 25(OH)D
5–20 pMa
0.02
Total 1,25(OH)2D
50–198 pMb
Free 1,25(OH)2D
325–525 fMc
0.5
TBG
241–722 nMb
Total T4
58–154 nMb
Free T4
11–23 pMb
0.02
Total T3
1.1–2.8 nMb
Free T3
3.0–6.8 pMb
0.3
SHBG
16.5–55.9 nMb
(male)
Male total T
9.2–31.8 nMb
Male free T
30–87 pMb
0.3
Male total E2
28–156 pMb
Male free E2
<1.7 pMd
1
SHBG
24.6–122.0 nMb
(female)
Female total T
0.3–1.7 nMb
Female free T
<15 pMb
0.9
Female total E2
46–609 pMb
Female free E2
1.6–18.5 pMd
3
aNielson et al. (88).
bhttps://www.labcorp.com/test-menu/ search.
cBikle et al. (89).
dhttps://www.questdiagnostics.com/testcenter/TestCenterHome.action.
Vitamin D binding protein (DBP), thyroxine binding globulin (TBG), and sex hormone
binding globulin (SHBG) are tabulated with their serum concentration levels. The
respective metabolite concentrations total, free and percent free are also presented.
Albumin can bind all the hormones listed and transthyretin can bind T4 albeit at lower
affinity compared to their primary carrier proteins. The bioavailable concept has been
applied to these hormones and comprise the sum of albumin-bound-hormone and
free hormone.
leading to inaccurate calculated free 25(OH)D levels in
those serum samples.
Direct measurement of the various free hormones of clinical
importance have been developed (86,87). However, this aspect
of clinical chemistry remains challenging due to (i) the low
concentration of these metabolites in the serum (Table 3) and (ii)
variability of serum composition (e.g., lipids and other potentially
interfering molecules) between patients that can introduce error
into the measured value. Recently, an ELISA based method for
detection of free 25(OH)D has been developed (90) and has been
utilized in a number of clinical studies that we will summarize in
the clinical studies portion of this review. Most recently, a high-
throughput method to measure bioavailable 25(OH)D has been
developed (91). However, procedures to ensure accuracy and
precision across a wide range of sample conditions both for both
of these assays have not been yet developed; as a consequence,
these assays are deemed for research use only.
MOLECULAR BIOLOGY AND
BIOCHEMISTRY OF DBP
The vitamin D binding protein (DBP) is a multi-functional
protein also known as Group Specific Component (GC)
[reviewed in Chun (92) and Delanghe et al. (93)]. DBP is
a member of the albumin superfamily of proteins. DBP is a
moderately abundant protein (∼5µM in humans) in serum of
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Chun et al. Free 25D
TABLE 4 | Molecular biology of most common DBP polymorphisms.
SNP name GC name Codon variant Amino acid variant
rs4588 GC1 ACG Thr-436
GC2 AAG Lys-436
rs7041 GC1F GAT Asp-432
GC1S GAG Glu-432
Two single nucleotide polymorphisms (SNP) account for three of the major forms of DBP
(originally known as GC-globulin). Table also includes the specific codon and amino acid
variation that define these variants.
vertebrates. In humans the GC/DBP 13 exon gene is located at
4q13.3. Its gene product, DBP, is highly expressed in the liver and
exported into the circulatory system. DBP contains 474 amino
acids of which the 16 N-terminal amino acids function as a
signal peptide. DBP can be glycosylated (94) to varying degrees
depending on genotype. The glycosylation pattern has been
suggested to be structural basis for the macrophage activating
factor activity of DBP (95,96).
DBP’s most well-characterized role is that of a carrier protein
for vitamin D metabolites. Its rank order avidity for the
vitamin D and it metabolites are (10,11,89,97) as follows:
24,25(OH)2D>25(OH)D>1,25(OH)2D>vitamin D. DBP binds
vitamin D3 and its metabolites with greater affinity than vitamin
D2 and it metabolites (97). DBP can also bind actin (98). This
biological action is suggested to be that of scavenging of exposed
actin, preventing overzealous extracellular polymerization after
tissue injury (99). DBP can also bind circulating fatty acids (100)
and C5a des Arg, the latter of which enhances complement
activation (101).
DBP migrates at ∼52–59 kDa in electrophoretic gels (102).
It was variations in DBP mobility in isoelectric focusing (IEF)
gels that garnered initial research attention prior to its function
being determined. One banding pattern was termed GC-1F
(faster), another GC-1S (slower), and still another GC2 (103).
GC2 migrated less rapidly toward the anode compared to either
GC1 forms. The different forms, due to single amino acid
differences (Table 4), were (i) used to determine allelic frequency
for samples worldwide (104) and (ii) found to associate with the
racial background of the source human serum. In the Kamboh
study, black subjects were more likely to have the GC1F forms
(67–79% of alleles in USA blacks) while white subjects more
frequently yielded the GC1S pattern (49–57% of alleles in USA
whites). The GC2 allele was observed were more frequently seen
in white subject samples (21–31% in USA whites) compared to
blacks (8–13% in USA blacks). These three classic forms (GC1F,
GC1S, and GC2) account for the vast majority of the variation
DBP across human populations. Since these early studies, many
other SNPs (105) have been found in GC of which a small
percentage encodes a missense mutation that changes the amino
acid code.
Affinity Differences and DBP Genotype
The biological significance of DBP’s different allelic forms is
unclear. Conceptually, if the different genotypic forms of DBP
had different affinities for vitamin D metabolites, then the
levels of those metabolites could be influenced by the genotype.
However, in terms of experimentally measured differences in
affinity for vitamin D metabolites, one study reported large
differences (106) but three others did not (107–109). Media
supplemented with serum that contained different alleles of
DBP had differing impacts on assessed immunological readouts
consistent with affinity differences in three in vitro studies
(110–112). The mechanism for the observed differences were
not investigated in those studies; thus, non-affinity dependent
mechanisms cannot be ruled out. Using an ELISA based assay to
detect free 25(OH)D, some small differences in percent free were
detected between the genotypes (113). However, these differences
were smaller in magnitude than the differences anticipated
if the report of large affinity difference among genotype
was accurate.
DBP Serum Concentration and DBP
Genotype
The other mechanism by which DBP genotype could impact
free 25(OH)D levels is through differentials in the genotype-
dependent DBP concentration. It is on this basis that Powe
et al. (114) proposed how black Americans, with lower total
serum 25(OH)D levels and substantially lower DBP levels
compared to white Americans, could ultimately have similar free
25(OH)D levels. However, it was eventually determined that the
monoclonal antibody-based ELISA used in their study was less
sensitive to the GC1F DBP, the form most frequently found in
blacks (88,115). This resulted in an underestimation of serum
DBP levels in black subjects and a consequent overestimation
of the calculated free and bioavailable serum 25(OH)D levels in
their serum. Nonetheless, there is evidence of some genotypic
effect on DBP concentrations. One early study (116) of Danish
women found DBP concentration ranked according to the
presence of GC1 alleles (GC1-GC1 >GC1-GC2 >GC2-GC2).
Consistent with those findings was a study in a population male
subjects with modest racial diversity where GC2/GC2 subjects
exhibited the lowest serum DBP concentration (88).
In another recent study utilizing serum samples from both
women and men with modest racial diversity, the presence of the
GC2 allele in one of three allelic combinations resulted in lower
DBP levels compared to the three allelic combinations without
GC2 (113). Currently, it is thought that DBP’s role in determining
free 25(OH)D levels is largely through its concentration [i.e.,
for a fixed total 25(OH)D, more DBP yields less free 25(OH)D]
and perhaps to a small degree by genotype. As such, in most
cases total 25(OH)D and free 25(OH)D are highly correlated.
However, there are some conditions where DBP levels diverge
from typical levels. For instance, patients with liver disease
have lower levels of DBP while pregnant women have higher
DBP levels (89,117) resulting in higher free 25(OH)D levels
in those with lower DBP concentrations. In the more extreme
case of patients before [lower DBP, lower 25(OH)D] and after
[higher DBP, higher 25(OH)D] liver transplants, the percentage
of free 25(OH)D relative to total 25(OH)D is higher before
transplantation. Recently, a case report described a subject with
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Chun et al. Free 25D
a homozygous deletion of DBP (118). Compared to unaffected
and heterozygous siblings, the affected patient had no DBP and
very low serum levels of 25(OH)D and 1,25(OH)2D. Despite the
extremely low serum levels of vitamin D metabolites, the subject
had normal calcium balance with only relatively small alterations
in bone health and mineral metabolism. This case report is the
strongest evidence to date that the total serum 25(OH)D and
1,25(OH)2D level can be disconnected from “normal” vitamin D
status. One explanation is that albumin, or some other chaperone
in the serum, assumes the role of delivering 25(OH)D and
maintaining viable levels of free 25(OH)D and 1,25(OH)2D,
as was measured in this subject, to target tissues for further
metabolism and action.
ANIMAL STUDIES OF FREE 25(OH)D AND
BONE
Dbp heterozygous and homozygous knockouts in mice do
not have any obvious bone phenotype relative to wild type
(119). However, with the loss of a single copy of Dbp the
levels of circulating total serum 25(OH)D and 1,25(OH)2D are
diminished and when both copies were absent, the levels of serum
25(OH)D and 1,25(OH)2D were extremely low, phenocopying
the DBP−/−subject just described above. Interestingly, neither
DBP+/−or DBP−/−mice exhibited any skeletal abnormalities
or problems with calcium and phosphate balance (119). These
findings support the hypothesis that free hormone levels alone
are adequate for sustaining skeletal health. Consistent with this
was another study with Dbp homozygous knockouts where the
levels of 1,25(OH)2D was measured in the intestinal tissues (120).
Even though the double knockout animals had very low total
serum 1,25(OH)2D levels compared to wild type, the levels of
1,25(OH)2D measured in the intestinal tissues were very similar.
These findings suggest that trace amounts of free metabolites
and/or enhanced local conversion of 25(OH)D to 1,25(OH)2D
are sufficient as long as sufficient amounts substrate 25(OH)D
are available to the host. The fact that mice with no DBP are still
viable suggests that albumin, though having a much lower affinity
for vitamin D metabolites, could serve as the carrier protein
in place of DBP. When Dbp null mice were raised on vitamin
D3-free diets, they developed secondary hyperparathyroidism
and bone mineralization defects much more rapidly than paired
wild-type mice (119). This result indicated that DBP’s function
is to maintain a stable reservoir of circulating extracellular
vitamin D metabolites. Owing to its higher affinity for vitamin
D metabolites, DBP is more effective in this role than albumin,
despite both DBP and albumin being filtered into the urine and
reclaimed by megalin.
Another test of the biological impact of free 25(OH)D in mice
(121) was based on the difference in affinity of DBP for D2 or
ergocalciferol (vitamin D found in fungi) vs. D3 or cholecalciferol
(vitamin D found in animals) forms of vitamin D metabolites.
Mice raised on diets containing exclusively D2 or D3 would result
in the mice having only 25(OH)D2 and 25(OH)D3 circulating in
their serum. Since DBP affinity for D2 forms is lower relative to
D3 forms, the free 25(OH)D2 levels were expected to be higher
in animals raised on D2 diets. In this study, mice were placed
on D2 or D3 diets (1,000 IU/kg) beginning at week 3 and tested
at week 8 and week 16 for 25(OH)D3 and 25(OH)D2 serum
levels and bone phenotype by histomorphometry. These mice
had similar total 25(OH)D levels at week 8 (26.6 ±1.9 ng/ml
25(OH)D2 vs. 28.3 ±2.0 ng/ml 25(OH)D3) and at week 16 (33.3
±4.4 vs. 31.7 ±2.1 ng/ml). However, as anticipated, they differed
in their free 25(OH)D levels with free 25(OH)D2 greater than
free 25(OH)D3 at week 8 (16.8 ±0.65 vs. 8.4 ±0.63 pg/ml,
P<0.001) and at week 16 (17.4 ±0.43 vs. 8.4 ±0.44, P<
0.001). Histomorphometric analysis of their bones detected that
at week 8, the D2 fed mice had significantly higher osteoclast
surface/bone surface, eroded surface/bone surface, and mineral
apposition rate (high bone turnover) compared with mice raised
on the D3 diets. Additionally, osteoblast surface/bone surface, an
index of bone formation, was higher in week 8 D2 in females
only. The reason underpinning this sexual dimorphism in bone
formation rates remains unknown. The bone phenotype at week
16 revealed significantly higher bone volume/total volume and
trabecular number in the D2 mice relative to the D3 mice.
Taken together, despite similar total serum 25(OH)D levels, bone
phenotype differences were observed in association with different
free 25(OH)D levels (higher in D2 mice) suggesting the relevance
of free 25(OH)D to bone health.
To the best of our knowledge, there have been no
studies on the effects of free 25(OH)D and the bone health
of animals besides the studies in mice summarized above.
However, the existence of nocturnal bats that roost in dark
locations (122) could be informative. These bats have very
low total serum 25(OH)D (<5 ng/ml) as their normal state
suggesting that “deficient” (by human standards) total 25(OH)D
levels presumably still yields adequate free 25(OH)D levels to
sustain normal bone and mineral homeostasis for this species.
Interestingly, these bats are fully capable of attaining higher
25(OH)D levels when housed in conditions that expose them to
sunlight. Perhaps these animals have lower DBP concentrations
or metabolite affinities for DBP to compensate for their naturally
low total serum 25(OH)D levels. A test of these animal’s
serum with the free 25(OH)D assay could be informative.
Other possible adaptations that permit compensation to the
very low circulating levels of 25(OH)D include: (1) a highly
efficient CYP27B1; (2) hyper-sensitive VDR to ligand; (3)
heightened transactivation potential of VDR-interacting co-
activators; and/or (4) diminished functional activity of the
CYP24A1 catabolic machinery. There is one study comparing
DBP affinity for 25-hydroxyvitamin D among several animals;
these investigators found the DBP from rat and cattle exhibited
higher affinity to 25(OH)D compared to horse and rhesus
monkey with humans having the lowest affinity of species tested
(123). What the levels of free 25(OH)D are and its importance to
these species have not yet been examined.
HUMAN STUDIES OF FREE 25(OH)D
Revitalized interest on the impact of vitamin D on human
health beyond bone was spurred by studies that investigated
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Chun et al. Free 25D
vitamin D’s regulatory role in the adaptive and innate immune
system (26,29,124). Three in vitro studies have demonstrated
that decreasing free 25(OH)D by increasing DBP in the culture
media diminished immune functions of adherent monocytes
(110), dendritic cells (111), and T-lymphocytes (112). Because
of these findings in vitro, the parameter of free 25(OH)D
began to receive greater interest in clinical research. In
non-bone health association studies, the results have been
mixed. In some studies free serum 25(OH)D levels were
inversely associated with coronary artery disease (125), pediatric
inflammatory bowel disease (126) and ulcerative colitis (127),
insulin sensitivity (128), reduction in lipid markers in statin
patients (129), and acromegaly (130). However, in some other
studies free 25(OH)D was inferior in asthma (131) and no
better than total 25(OH)D for colorectal cancer in African-
Americans (132).
Concerning bone and mineral health, a 2011 report (133)
was the first to utilize free 25(OH)D and the related concept
bioavailable 25(OH)D (sum of free 25(OH)D and albumin-
bound 25(OH)D) for analytical purposes. They found that these
two metrics of serum vitamin D status were more closely and
directly associated with BMD in the individual than total serum
25(OH)D. This group then followed up with another report
(134) that showed that compared to measures of total 25(OH)D
bioavailable 25(OH)D had a significant direct association with
serum calcium (corrected for the serum albumin level) and a
significant inverse association with PTH. These investigators
suggested that this could address the long-standing paradox
of how black Americans with lower total serum 25(OH)D
levels have higher BMD and similar PTH levels compared
to white Americans. In additional work, they measured DBP
serum concentrations in black Americans and found them
to be lower than those in white Americans. They concluded
that the resultant higher bioavailable 25(OH)D may account
for the nominal differences in BMD and serum PTH (114)
despite what appeared to be sub-optimal total serum 25(OH)D
levels; unfortunately, a high-throughput assay to directly measure
free 25(OH)D was not available at that time. Thus, in these
reports, measured DBP, albumin and total 25(OH)D values were
input into mathematical equations (11,84) to calculate values
for free 25(OH)D and bioavailable 25(OH)D. Disappointingly,
one of the popular ELISA kits for DBP quantitation at the
time relied upon a monoclonal antibody that turned out to
possess reduced sensitivity to the DBP polymorphism (GC1F)
most frequently found in black Americans (88,115). Because
of this characteristic of the ELISA, the DBP concentration
was under-reported for these subjects leading to an over-
estimation of the calculated free and bioavailable 25(OH)D. The
manufacturer of this monoclonal ELISA has re-designed their
ELISA and released a new version in January 2017 addressing
this problem (135). Even with these complications, these early
studies sparked interest in examining bone and mineral health
by markers of vitamin D status other than the traditional total
serum 25(OH)D.
Investigators have continued to use calculated free and
bioavailable 25(OH)D in serum in association studies. In light
of the difficulties with the monoclonal antibody-based ELISA
for DBP, immunological methods based on polyclonal antibodies
(less influenced by DBP polymorphisms) and techniques
independent of antibodies entirely (mass spectrometry) can
be employed to measure DBP for calculation of bioavailable
25(OH)D in serum (88,115). Additionally, an ELISA method
has been developed that measures free 25(OH)D directly (90)
such that some investigators use this assay exclusively in their
studies though many also include data from calculated free
and/or bioavailable 25(OH)D.
HUMAN CLINICAL STUDIES OF FREE
25(OH)D AND BONE
This section surveys the recent literature examining whether free
(directly measured or calculated using the polyclonal DBP assay)
vs. total 25(OH)D is more consistently associated with various
measures of bone health, including intestinal calcium absorption,
parathyroid hormone secretion, and bone mineral density.
Intestinal Calcium Absorption
To our knowledge, there has only been one study to date
examining the relation between free and total 25(OH)D with
intestinal calcium absorption. Aloia et al. randomized 71 adults
to receive either placebo, 800, 2,000, or 4,000 IU/days of
vitamin D3 over 8 weeks. At both baseline and follow-up,
neither free nor total 25(OH)D nor 1,25(OH)2D in the serum
was associated with intestinal calcium absorption efficiency
(136). This supports the concept that VDR-directed increases in
intestinal calcium absorption are controlled locally, outside of the
serum compartment.
Parathyroid Hormone
Multiple investigators have assessed whether free vs. total
25(OH)D is more strongly correlated with the serum PTH level,
with results being inconsistent. For example, in an analysis of
155 subjects that included 24 cirrhotics and 20 pregnant women,
Schwartz et al. reported that both free and total serum 25(OH)D
were similarly, inversely correlated with the serum PTH (117).
Similar findings have been reported in: (1) healthy pre- (137)
and postmenopausal women (136); (2) individuals with obesity
(138); (3) patients cirrhosis of the liver (139); (4) children (2–18
y/o) in Spain (140); (5) blacks and whites in a supplementation
study (placebo, 2,000, 4,000 IU/days for 16 weeks) (141); (6)
a RCT of prediabetics (142); and (7) pregnant white women
in Germany (143). While the above studies reported similar
correlations between serum free and total 25(OH)D with serum
PTH, others have favored free 25(OH)D. For example, Schwartz
et al. conducted a 16-weeks trial in which 81 older women
and men received vitamin D3 at doses of 800, 2,000, or 5,000
IU/days or 50,000 IU/weeks. At the end of the study, free, but
not total serum 25(OH)D was inversely associated with the
serum PTH; however, free 25(OH)D explained only a small
amount of the variability in PTH [R2=0.08; (144)]. In two,
smaller trials – one in which 38 participants received 500,000
IU of vitamin D2 or D3 over 10 weeks (145) and another in
which 35 participants received 2,400 IU/day of vitamin D3 or 20
mcg/day of 25-hydroxyvitamin D3 (146)—Shieh et al. reported
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Chun et al. Free 25D
that longitudinal increase in free 25(OH)D was significantly
associated with concurrent decrease in serum PTH during the
first 8–10 weeks of supplementation (when 25(OH)D levels
change most rapidly), whereas increase in total 25(OH)D was
not. In adults with primary hyperparathyroidism, Wang et al.,
similarly found that the free serum 25(OH)D was inversely
correlated with circulating PTH levels, but total 25(OH)D was
not (147). Further complicating the picture are studies favoring
total 25(OH)D over free 25(OH)D. In a cohort of Hungarian
adults assessed at the end of winter total, but not free 25(OH)D,
was inversely correlated with PTH (148). In a study of UK whites
and south Asians, total, but not free 25(OH)D, was inversely
correlated with PTH (149). In a study of pregnant adolescents
(13–18 y/o), the inverse association of PTH with free 25(OH)D
was weaker than that observed total 25(OH)D (150).
Bone Mineral Density
While a change in the serum PTH level is the outcome that has
been most frequently tested in relation to free vs. total 25(OH)D,
some cross-sectional studies have examined bone mineral density
(BMD) as well. As was the case with changes in the serum
PTH, results with BMD have been inconsistent. For example,
Jemielita et al. reported that in 304 adults, neither total nor free
serum 25(OH)D at a single point in time was associated with
BMD assessed by DXA, or peripheral quantitative CT (151). In
contrast, in a cross-sectional analysis comparing the correlations
between free vs. total serum 25(OH)D with BMD and composite
indices of femoral neck strength, Alwan reported that higher free
25(OH)D levels were correlated with greater BMD (lumbar spine,
femoral neck, total hip), and femoral neck strength (r=0.24–
0.34, p<0.05), but total 25(OH)D was not (152). On the flip
side, Michaelsson et al. reported that in women from Sweden
(mean age 68 years) higher total, but not free, serum 25(OH)D
was associated with greater BMD (153).
CHALLENGES AND PROSPECTS
Undoubtedly, further clinical studies should be conducted using
bone/mineral outcomes as well as non-skeletal health readouts
to assess the utility of free 25(OH)D. As described in the
above section pertaining to bone and mineral health, human
studies comparing whether free vs. total serum25(OH)D is
more frequently associated with intestinal calcium absorption,
parathyroid hormone secretion, or bone mineral density have
yielded inconsistent results ranging from no difference, to those
favoring either free, or total 25(OH)D. We propose that there
are two major challenges that contribute to these inconsistencies.
First, is the lack of a specific “readout” of vitamin D bioactivity.
While circulating PTH levels are influenced by vitamin D status
(154–156), it is also regulated by the calcium sensing receptor.
Additionally, attempts to associate BMD with vitamin D status
are complicated by the fact that BMD in adults is principally
determined by attained peak bone mass that is partly dependent
on vitamin D status (157). Thus, it is difficult to discern the
relative importance of free vs. 25(OH)D in human cross-sectional
studies using these parameters. As such, even when studies
report statistically significant correlations, the r values tend
to fall in the range (−0.3–0.1 and 0.1–0.3) that are deemed
“weak relationship” by statisticians. Second, many human studies
do not employ subjects who are 25(OH)D deficient. These
subjects would have the most to gain physiologically from
treatment to return 25(OH)D in the serum to normal. Ideally,
supplementation studies must include subjects that are clearly
at insufficient levels and then have it demonstrably shown that
their levels are raised into the sufficient range. It is through
longer-term longitudinal analyses of intra-individual changes in
serum total and free 25(OH)D after an aggressive vitamin D
supplementation regimen that would have a greater likelihood to
detect any associations.
Lastly, there is currently only one method of direct
measurement of free 25(OH)D (90) with reasonable throughput.
Though very promising, this assay is based on antibody
interaction with 25(OH)D and needs further validation on
the wide variety of sample quality (i.e., time from collection
to testing, temperature of storage, variability of potentially
interfering serum components among patients, etc.) encountered
in clinical laboratory practice (158). In the future, perhaps a mass
spectrometry-based method could be developed as is occurring in
the measurement of other free hormones such as estradiol (159),
thyroid hormone (160), and testosterone (161).
AUTHOR CONTRIBUTIONS
JA developed the overall organization of and approved this
publication. JA, RC, AS, CG, and MH wrote sections of this
review. VY and JW participated in copy editing.
FUNDING
This work was supported by NIH 5R01AR063910-05.
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02360
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2019 Chun, Shieh, Gottlieb, Yacoubian, Wang, Hewison and Adams.
This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner(s) are credited
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REVIEW
published: 10 January 2020
doi: 10.3389/fendo.2019.00910
Frontiers in Endocrinology | www.frontiersin.org 1January 2020 | Volume 10 | Article 910
Edited by:
Giacomina Brunetti,
University of Bari Aldo Moro, Italy
Reviewed by:
Jan Josef Stepan,
Charles University, Czechia
Xiangbing Wang,
Rutgers, The State University of
New Jersey, United States
Janice Schwartz,
University of California, San Francisco,
United States
*Correspondence:
Roger Bouillon
Roger.bouillon@kuleuven.be
†ORCID:
Roger Bouillon
orcid.org/0000-0002-6446-3763
Frans Schuit
orcid.org/0000-0003-3225-6695
Leen Antonio
orcid.org/0000-0002-1079-2860
Fraydoon Rastinejad
orcid.org/0000-0002-0784-9352
Specialty section:
This article was submitted to
Bone Research,
a section of the journal
Frontiers in Endocrinology
Received: 11 July 2019
Accepted: 13 December 2019
Published: 10 January 2020
Citation:
Bouillon R, Schuit F, Antonio L and
Rastinejad F (2020) Vitamin D Binding
Protein: A Historic Overview.
Front. Endocrinol. 10:910.
doi: 10.3389/fendo.2019.00910
Vitamin D Binding Protein: A Historic
Overview
Roger Bouillon 1
*†, Frans Schuit 2†, Leen Antonio 1,3† and Fraydoon Rastinejad 4†
1Laboratory of Clinical and Experimental Endocrinology, Department of Chronic Diseases, Metabolism and Ageing, KU
Leuven, Leuven, Belgium, 2Gene Expression Unit, Department of Cellular and Molecular Medicine, KU Leuven, Leuven,
Belgium, 3Department of Endocrinology, University Hospitals Leuven, Leuven, Belgium, 4Nuffield Department of Medicine,
Target Discovery Institute, University of Oxford, Oxford, United Kingdom
Vitamin D and all its metabolites are bound to a specific vitamin D binding protein,
DBP. This protein was originally first discovered by its worldwide polymorphism and
called Group-specific Component (GC). We now know that DBP and GC are the same
protein and appeared early in the evolution of vertebrates. DBP is genetically the oldest
member of the albuminoid family (including albumin, α-fetoprotein and afamin, all involved
in transport of fatty acids or hormones). DBP has a single binding site for all vitamin
D metabolites and has a high affinity for 25OHD and 1,25(OH)2D, thereby creating a
large pool of circulating 25OHD, which prevents rapid vitamin D deficiency. DBP of
higher vertebrates (not amphibians or reptiles) binds with very high affinity actin, thereby
preventing the formation of polymeric actin fibrils in the circulation after tissue damage.
Megalin is a cargo receptor and is together with cubilin needed to reabsorb DBP or the
DBP-25OHD complex, thereby preventing the urinary loss of these proteins and 25OHD.
The total concentrations of 25OHD and 1,25(OH)2D in DBP null mice or humans are
extremely low but calcium and bone homeostasis remain normal. This is the strongest
argument for claiming that the “free hormone hypothesis” also applies to the vitamin D
hormone, 1,25(OH)2D. DBP also transports fatty acids, and can play a role in the immune
system. DBP is genetically very polymorphic with three frequent alleles (DBP/GC 1f, 1s,
and 2) but in total more than 120 different variants but its health consequences, if any,
are not understood. A standardization of DBP assays is essential to further explore the
role of DBP in physiology and diseases.
Keywords: vitamin D, vitamin D binding protein (DBP), megalin, actin, 25-hydoxyvitamin D, 1,25-dihydoxyvitamin D
INTRODUCTION
The vitamin D binding protein (DBP) is a multifunctional protein that is well-conserved in the
evolution of vertebrates. We review the history of the discovery of this gene and protein and
summarize its main functions as known today. The major milestones in the discovery of this gene
and protein and in the elucidation of its function are summarized in Table 1.
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Bouillon et al. Vitamin D Binding Protein: A Historic Overview
TABLE 1 | Major milestones in the discovery of the role of the vitamin D binding
protein.
1 Antirachitic activity in serum migrates with α-globulin mobility (1)
2 Radioactive vitamin D or 25OHD migrate with different
electrophoretic mobility in serum from mammals, birds,
amphibian or fish (αor β-globulin or albumin mobility)
(2)
3 Discovery of a major serum protein with genetically different
polymorphism called, Group-specific Component (GC)
(3)
4 Identity of GC with the vitamin D binding protein of human
serum
(4)
5 Purification and characterization of human DBP and other
species, including high affinity-high capacity properties van
DBP from higher vertebrate species
(5–7)
6 Immunoassays of DBP in humans and other species (8)
7 DBP is a member of the albuminoid family (9)
8 Description of the gene (and protein) structure of GC/DBP (10)
9 DBP binds actin and plays a crucial role in depolymerization of
extracellular actin filaments
(11)
10 Sex hormones regulate the concentration of DBP in a
species-specific way. The concentration of total 1,25(OH)2D
fluctuates in line with the total concentration of DBP so that
free 1,25(OH)2D is feedback regulated
(12,13)
11 Free 25OHD concentrations are extremely low (<0.1 % of total
concentration) and not feed-back regulated
(14)
12 DBP binds to megalin at the luminal site of renal tubuli and
thereby avoid urinary loss of 25OHD
(15)
13 DBP null mice are viable and healthy but have extremely low
total concentrations of 25OHD and 1,25(OH)2D
(16)
14 DBP binds fatty acids, and unsaturated fatty acids impair the
binding of 1,25(OH)2D and 25OHD to DBP
(17,18)
15 DBP binds to membranes proteoglycans of leucocytes and
thereby enhances complement C5a-stimulated chemotactic
activity in activated neutrophils
(19)
16 Crystal structure of human DBP (20)
17 DBP can be measured by mass-mass spectrometry (21,22)
18 First human subject with homozygous deletion of the GC gene (23)
DISCOVERY OF GC PROTEIN AS AN
α-GLOBULIN AND ITS IDENTITY WITH THE
SERUM VITAMIN D BINDING PROTEIN
In 1961, when serum proteins were still mainly characterized
by their electrophoretic mobility as α,β, or γglobulins, further
identification of the major proteins led to the discovery a highly
polymorphic protein with genetically defined small differences in
electrophoretic mobility, and therefore named “Group-specific
component” or GC (24). Initially, only GC1 and GC2 were
identified, but later on GC1 was found to be a mixture of
GC1f (fast) and GC1s (slow), because GC1f has a slightly faster
electrophoretic mobility than GC1s (Figure 1). Using polyclonal
antibodies to detect GC and due to improved sensitivity for
detecting small differences in the isoelectric point by isoelectric
focusing of sera from subjects from around the world, more
than 120 different variants were detected (Figure 1) (25–27).
This technique allowed using this protein to study genetic links
between populations and to use it in forensic medicine or
paternity disputes (28).
Many authors studied independently the electric mobility
of “vitamin D” activity in serum either by measuring the
anti-rachitic activity, or later on, by using radiolabeled vitamin
D or 25-hydroxyvitamin D (25OHD). Based on such data,
lipoproteins were found to be the main transport mechanism in
cartilaginous fish and amphibians (29). In most other vertebrate
species, an α-globulin was the major transport protein in
mammals and a β-globulin mobility was found in birds, whereas
in some species, radioactive vitamin D (metabolites) migrated
with albumin mobility (29,30). The first major link between
GC and DBP was made by Daiger et al. Indeed, from the
perfect parallelism between the electrophoretic mobility of GC
(measured by immunologic techniques) and the mobility of
[14C]vitamin D, they concluded that GC is the major transport
protein for vitamin D (4). Peterson et al. had reported the
purification of small amounts of human DBP and mentioned
that GC was only a minor contaminant of “his” protein (31).
However, the fact that the GC protein was identical to DBP
was soon confirmed by three different laboratories reporting the
purification of DBP from human serum, monitored by prior
addition of [3H]25OHD (5–7). We saturated the binding capacity
of human serum by adding, in addition to radiolabeled 25OHD,
sufficient stable 25OHD to avoid elimination of apoprotein
during the purification process, thereby achieving a higher
recovery rate (5). All these groups were able to show that
the protein they isolated based on its binding of [3H]25OHD
was immunologically and electrophoretically identical to GC
protein. The three groups initially applied different names,
such as transcalciferin (5), but soon agreed upon DBP as
abbreviation for the serum vitamin D binding protein (8). DBP
was subsequently also purified from rat (32), chick (33) and many
other species.
In summary, GC was discovered in the early 1960s as a
polymorphic serum protein and at the same time a serum protein
(DBP) was identified transporting vitamin D and its metabolites.
In 1985 and 1986 GC/DBP were found to be the same protein.
GENE CODING FOR GC/DBP
From early onwards, the three major forms of GC were shown to
be the result of a pair of co-dominant autosomal alleles, resulting
in homozygous GC1f-1f, GC1s-1s, GC2-2 or a mixture of these
genes in most subjects. The gene for DBP/GC is localized on
human chromosome 4q11-q13 as shown by in situ hybridization
techniques, whereas the gene is localized on chromosome 5 or 13
in the mouse and rat, respectively (34). The gene is positioned
close to the genes for albumin, α-fetoprotein and afamin
(also known as a-albumin), with a centromere-DBP-albumin-α-
fetoprotein-afamin-telomere orientation. Their protein products
are mainly synthesized and secreted by hepatocytes. The DBP
gene is also expressed in kidney, testis, endocrine pancreatic cells,
and fat cells (35). Genetic analysis of the evolution of these sets
of genes indicates that DBP might well be the oldest member of
the family (Figure 2). Human and rat DBP have 13 introns and a
42 kb gene structure. The human gene codes for a 1690 nucleotide
mRNA and a 458 amino acid long single chain protein, preceded
by a 16 amino-acid signal propeptide.
In summary, the structure of the GC/DBP protein was
identified, located on human chromosome 4 close to the other
members of the albumin gene family.
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Bouillon et al. Vitamin D Binding Protein: A Historic Overview
FIGURE 1 | DBP/GC polymorphism as revealed by measurement of their isoelectric point and protein structure.
FIGURE 2 | Gene and chromosome structure of GC/DBP and adjacent albuminoid family genes.
EVOLUTION OF VERTEBRATE GC/DBP
One of us (FS) studied the vertebrate GC gene and evolution of
the predicted DBP protein primary structure, whereas another
co-author (FR) evaluated the 3D structure of GC/DBP and
its interaction with the vitamin D metabolites. The National
Center for Biotechnology Information, U.S. National Library
of Medicine (NCBI Gene) listed on June 7th 2019 GC gene
sequences of 210 vertebrates (nine bony fishes, one amphibian, 14
reptiles, 59 birds and 127 mammals). In all clades, we ascertained
the correctness of the searched gene not only by sequence
homology but also by syntheny with its two neighboring genes,
SLC4A4 and NPFFR2. A complete domain 1 primary structure
(AA1-110) of DBP was found in 191 of these sequences. Here
we summarize the essential conclusions, as a detailed analysis
will be presented in a separate paper. First, in all major
clades of vertebrates the predicted GC/DBP gene can be found,
whereas [as expected from the literature (36)] the paralogous
albumin gene is absent all fish, amphibia, lizards, snakes, and
turtles. Second, by far the best conserved part of the primary
structure of DBP (virtually 100% conserved in all species with
a complete sequence) was a series of 28 cysteines that are
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Bouillon et al. Vitamin D Binding Protein: A Historic Overview
ordered over the three protein domains as a typical repetition
of “.....C....CC....C.....”. Two of these repetitions are present in
the domain 1 primary structure. The evolutionary importance of
cysteine pairs to form disulfide bridges in the primary structure
was already suggested earlier, based on fewer gene and protein
(six species for GC/DBP) structures (9,37). Third, the highest
protein homology was present in domain A, reflecting its crucial
role to transport vitamin D and its metabolites, which are mainly
hydrophobic. For instance, 25OHD3has only two polar hydroxyl
groups (on C3 and C25). These two critical recognition features
involve in mammals residues Tyr-32 and Ser-76 to recognize the
25-OH and 3-OH moieties, respectively, which are located at two
extreme ends of its ligand. Tyr-32 is not only well-conserved
among mammalian species but also in bony fishes; however,
the reptilian branch of vertebrates evolved to a different well-
conserved asparagine. For residue 76, the conservation is the OH-
group from either a serine or the slightly larger residue threonine.
Moreover, amino-acid residues 8, 12, 24, 35, 68, and 107 that
contribute to van der Waals interactions with vitamin D or its
metabolites are also highly conserved among mammals and often
substituted conservatively by other hydrophobic amino acids
(Ile-12 or Val-12; Leu-107, or Met-107) that would form similar
van der Walls interactions with the ligand. This conservative
nature of the amino acids involved in the binding cleft of
DBP explains the high affinity of DBP for 25OHD in all clades
of vertebrates.
In summary, GC/DBP is found in nearly all vertebrate species
with well-conserved structure over the 500 million span of
vertebrate evolution. This is especially the case for the ligand
(25OHD) binding cleft.
DBP: ORIGIN, TURNOVER, AND SERUM
MEASUREMENTS AND CONCENTRATION
Origin and Turnover
Vitamin D binding protein, as well as its close family members,
is mainly produced in the liver, although the gene and protein
are also expressed in very low concentrations in other tissues as
mentioned above. The pool of DBP in rabbits is about 54 mg/kg
with a half-life of 41 h and distributed between the intravascular
pool (1/3) and the extravascular pool (2/3). The distribution is
largely limited to the extracellular volume, in line with albumin
(38). The half-life of DBP in human plasma is about 1.7 days
and thus markedly shorter than the half-life of 25OHD, which
is estimated at about 15 days, based on studies with deuterium
labeled 25OHD in healthy subjects from the UK and Gambia
(39). The estimated daily production of DBP is about 700–900
mg/d for an adult person (10 mg/kg/d). In comparison, the
total body albumin is about 280 g for a normal adult and thus
more than 300 times greater than that of DBP. About 40% of
albumin is intravascular, with the remaining 60% distributed in
the interstitial space of various organs (primarily muscle, adipose
tissue, connective tissue, and skin) with an average interstitial
concentration of about 60–70% of that of plasma. Rabbit and
human data suggest a similar compartmentalization of DBP
(38,40). The absolute synthesis rate of albumin is about 150
mg/kg/day or 10.5 g/day for a 70 kg human. About 8.5% of
plasma albumin and 4% of the total body albumin are synthesized
each day, corresponding to a total body albumin turnover time
of about 25 days or a half-life of 17.3 days. This is about 10
times longer than that of DBP but shorter than other circulating
proteins (hemoglobin has a life span of 120 days) and similar to
that of γ-globulins (41). The mRNA of DBP in liver is rather low
(similar to the situation of albumin mRNA), suggesting a slow
turnover of this mRNA comparing with the protein turnover. The
clearance site of DBP is not fully understood, but DBP is partially
filtered in the glomerulus and reabsorbed in the tubuli mediated
by a carrier receptor mechanism (megalin, see below), followed
by intracellular degradation. In rabbits, DBP holoprotein and
apoprotein are cleared at about the same rate (42). The half-life
of DBP is thus markedly shorter than the half-life of 25OHD and
this implies that 25OHD is recirculated after degradation of DBP.
Removal of sialic acid does not changes its plasma clearance (40).
Protein Structure and Polymorphisms of
DBP
The mature DBP structure of humans is 458 amino acids (AA)
long, whereas rat, mouse, and rabbit DBP all are 460 AA long.
DBP has a highly conserved number of cysteines and disulfide
bridges. It has three domains, much like albumin, and these
domains are probably the results of gene duplication of a singly
common ancestor structure (34). Unexpectedly, the 3D structure
of DBP differs substantially from that of albumin (20). The A
domain has a cleft structure allowing to bind 25OHD with high
affinity. The structure of the holoprotein (DBP plus 25OHD
or analogs) confirms the predicted AA sequences (AA 35–
49) responsible for binding of 25OHD and all other vitamin
D metabolites. Indeed, there is only a single binding site for
all D metabolites. DBP has the highest affinity for 25OHD-
lactones, followed by 25OHD =24,25(OH)2D>1,25(OH)2D
(5,6,43). The binding site of DBP for 25OHD is show in
Figure 3. The structure of DBP with a vitamin D with a pentanor
side chain modification, known from in vitro binding studies
to have a high DBP affinity, allows to explain why such analog
has a higher affinity (for detailed discussion see (20,34). The
binding site of vitamin D for DBP is totally different from
that of the binding site of the vitamin D receptor (VDR) (44).
The main characteristics of DBP are summarized in Table 2.
Human DBP has an isoelectic point (IEP) of about 4.89, but this
varies according to DBP/GC genotype. The stability of DBP at
high temperature is markedly enhanced by binding to 25OHD.
The holoprotein (DBP.25OHD complex) has a different IEP
compared with the apoprotein, and this indicates that the protein
undergoes a structural modification when bound to vitamin D
metabolites (5,45). DBP is highly polymorphic as it was originally
discovered by this characteristic and therefore received its initial
name of group-specific component. The three most common
alleles and protein structures are shown in Figure 1. GC1 (1f
or 1s) has a high degree (about 10–25%) of O-glycosylation in
threonine position 436 with a linear trisaccharide (NeurNAc-
Gal-GalNAC) whereas residue 434 is much less glycosylated (1–
5%) by a disaccharide (without the final sialic acid). DBP/GC is
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Bouillon et al. Vitamin D Binding Protein: A Historic Overview
FIGURE 3 | Crystal structure of human DBP in combination with 25OHD or actin. In addition the main amino acids involved in the binding of 25OHD to the cleft in the
A domain of human DBP as shown.
similarly (poorly) glycosylated on AA 434 but not on AA 436
(being lysine rather than threonine) in DBP/GC1. The terminal
sialic acid of DBP/GC 1 can be present or absent and therefore
both DBP/GC1f and DBP/GC1s are present in serum in double
bands with a very small difference in isoelectric point (Figure 1).
Neuraminidase treatment can remove sialic acid and thereby
eliminate this double band on isoelectric focusing (45,46). The
genetic or molecular (pre-or posttranslational) origin of the large
number (>124) of variants of DBP in humans is largely unknown
(34,47) and the implication for the functions of DBP (see below)
is unknown. The most common genetic variants (GC1s/1f/2)
are due to polymorphisms in the third domain, whereas the
few other variants are due to polymorphisms in the second
domain [reviewed in (34)]. The best-known variant (GC1A1) is
one found in Aboriginals and some South African blacks (48).
Genetic polymorphism of DBP has also been documented in
other species such as rats (32,49), monkeys (50), swine, rabbits
(24), chicks, and horses.
Measurement of DBP
DBP is usually measured by one of the many methods based on
specific anti-DBP antibodies. This frequently requires species-
specific antibodies when measured in different species. In the
author’s laboratory, polyclonal antibodies were generated for
DBP from humans, rats, chicks, rabbits, mice, guinea pigs,
and dogs. The binding capacity of serum for 25OHD can also
be estimated by using radiolabeled 25OHD using a Scatchard
plot to calculate its affinity and binding capacity. In most
cases, the measured binding capacity was lower than the
immunoassay values (51) but later technological improvements
allowed to generate very similar concentrations between binding
and immunoassays.
We used radial immunodiffusion as this avoids the
need for major dilution of serum samples, but others
used a wide variety of other assay methods, such as rocket
electrophoresis, turbidimetry, or nephelometry, ELISA or even
radioimmunoassay. More recently, DBP has also been measured
by tandem mass spectroscopy after prior peptide digestion.
This method requires specific synthesis of a mixture of labeled
peptides common to all GC/DBP isoforms and in addition other
peptides which are genotype-specific (21,22,52,53). Based on
aclose collaboration between a team of the Leuven University,
NIST (Karen Phinney and Lisa Kilpatrick) and Dr. Hoofnagle’s
group in Seattle, a reference standard for DBP (based on protein
purified from homozygous GC1f, GC1s, and GC2 volunteers)
and a reference method for an MS/MS based assay of DBP has
been developed (53). An Elisa technique by R&D (54,55) used
monoclonal antibodies and was widely used to measure serum
DBP concentration. The results obtained with this method
surprised many experts as this assay showed race- and DBP/GC-
specific results, whereby blacks or African-Americans (mostly
having DBP/GC1f genotype) had markedly (−50%) lower
serum DBP than whites (54). The authors, therefore, concluded
that free 25OHD concentrations in African Americans were
similar to those in Whites and that this could explain the
paradox of “low” vitamin D status but solid bones and low
fracture rate in African-Americans. These data and at least 50
other manuscripts using this assay all seemed to conclude that
“we” all used wrong methods (total 25OHD) instead of free
25OHD to estimate the real vitamin D status (56). However,
previous studies using polyclonal antibodies did not find racial
differences in serum DBP (57). Extensive studies thereafter,
using polyclonal and mass spectrometry based assays (22,58,59)
all convincingly demonstrated that the R&D monoclonal DBP
assay discriminates against DBP/GC 1f and that all results in
genetically heterogeneous populations should be “retracted” or
re-interpreted. The senior author of the Powe et al. paper later
on agreed that the DBP results, based on the monoclonal R&D
assay, from these studies were wrong (60).
The concentration of DBP in normal human serum is in
the µmolar range (∼6µmol/l or about 300 ml/l) but different
laboratories have reported mean concentrations varying between
200 and 600 mg/l. Most of these differences are probably
due to lack of standardization of DBP assays and reference
material. Hopefully, this will soon be corrected by using the
NIST reference preparation (53). Most assays use polyclonal
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TABLE 2 | Major characteristics of the human vitamin D binding protein.
Gene - Located on chromosome 4q11–q13, close to albumin, α-
fetoprotein, and afamin genes and in syntheny with its two
neighboring genes, SLC4A4 and the neuropeptide receptor
2, NPFFR2
- Autosomal co-dominant gene transmission
- Highly polymorphic gene/protein with more than 120
variants in human populations around the world.
Polymorphisms also found in several other species
including rodents
Protein - 458 amino acids−58 kD–preceded by 16 amino acid signal
propeptide
- Three domains: a highly evolutionary conserved A domain
with a cleft able to bind all vitamin D metabolites. The B and
C domains can bind actin with high affinity
- Single binding site at the surface of the A domain, creating a
cleft for all vitamin D metabolites
- Isoelectric point 4.6–5.0 depending on gene polymorphism
or posttranslational modifications
- Pool size: 2.8 g
- T½: 1.7 days
- Daily production: 10 mg/kg
Serum
concentrations
-µmolar concentration (∼200–600 mg/l) in normal adults
- Mainly apoprotein configuration and <5% as holoprotein
Biological
functions
- Binding/transport of all vitamin D metabolites with high
affinity (25OHD lactone >25OHD =24,25(OH)2D=
25,26(OH)2D>1,25(OH)2D>vitamin D, and vitamin D3
metabolites >vitamin D2metabolites)
- Binding of actin monomers and enhanced clearance of
fibrillar actin
- Binding of fatty acids and especially unsaturated fatty acids
- Binding to megalin-cubilin receptor complex
- Binding to membrane of leucocytes and activation of
complement C5 system
Clinical
implications
- DBP concentration and affinity define the free concentration
of all vitamin D metabolites
- Low concentration in fetus and neonates
- Increased serum concentration when exposed to estrogens
- Decreased serum concentration in case of liver diseases,
nephrotic syndrome, malnutrition, severe acute trauma, or
disease
- Complete genetic absence leads to very low serum
concentrations of all vitamin D metabolites without a
clinical phenotype
antibodies and therefore the effect of protein polymorphism in
the final measurement is minimal. As mentioned above, one
assay, however, used monoclonal antibodies and reported mean
DBP concentrations that were very different according to the
genetic (or racial) differences in DBP (54). Other monoclonal
antibodies, however, do not discriminate GC/DBP isoforms (61,
62). When measured with a variety of polyclonal antibodies, DBP
concentrations in humans with GC/DBP2 genotype are slightly
(10–20%) lower than in GC/DBP 1 carriers. This was already
know in 1966 and confirmed in many later studies [reviewed
in (63)].
DBP circulates in serum in much higher concentrations than
the combined concentration of all vitamin D metabolites, as
serum DBP concentration is in the mid µmolar concentration
whereas the serum concentration of the major metabolite,
25OHD, is usually below 100 nmol/l. This implies that <5, and
usually <2% of DBP is a holoprotein (DBP plus vitamin D
metabolite) and nearly all DBP circulates as apoprotein. This is
quite different from the other binding proteins for ligands of
nuclear receptors. The DBP gene transcription is regulated by a
balance between hepatic nuclear factors (HNF) 1αand 1β, as is
the case for other liver abundant proteins. However, unlike the
regulation of albumin, HFN1αis a positive regulator and HFN1β
is a dominant negative regulator of DBP expression (64). DNA
methylation of the promotor region can also modify the DBP
gene expression (65). DBP is already expressed in the yolk sac
(as documented in animals) and later on in the fetal liver and
thus appears in the fetal circulation. Indeed, DBP can be found in
serum of human fetuses in the 3th trimester and its concentration
in (human) cord serum is only about half that of maternal serum
as reported by several authors (13,34).
Regulation by Hormones and Other
Factors
The concentration of DBP in human serum is in the
µmolar (micromolar) range as measured by immunoassay
or, more recently, by MS/MS (34). In humans, exposure
to estrogens increases serum DBP but androgens have no
effects. Decreased serum DBP concentrations are found in
patients with liver cirrhosis, malnutrition, peritoneal dialysis, and
nephrotic syndrome (see also below DBP-megalin interaction).
The concentration in the human fetus and cord serum is lower
than in adult serum. Exposure to estrogens increases the serum
concentration of DBP (e.g., due to intake of contraceptive estro-
progestogens or during pregnancy). Therefore, the serum DBP
concentration in pregnant women at the time of delivery is
about twice the concentration found in cord serum (13,14).
Exposure to or loss of androgens does not change serum
DBP concentrations in humans. Vitamin D deficiency or
excess, or vitamin D resistance, idiopathic hypercalcemia of
infancy, or osteoporosis and many other diseases (such as
hyperparathyroidism or hyperthyroidism, sarcoidosis, cancer,
Addison’s disease, or growth hormone deficiency) have no effect
on serum DBP concentration. DBP is slightly increased in
acromegaly (66) and in some inflammatory diseases (such as
in rheumatoid arthritis) (67). DBP concentrations are slightly
decreased in serum of type 1 diabetes patients (68) and even
more severely in BB or streptozotocin-induced diabetic rats (69),
but better diabetes control can restore these concentrations (70).
Decreased concentrations of DBP are also found in patients with
a variety of kidney diseases or in case of renal loss of DBP.
Patients with nephrotic syndrome therefore not only lose massive
amounts of albumin but also large amounts of DBP (having a
lower molecular weight than albumin). This urinary loss of DBP
also entrails urinary loss of 25OHD and thus results in vitamin
D deficiency (71). Low DBP concentrations are also found in
animals or patients with genetic or acquired loss of megalin or
cubilin, wo proteins functioning as cargo receptor to reabsorb
serum proteins, filtered in the glomerulus and recovered in the
renal tubuli (15) (see below). The genotype of DBP also has a
small effect on the serum DBP concentrations as subjects with
homozygous GC2-2 genotype have about 5–10% lower serum
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Bouillon et al. Vitamin D Binding Protein: A Historic Overview
DBP concentrations compared to GC1 carriers (63). Whether this
is due to a lower hepatic synthesis or more rapid clearance (lower
glycosylation) is unknown.
DBP is a positive acute phase reactant after infections or minor
trauma (72,73). Severe trauma (e.g., hip fracture) or severe illness
(such as patients requiring intensive care treatment) decreases
serum DBP by more than 10%. Whether this is due to an
increase in distribution volume, decreased synthesis or increased
clearance as DBP or DBP-actin complex is not fully understood.
Indeed, major tissue injury causes the release of intracellular actin
into the blood stream, creating actin-DBP complexes that are
rapidly cleared from the circulation (74), so that the positive
effect of increased synthesis is largely compensated by even
more rapid destruction and thus resulting an decreasing DBP
concentrations. During ICU stay, serum DBP slowly increases
and reaches normal levels again after about 10 days (Ingels
et al., submitted). The serum concentration of DBP has a modest
influence on the half-life of 25OHD as demonstrated by using
deuterium-labeled 25OHD in healthy men living in the UK
or The Gambia (39). This is not surprising as lower DBP
concentrations, while serum 25OHD remaining similar, generate
higher free 25OHD concentrations and a slightly higher catabolic
rate. This study also showed that the half-life of 25OHD2was
slightly shorter than that of 25OHD3, in line with lower affinity
of 25OHD2for DBP.
Evolutionary Aspects of DBP
Early in the evolution of vertebrates, after whole genome
duplication, all elements of the vitamin D endocrine function
became gradually operational (75). The vitamin D receptor,
(VDR), coming from an ancestor nuclear receptor involved in
detoxification, acquired a critical role in maintaining a normal
calcium homeostasis and bone metabolism as to cope with an
environment with lower access to calcium and a need for solid
but light weighted bone in a terrestrial rather than an aquatic
milieu. The CYP P450 gene family was broadened to include
several genes with specific role in the activation and degradation
of vitamin D (CYP2R1, CYP27B1, and CYP24A1). Finally, a
specific transport protein for the lipid soluble vitamin D and
its metabolites in serum is found in most fish and all terrestrial
vertebrates studied so far. Not discussed here is the broad range
of genes that are under the direct or indirect control of the
vitamin D hormone (76) and which may explain why a poor
vitamin D status not only results in a skeletal/calcium but also
extra-skeletal phenotypes.
The VDR and the crucial CYPs for the metabolic activation
of vitamin D are already present in cartilaginous fish. However,
based on gel electrophoresis of radiolabeled 25OHD, Hay and
Watson (29) concluded that the vitamin D metabolites in fish
with a cartilaginous skeleton are transported by lipoproteins and
not by a specific DBP as in higher vertebrates. Allewaert et al.,
however, found two vitamin D binding proteins in fish (carp) but
only one resembles DBP (high 25OHD affinity and actin binding)
(77). In amphibians (n=12 species), Hay concluded that
vitamin D was transported by lipoproteins. Allewaert, however,
found in sera of amphibia (two rana species, Bufo marinus and
salamandra) and reptiles, a 25OHD-binding protein with high
affinity for 25OHD but with a low concentration (about 0.03
µmol/L or about 100-fold lower than in higher vertebrates) (78).
DBP from reptiles is able to bind actin (as in higher vertebrates,
see below), as revealed by sucrose gradient ultracentrifugation,
but DBP from all amphibian binding proteins did not show actin
binding. In reptiles (iguana, varanus, python, and geomyola), a
DBP like protein able to bind 25OHD and actin was found as
in chicks and mammals (78,79). In turtle, trachemys scripta (a
member of reptiles), DBP binds thyroxine as well as vitamin D
(80). The situation in birds is complex as most species use a
β-globulin, and a minority use a protein with an α-globulin or
albumin mobility. DBP is present in bird serum and chick (gallus
domesticus) but chick DBP has a slightly higher molecular weight
than human DBP. Although avian DBP has a β-globulin mobility,
it has a high degree of homology with human or rat DBP at
protein and gene level (33).
Older studies, before the use of gene structure to define
protein structures, revealed that mammals (65 species of 14
mammalian orders, including marsupials) use a transport protein
with α-globulin (n=65) or albumin mobility (n=7 such as
pacific dolphin or orcas, and three species of new world monkeys)
(29). The DBP concentration in mammals (other than humans)
are also in the µmolar range (as in birds). DBP levels are very low
in rat fetuses and at birth. Thereafter, its concentration increases
more than five-fold in adult rats. After rat “puberty,” androgens
increase serum DBP whereas the DBP concentration does not
change when rats are exposed to endogenous or exogenous
estrogens (12). Vitamin D deficiency does not influence the
concentration of DBP in rodents (nor in humans). Serum DBP
concentrations in rats and mice increase when these rodents
are exposed to androgens, contrary to what happens in humans
and birds. Indeed, mouse DBP as measured by a mouse specific
immunoassay, is higher in adult males compared to adult
females. The DBP concentration did not markedly vary among
different mouse strains [6–10 µmol/l in males and 5–9 µmol/l
in females (81)]. Cortisol binding protein in rats, however,
increases when animals are exposed to estrogens (as in humans).
In guinea pigs, DBP concentrations decrease during pregnancy
by nearly 50% (contrary to humans). Fetal and neonatal DBP
concentrations of DBP are very low, similar to what has been
observed in rats, despite the much greater maturity of guinea pigs
at time of birth. Therefore, when comparing different species,
we can conclude that DBP is already expressed early during fetal
life but the DBP concentration at birth is much lower than later
in life. Sex hormones change the concentration of DBP in all
species and usually estrogens increase DBP concentrations except
in rodents where androgens increase serum DBP.
DBP is found in all primates and migrates as an α-protein
(as in humans), except in two New World strains. Their DBP
is immunologically identical to that of other primates but has
a different mobility. The genetic and molecular basis of this
difference is not fully explained so far, as the molecular weight
and isoelectric point are identical in these New World monkeys
and humans (50).
The main lesson from all these studies is that vitamin D or its
metabolites bind to lipoproteins in all vertebrates but from bony
fish onwards, a protein of the albuminoid family, DBP, becomes
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Bouillon et al. Vitamin D Binding Protein: A Historic Overview
the major transport protein with high affinity for 25OHD. The
electrophoretic mobility of this protein however can vary from α-
to β-globulin or albumin mobility. DBP concentrations are low in
amphibia and reptiles (0.03 µmolar concentration) but are high
in birds and mammals (∼5µmolar concentration).
In summary, GC/DBP is mainly produced in the liver and
circulates in high concentrations in serum of birds and mammals.
It is usually measured by immune assays but now also by mass
spectrometry. Its concentration in serum is stable and regulated
by sex steroids. Decreased concentrations are found in liver or
kidney diseases. Total (genetic) absence of DBP was so far only
found in a single adult human without generating a bone or other
clinical phenotype.
FUNCTIONS OF DBP (TABLE 2)
Vitamin D Transport
DBP Is the Major Transport Protein for All Vitamin D
Metabolites
DBP, as its name suggests, is the major binding/transport protein
for all vitamin D metabolites. There is only a single binding
site in its A domain. Vitamin D is produced in the skin by
photochemical transformation of 7-dehydrocholesterol (7DHC)
into pre-vitamin D, followed by slow equilibrium with vitamin
D itself. Vitamin D then binds to DBP and thereby drives the
equilibrium in favor of vitamin D itself. This DBP-bound vitamin
D is then rapidly taken up by the liver, and hydroxylated by
CYP2R1 and possibly some other 25-hydroxylases, whereby the
25-hydroxylated metabolite is secreted into the circulation. It is
then again, and now with higher affinity, bound to DBP. Vitamin
D from dietary origin is absorbed in the intestine with about
70% efficacy in normal subjects. Although this process was for
a long time considered to be a passive transport of a lipophilic
molecule to access the lipid layer of the intestinal cells (82), more
recent data suggest that vitamin D absorption and secretion from
and into the lumen of the gut is medicated by carrier proteins
that are also involved in cholesterol transport in the intestine
(83). To what extent vitamin D esters are first digested and then
absorbed or lost in the feces is still a point of discussion [see
Solanum malacoxylon being a 1,25(OH)2D-glucuronide causing
severe generalized calcinosis in grazing cattle in Argentina (84,
85)]. After passage thought the intestinal cell, vitamin D is
transported by chylomicrons and thus uses the lymph pathway
before it arrives in the general blood circulation (82). In that
circulation, chylomicrons (including vitamin D) are mainly taken
up by fat cells or by the liver, but there is also a gradual shift
of vitamin D from chylomicrons to DBP. The metabolism and
transport of vitamin D resembles the metabolic fate of iodine
(also a nutritional product with only intermittent supply and
essential for the generation of an essential hormone; Figure 4).
In both situations, the prohormone, 25OHD, and thyroxin (T4)
are bound with high affinity to a specific serum transport
protein, DBP and thyroxin-binding globulin (TBG), respectively.
Therefore, there is a large pool of circulating precursor with
a long half-life (2 and 1 week, respectively), so that transient
loss of supply of the nutrient (vitamin D or iodine) does not
immediately cause lack of hormonal effect of the real ligand for
their nuclear receptor [1,25(OH)2D and VDR, T3and thyroid
hormone receptor, respectively (Figure 4)]. The affinity of DBP
for 25OHD is much higher than for the VDR, thereby keeping
this precursor preferentially in the serum pool, and the same is
true for T4. By contrast, the active hormones are ligands for their
nuclear receptors to which they bind with high affinity, whereas
their affinity for the serum binding protein is much lower. In
consequence, the distribution volume of 25OHD is very close to
that of albumin and DBP (∼extracellular volume), whereas the
distribution volume of 1,25(OH)2D and T3is close to that of the
intracellular volume.
Whether the role of DBP is essential for the overall metabolism
and action of the vitamin D endocrine system can best be
evaluated by data generated in animals or humans with total
lack of DBP. Absence of other transport proteins for ligands of
nuclear receptor are well-known and absence of TBG, cortisol,
or sex hormone binding proteins does not create hormonal
dysfunctions. This is explained by the very low total serum
concentrations of the ligands for these binding proteins, so
that the free (=not bound to their serum binding protein)
hormone or hormone precursor is virtually normal. Therefore,
the target tissues “see” normal free hormone concentrations (and
in fact the concentration of these hormones are virtually normal
when measured in tissues (86). Such data are the strongest
arguments in favor of the free hormone hypothesis, posing it
that the unbound ligands and not their total concentrations are
physiologically important. For a long time, out of hundreds of
thousands of human sera, not a single case of complete DBP
deficiency was found, suggesting even that this protein might
be essential for normal life. DBP null mice, however, did not
have a phenotype and their concentrations of “total” 25OHD
and 1,25(OH)2D were extremely low (close to the detection
limit) (16). Nevertheless, such mice did not have rickets or
another phenotype when kept on a vitamin D replete diet.
In line with the role of DBP as responsible for generating a
pool of circulating precursor, DBP null mice are much more
prone to vitamin D deficiency when kept in conditions of
vitamin D deficiency (no UVB light exposure and no vitamin
D in their diet). In addition, these mice were more resistant
to the toxic effect of exposure to high doses of vitamin D,
probably by more rapid catabolism of vitamin D and its
metabolites. In 2019, the first case of true total DBP deficiency
was described in a 58-year old Canadian woman, with very
low serum concentrations of 25OHD and 1,25(OH)2D and
absence of serum DBP (all measured by gold standard technology
of liquid chromatography-tandem mass spectrometry) (23).
Nevertheless, no history of rickets or signs of osteomalacia were
present and the patient had normal serum calcium and PTH
concentrations. She was found to have a homozygous deletion of
the complete GC gene region and part of the adjacent NPFFR2
gene. This latter gene codes for a GPCR involved in nociception
and hypothalamic-pituitary axis (87). This rare event may be
explained by parental consanguinity with Lebanese background.
Both genes are known to be closely linked together during
the whole evolution of vertebrates (see above in chapter on
genetic evolution of GC/DBP). The sibling with only one mutated
allele had about half of the normal DBP concentration (in
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FIGURE 4 | Comparison of the transport and metabolism of vitamin D endocrine system with that the iodine-thyroid system.
line with the co-dominant inherence of both GC alleles) and
serum 25OHD and 1,25(OH)2D concentration were well-below
the normal concentrations in the healthy other sibling. The
patient also had a debilitating ankylosing spondylitis, a known
autoimmune disease. It is rather unlikely that this is related to
the absence of DBP, as this phenotype is not found in DBP
null mice. It may be related to the loss of the adjacent gene or
due to repeated exposure to very high doses of vitamin D as
to “correct her apparent vitamin D deficiency” although these
interventions failed to increase serum vitamin D metabolites
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Bouillon et al. Vitamin D Binding Protein: A Historic Overview
(23). Similarly, absence of albumin in humans is not causing a
major phenotype.
Affinity of DBP for Vitamin D Metabolites
There is clearly only a single binding site for all vitamin D
metabolites in DBP (Figure 3), in contrast to albumin which
has several low affinity binding sites. 25OHD binds with high
affinity but the absolute value differs slightly between species,
and is dependent on the buffer medium, pH and temperature
(88). Indeed, the affinity of DBP is higher (about 10 times) in
barbital buffer at pH 8.6 compared to their affinity measured
in different buffers at pH 7.4 (88). The affinity of DBP is
highest for 25OHD-lactones, followed by about equal affinity for
25OHD and 24R,25(OH)2D or 25S,26(OH)2D. Its affinity for
1,25(OH)2D is about 10–100 lower compared to that of 25OHD.
The lowest affinity is for vitamin D itself. The structure of the
cleft on human DBP largely fits with these different affinities (20).
Natural variants of the side chain such as in vitamin D2and its
metabolites may affect the binding for DBP. For humans and
most mammals the difference in affinity is small, with about 20
% lower affinity for 25OHD2compared to 25OHD3(89,90). In
birds, DBP has a much lower affinity for 25OHD2compared to
25OHD3and this is supposed to be one of the main reasons
for the low biological activity of vitamin D2for the prevention
of rickets in birds (91,92). The relative difference in affinity
between 25OHD2and D3was greater when measured in diluted
serum (10-fold difference) (92) than when measured for purified
chick DBP (three-fold difference) (33). Vitamin D2is also largely
ineffective in comparison with vitamin D3to promote the
intestinal absorption of calcium-47-isotope in Cebus Albrifons
monkeys but for so far unknown reasons (93). In contrast to
DBP, VDR does not discriminate between 1,25(OH)2D3and
1,25(OH)2D2. Most enzymes involved in vitamin D metabolism
also do not discriminate between D2and D3or its metabolites
except for some non-CYP2R1 25-hydroxylases (94).
The affinity between DBP and its major metabolites has
usually been studies by using [3H]labeled vitamin D metabolites
and either diluted serum or purified DBP. We also estimated
the affinity (Ka) in rat serum as the ratio of “on rate” over “off
rate” by measuring the association and dissociation rate constant
(95). The dissociation rate constant was highly temperature
dependent, being about 1 day at 4◦C and between 2 and 12 min
at 37 and 24◦C, respectively. The half-association time was about
1 min at 4◦C and even more rapid at higher temperature. The
best estimation of the Ka based on these rate constants was very
similar to the Ka measured by direct binding assay, charcoal
precipitation of free ligands and Scatchard plot analysis. As the
affinity is dependent on temperature, pH, buffer conditions, and
methodology, variable data have been generated but there is
general agreement that human DBP has a very high affinity for
25OHD, with a Ka of around 1.5 ×108M−1(34,88) at 37◦C.
Whether the different isoforms of DBP have different affinities
for 25OHD is a matter of debate as one group (96) concluded
that DBP/GC2 has only half of the affinity of DBP/GC2s and this
isoform had again only half the affinity of DBP/GC1f. Three other
laboratories, however, independently, could not find significant
differences in affinity for 25OHD when measured by Scatchard
plot using highly labeled [3H]25OHD rather that vitamin D3
itself used by Arnaud and Constans (34,60). When we compared
the affinity of 20 human samples, only a very minor difference in
affinity was found with a Ka of 1.63 (±0.29) ×10−10 M for GC1s
homozygotes compared to 2.11 (±0.25) ×10−10 M for GC1f,
and intermediate values for DBP/GC2 homozygotes (79). The
rank order of these relatively small differences was, moreover,
different when the measurements were done in a phosphate
buffer compared to a Hartman buffer (both at pH 7.4) (33). A
rare variant of DBP/GC (GC Aborigine or GC1A1) as found
in Aboriginals and some blacks from South Africa (48) have
similar Ka values (88). The affinity of DBP from different species
as measured in the author’s laboratory is shown in Table 3.
The highest affinity for 25OHD is found in serum from rats
[confirmed by (49)] and 5 species of amphibians (79). Lowering
the pH rapidly decreased the affinity (to very low levels at pH
5), but the affinity increased at pH 8–10 with the highest values at
about pH 8.6 (about 10-fold higher in mammalian DBP but not in
DBP from chicks or toads). The affinity of DBP for 1,25(OH)2D
is about 1.5 ×107M−1, with little difference between DBP/GC
genotypes and only a minor increase in affinity at higher pH (88).
The affinity of purified DBP from human, rat, and chick serum
was very similar to that measured in (highly) diluted serum. The
variation of the assay results are about 25% of the prefix of the
Ka value. When comparing methods to separate bound and free,
charcoal or filter assays generated similar results (67,88,97).
The affinity of DBP for 1,25(OH)2D is about 30–50-fold lower
than for 25OHD in all species studied so far. The Ka at pH 7.4
and 4◦C is about 1-5-2 ×107M−1in most species but about
10 times higher values were measured in diluted rat (49,88)
and amphibian sera (Table 3). In chicks (Galllus domesticus),
the Ka of DBP for 25OHD at 4◦C and pH 7.4 is 1 ×109M−1
(33). Bouillon et al. could not find a specific vitamin D3-binding
protein as described before by Edelstein et al. (33,98). Indeed,
labeled vitamin D behaved similarly to labeled 25OHD on simple
and crossed immunoelectrophoresis of chick DBP. In DBP-free
serum, both metabolites were bound to albumin.
DBP and Concentration of Unbound Vitamin D
Metabolites: Free or Bioavailable D Metabolites
As the DBP has a high affinity for most vitamin D metabolites,
and also has a much higher molar concentration compared
to its ligands, the free concentration of all its metabolites
is very low, in fact much lower than that of other ligands
of nuclear receptors. The free concentration of 25OHD and
1,25(OH)2D were first calculated1based on the measurements
of total concentrations of the metabolites, total concentration
of DBP, the best estimation of the affinity and the law of mass
action (13,14). Free hormones such as cortisol, thyroxin, and
1The formula used to calculate free hormone concentration usually refers to
Vermeulen et al. (99). In reality, however, the real inventor of the formula is
Walter Heyns from the Leuven laboratory of endocrinology (see his formula for
calculating free cortisol (100) who taught Vermeulen’s assistant (L. Verdonck),
during a working visit in Leuven, how to use his formula to calculate free
testosterone concentrations. The Vermeulen group later independently validated
this calculation by comparing the results with direct measurements of free
testosterone (Walter Heyns and Alex Vermeulen, personal communication 2018).
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TABLE 3 | Affinity of DBP from different species for the major vitamin D
metabolites*.
Species 25OHD 1,25(OH)2D Comments
Human 5 ×108M−11.5 ×107M−1Variation between 3 and 10
×108, depending on buffer
and DBP/genotype
Monkeys 5 ×108M−11.5 ×107M−1** Measured in old and new
world monkeys. In Cebus
albicans, DBP migrates with
albumin mobility but has a
similar Ka
Rat 6 ×108M−11.4 ×108Measured in three strains
Rabbit 1.5 ×109M−1NM
Chick 4 ×108M−11.7 ×107M−1
Reptiles NM NM
Amphibia 2–14 ×1010 M−12.6 ×108M−1Measured in 5 species
*Measured in diluted serum at pH 7.4 and 4◦C, unless specified otherwise; all values are
expressed as Ka at M−1values.
**The Ka for 1,25(OH)2D varied between 1.2 and 2.6 ×107, depending on buffer and
DBP/GC genotype.
NM, not measured.
sex hormones are usually a better marker of their biological
activity than their total concentrations (101). These free hormone
concentrations are also better feedback-controlled than the total
hormone concentrations. The best example is that of extreme
deficiency of transport proteins of one of these ligands, resulting
in a dramatic decrease in total but keeping free hormones within
the normal range. Is this also applicable to the major vitamin D
metabolites? Two different terminologies are frequently used for
free hormones: some define free hormone as non-protein (either
to the specific binding protein and albumin) bound hormone,
whereas bioavailable hormone is the combination of free and
loosely albumin-bound hormone.
The first major difference with other transport proteins is
the combination of high affinity and very high concentration of
DBP so that the free concentration of 25OHD and 1,25(OH)2D
are very low in absolute and relative concentrations. The free
25OHD and 1,25(OH)2D concentrations are about 10 and 1
pmol/l (14). This also means that the free 25OHD concentration
is <0.1% of total 25OHD and that free 1,25(OH)2D is about 1%
of its total concentration. Due to difference in affinity, the molar
ratio of total 25OHD over total 1,25(OH)2D is about 500 but
free 25OHD is only 10 times higher than free 1,25(OH)2D. In
Figure 5, a few formulae used to calculate the concentration of
free vitamin D metabolites are shown. All formulae use the law
of mass action as published by Vermeulen et al. (99) (see also
footnote 1) for androgens and by Coolens et al. (100) for cortisol.
The simplest way to express free hormone (vitamin metabolite)
concentration is a calculation of the free hormone/metabolite
as a molar ratio of molar concentration of hormone/metabolite
over molar concentration of its binding protein (DBP). As DBP
has such a high capacity, the concentration of the apoprotein is
indeed virtually also close to that of the total DBP concentration.
Such molar ratio of 1,25(OH)2D:DBP is nearly identical (r>
0.95) to that of calculated free 1,25(OH)2D (14).
Free 25OHD is not feedback-regulated in humans nor in
animals. Increased intake of vitamin D or higher sun exposure
increase total as well as free 25OHD concentrations, and vitamin
D deficiency does not change DBP concentrations whereby total
and free 25OHD concentrations decrease to about the same
degree. In most studies, increase in DBP by estrogen intake
does not increase serum 25OHD (14,34). Whether free 25OHD
is a better predictor than total 25OHD for health outcomes is
controversial and will be discussed elsewhere in this Journal and
has recently been reviewed several times (34,60). Free 25OHD
can be calculated based on DBP concentrations and the affinity
of DBP for 25OHD but the DBP assay is not standardized
yet and the exact value of the Ka value at 37◦C is also not
perfectly known (see above). Therefore, the predictive value of
calculated free 25OHD is yet unknown. Free 25OHD can also
be directly measured by ultrafiltration or dialysis (43,102) or by
ELISA (103). Dialysis and ultrafiltration are technically difficult
and face problems due to interference of adsorption of very
low vitamin D metabolites to glass or plastic tubes, and even a
minute impurity of labeled metabolites represent concentrations
potentially higher than the free metabolites. In normal subjects,
the directly measured free 25OHD concentration has a strong
correlation with the calculated free concentrations (taking into
account a variation coefficient of about 10% of all measurements).
In the MrOs study on elderly men, calculated free 25OHD
(using polyclonal DBP assays) correlated strongly with directly
measured free 25OHD (r=0.80–0.83) (22,59). Moreover,
the calculated or measured free 25OHD concentration strongly
depended on total 25OHD (r=0.96 and 0.83, respectively).
Of course, as for other free hormones, the direct measurements
are especially important in case of abnormal concentrations or
affinity of the binding proteins. This aspect has not yet been
fully evaluated for free 25OHD. Free 1,25(OH)2D concentration
probably “behaves” more like free thyroxin, cortisol or sex
steroid hormones. Indeed, the total 1,25(OH)2D concentration
correlates with DBP concentrations in most studies whereas this
is not the case for total 25OHD. Increased DBP by estrogen
exposure or early pregnancy increases DBP and total 1,25(OH)2D
very similarly, so that the free concentrations remain unchanged
(14). Similarly, in sexually mature hens, DBP and 1,25(OH)2D
increase several fold but 1,25(OH)2D increases more than the
DBP so that the free 1,25(OH)2D concentration is high, in
line with the high calcium demands for egg shell calcification.
When, however, egg shell calcification is blocked by a thread
in the uterus, soft eggs are produced, calcium requirements
drop, and total and free 1,25(OH)2D fall back to the level of
immature chicks (104). Infusion of large amounts of human
DBP in blood of normal rats, increases combined rat and
human DBP concentrations and also increases total 1,25(OH)2D
concentrations (67). Total absence of DBP in mice results in
very low (nearly undetectable) total 1,25(OH)2D concentrations
but the tissue concentration of free 1,25(OH)2D is normal (86)
and serum calcium and PTH status remain normal. In the only
human case of biallelic mutation of the DBP/GC gene, resulting
in undetectable serum DBP concentrations, total 1,25(OH)2D
was nearly undetectable but without consequences for calcium
or PTH homeostasis (23). Also animal studies came to the same
conclusion as rabbits immunized against 1,25(OH)2D-conjugates
as to induce antibodies for immunoassays have a more than
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100-fold increase in total serum 1,25(OH)2D concentrations
without repercussions on calcium homeostasis (51). There are
at present no publications reporting direct measurements of free
1,25(OH)2D. Whether free 1,25(OH)2D is a better predictor than
total 1,25(OH)2D concentrations for health outcomes is unclear.
In the MrOs study, free 1,25(OH)2D concentrations were a
better predictor of inflammatory markers than total 25OHD,
total 1,25(OH)2D or free 25OHD (105). As the concentration
of DBP in maternal serum (at time of delivery) is about twice
the concentration of DBP in cord serum, the maternal:neonatal
ratio of DBP and vitamin D metabolites may be informative for
the relative importance of free vs. total vitamin D metabolite
concentrations. From previous genetic studies, it is well-
documented that there is no transplacental transfer of DBP/GC
(24). The total and free 25OHD concentrations show a high
correlation between the maternal and fetal compartments, with r
values usually above 0.6. The total 25OHD concentration is much
higher in the mother than in the neonate but the free 25OHD is
higher in cord serum. This probably reflects a good transplacental
transport and a contribution of fetal 25-hydroxylase activity to
the fetal vitamin D status. The correlation of total and free
1,25(OH)2D between both compartments is somewhat lower
than for 25OHD and the free 1,25(OH)2D concentration in the
fetus is slightly higher than in maternal serum. As fetal (and
placental) 1,25(OH)2D production may be partly responsible for
the higher neonatal ionized concentration (and placental calcium
transport), as shown in sheep by Care (106), such higher fetal
1,25(OH)2D may reflect its biological role.
The most important implication of the free hormone
hypothesis is that only the free or bioavailable
hormone/ligand/vitamin D metabolite has access to the
cytoplasm or nucleus of the cell. Indeed, cells have access to the
total concentration of ligands bound to serum proteins when
the cell uses specific receptors to internalize these complexes
(Figure 6). As example, cholesterol carried by lipoproteins
can be internalized by hepatocytes or other cells using specific
LDL or HDL receptors and, therefore, these cells have access
to the total concentration of cholesterol. “Free” or unbound
cholesterol” is not discussed or used for clinical purposes. Other
examples are shown in Figure 6. By contrast, most hormones
enter cells by diffusion and only the unbound hormone enters
most cells. This has clinical implications, as free hormone
measurements are the preferred technique to estimate thyroid
function. The real situation is probably slightly more complex
as excess free ligands in the “nutrient type” of cellular import
may influence cell function (e.g., iron overload). If cells express
receptors for some binding proteins such as receptors for the
sex steroid-binding protein or the presence of megalin in some
cells, then such cell membrane receptors allow the import of the
binding proteins (e.g., DBP) with all its ligands (see below). For
some hormones, specific membrane carriers (such as membrane
transporters for thyroid hormones) further complicate the
model. For most situations, however, the free hormone model
is physiologically and clinically relevant. We think that the
DBP∗1,25(OH)2D complex belongs to the hormonal type of
serum transporters as only free 1,25(OH)2D influences cell
function. Its free concentration in the picomolar range comes
close to the affinity of VDR for its major ligand. For 25OHD,
the question is different and highly relevant. Indeed, the free
25OHD concentration is around 10 pmol/l and this seems low
in comparison with the affinity of the major CYPs responsible
for its further metabolic activation. This raised the question
whether (free) 25OHD has real access to CYP27B1 or CYP24A1
in many cells and thereby has functional implications. The role
of megalin in the kidney tubuli will be discussed below (chapter
“DBP binds megalin”). Most other cells do not express or have
only minimal expression of megalin. Probably the best method
to explore the functional role of 25OHD in the local production
of 1,25(OH)2D can be found by deletion or overexpression of
CYPs (CYP27B1 or CYP24A1) in specific tissues and expose
such mice/animals to variable serum concentrations of 25OHD
and or DBP. Tissue specific deletion of CYP24A1 in mammary
cells had a clear effect on the risk of breast cancer (107).
Overexpression of CYP27B1 in bone cells seemed to be beneficial
for bone (108,109) and deletion of this gene in growth plate
chondrocytes generated bone effects as long as the growth plate
remained fully active (110). Therefore, from these few examples,
it seems that 25OHD has sufficient access to some target cells
and generate local actions without full understanding how such
low free or bioavailable concentrations can activate these genes
and cells.
When comparing four different species (humans, rats, guinea
pigs, and sheep) and calculating free 1,25(OH)2D based on total
serum 1,25(OH)2D and DBP concentrations in the maternal
and neonatal compartments, free 1,25(OH)2D is higher in the
fetus/neonate than in their mothers. The concentrations of total
and free 1,25(OH)2D concentrations show a good correlation in
all these species (111) so that it is likely that the fetal production
of 1,25(OH)2D contributes to this gradient and maybe also to the
greater concentration of calcium in the fetus compared to the
maternal calcium concentrations. Whether bioavailable (=sum
of free and ligands loosely bound to albumin) is more important
than free ligands is not fully understood and has been discussed
but not “solved” in other reviews.
Whether the free hormone hypothesis also applies to the
vitamin D endocrine system can be demonstrated by in vivo
data as summarized above but also by dedicated in vitro studies.
Addition of purified DBP or serum to culture media followed
by measuring the biological response of cells or tissue to
either 25OHD or 1,25(OH)2D has repeatedly shown that the
presence of DBP impairs the biological action, whether using
the expression of a natural gene/protein or using a genetically
engineered gene construct. As examples:
1. The proliferation of human lymphocytes, cultured in a DBP
free medium, can be inhibited by 50% by 1,25(OH)2D at
1010−12 concentrations, but required 100-fold higher molar
concentrations if purified DBP is added at the physiologic
concentration of 5 µmol/l (112). DBP counteracted the action
of several vitamin D analogs with the same rank order as
predicted on the basis of their affinity for DBP.
2. Resorption (measured by release of radioactive calcium) of
bone tissue explants (forelimb bones from 18d old fetal rats, in
vivo exposed to radioactive calcium) cultured in the absence
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FIGURE 6 | Model of nutrient and hormonal type of access of extracellular ligands to cells.
of DBP is stimulated by low concentrations of 1,25(OH)2D.
Adding purified rat or human DBP (5 µmol/l) impaired bone
resorption but rat DBP was much more potent, in line with its
6–10-fold higher affinity for 1,25(OH)2D (113).
3. Cathelicidin, a well-known target of the vitamin D hormone,
is stimulated by 1,25(OH)2D or 25OHD in human monocytes,
cultured in serum from DBP null mice. Adding serum from
normal mice however impaired this effect. The authors did not
observe a megalin-mediated action (see below) (114).
4. Human foreskin keratinocytes express mRNA for CYP24A1
when exposed to 1,25(OH)2D. This action was markedly
inhibited by culturing these cells in the presence of diluted
serum compared to an albumin only based medium. Similarly,
the production of radioactive 24,25(OH)2D from [3H]25OHD
was inhibited by DBP (115).
5. Dermal fibroblasts from a patient with biallelic mutations of
the DBP/GC gene or from normal volunteers were exposed
to exogenous 25OHD, either in the presence of 10% normal
serum containing DBP or 10% DBP deficient serum. The
uptake of 25OHD in all cells was markedly inhibited by the
presence of human DBP (23).
All these experiments clearly demonstrate that the presence of
DBP decreased the access and actions of vitamin D metabolites
in a wide variety of cells.
DBP Binds to Megalin-Cubilin Receptor
Patients with nephrotic syndrome loose massive amounts of
serum proteins in their urine, exceeding the liver capacity to
replace the daily loss. DBP has a smaller molecular weight than
albumin and is lost together with 25OHD and other vitamin D
metabolites. This renal loss of 25OHD frequently results in a poor
vitamin D status as first reported in 1977 (71) and subsequently
confirmed in many other studies. Megalin (global or kidney-
specific) knockout mice are unable to reabsorb several proteins
in the renal tubuli as it is a cargo receptor for several serum
proteins, allowing to prevent their urinary loss (15). Megalin is
a membrane receptor and requires cubilin as co-receptor (116),
and both are expressed in several tissues but mainly in the luminal
brush border membrane of the renal tubuli. Megalin is a giant
protein of 600 kDalton and belong to a family of low-density
lipoprotein receptors. Therefore, it is also known as LRP2. This
protein was initially identified as autoantigen in an experimental
model of membranous nephropathy, called Heymann nephritis
(117). Megalin is a well-conserved gene/protein, already present
in nematodes. In mammals, the expression of LRP2/megalin
is restricted to specialized absorptive epithelia in the brain,
the eye, the lung, the kidney, and the reproductive tissue.
Therefore, the major phenotype of a global megalin knockout
is characterized by major developmental brain abnormalities,
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Bouillon et al. Vitamin D Binding Protein: A Historic Overview
usually even lethal. Megalin binds and internalizes a large
number of low molecular weight proteins, responsible for
transporting a variety of lipids, vitamins, and hormones. This
includes DBP, the retinol-binding protein, and transcobalamin.
Thereby, megalin-mediated clearance of these binding proteins
from the tubular lumen serves to retrieve essential vitamins and
hormones bound to these carriers and to prevent uncontrolled
loss of essential metabolites by glomerular filtration. More than
20 other proteins, including thyroglobulin (118) are also known
to be ligands of these membrane receptors (119). In the absence
of one of these co-receptors (megalin or cubilin), many low
molecular weight proteins are lost in the urine, known as tubular
proteinuria. Loss of these receptors can be due to a variety of
acute, chronic or genetic diseases. In such diseases, DBP is also
lost in the urine together with 25OHD and this loss of vitamin
D metabolites may exceed the daily supply and synthesis of
vitamin D and therefore cause vitamin D deficiency and rickets
(15). This also implies that the renal tubuli, where CYP27B1 is
transforming 25OHD into 1,25(OH)2D, has access to 25OHD
by either the serosal site (and thus mainly access to non-DBP
bound 25OHD), or via the luminal site (where DBP in complex
with 25OHD is internalized by the megalin-cubilin receptor
complex). Thereafter, reabsorbed proteins are broken down in
lysosomes, leaving the luminal 25OHD available for metabolic
activation. Megalin is then recycled to the luminal membrane.
This phenomenon does not imply that the megalin pathway
is essential [as suggested by some expert reviewers (120)] for
the renal synthesis of 1,25(OH)2D, as megalin-deficient mice do
not develop rickets if the urinary loss of vitamin D metabolites
is compensated by sufficient intake. Neither do DBP-deficient
mice develop rickets if they have access to sufficient vitamin
D. Because the megalin receptor is a cargo receptor for a large
number of proteins, loss of one of these membrane receptors can
cause a variety of deficiencies, apart from vitamin D deficiency.
Whether megalin- or cubilin-mediated uptake of DBP is also
operational in other cells, such as placenta or parathyroid gland
is insufficiently documented so far. However, the gene expression
of megalin outside the kidney, brain, and eyes, is usually very
low. The lung may be an exception and is now known to be
an important target for vitamin D action (121). As discussed
above, such transport mechanism, if operational outside the
kidney, would expose cells expressing these cargo receptors to
DBP bound 25OHD rather than free 25OHD.
DBP Binds Actin
About 50 years ago, three major binding proteins were known,
the serum binding protein, now identified as DBP/GC, the
intracellular/nuclear VDR (now known to be a member of the
nuclear transcription factors), and an intracellular 6S binding
protein (MW about 90,000 Daltons), with preferential binding
for 25OHD and which was considered as a cellular “receptor”
for 25OHD (122,123). This protein was found in most tissues
but also in whey and milk of humans and mammals. In
1977, however, we demonstrated that the 6S 25OHD-binding
protein was in fact a complex of DBP with an ubiquitous
cytosolic heat-labile compound that was by itself unable to bind
vitamin D metabolites (11). This phenomenon was confirmed
by using antibodies against DBP (11,124). A few years later,
while trying to identify this intracellular compound by adding
different proteolytic enzymes and DNAase, Van Baelen et al.
(125), found that the addition of DNAase created a triple
complex DBP-unknown intracellular protein-DNAase. Based
on this observation, the Leuven group identified actin as the
cytosolic protein that binds with high affinity to create the so-
called intracellular 25OHD-binding protein/receptor. Actin-DBP
binding does not involve the presence or absence of vitamin
D ligands, or vice versa. This was confirmed by resolving
the 3-dimentional structure of DBP-actin by three different
groups (126–128).
Many actin-binding proteins regulate or control intracellular
actin polymerization or disassembly, including profilin (129).
The extracellular binding proteins are, however, mainly DBP
and gelsolin (also known as brevin). Profilin in also present
in platelets and can act as an actin sequestrant but with
a 1,000-fold lower affinity and therefore of probably minor
extracellular importance (130). Gelsolin-actin forms a 2-1
complex as gelsolin is able to sever actin polymers and then one
gelsolin binds and caps each filament fragment. Together with
profilin and gelsolin, DBP creates a well-coordinated strategy
with complementary mode of action as to rapidly remove actin
(polymers). These observations have several implications. First,
DBP is not found inside cells when plasma contamination is
carefully avoided during tissue preparation. Thus, it is rather
unlikely that extracellular DBP interferes with the key functions
of intracellular actin. Second, actin-DBP complexes only happens
when intracellular actin is released in the bloodstream. Indeed,
Van Baelen et al. (125) clearly showed that DBP was able to
depolymerize polymeric actin. Actin can easily switch from
monomeric into polymeric actin in the cytosolic milieu, thereby
helping in building the intracellular organizations of cells. In
the plasma milieu, actin monomers are rapidly transformed
into polymeric structures, which could result in clogging the
microcirculation much like fibrinogen/fibrin. This process could
be accelerated by the aggregation of platelets as actin-ADP binds
to platelet surfaces and acts as agonist for platelet aggregation
(131). DBP, however, is able to build a one-to-one complex with
actin and together with other actin severing/binding proteins
(e.g., brevin) in serum, allows to prevent the formulation of
actin polymers in the circulation (74,125). These proteins have
different roles in actin depolymerization by either severing or
capping the actin chains and creating actin-DBP complexes that
are more rapidly cleared from the circulation that DBP alone
(74). Actin and DBP are very tightly bound (Ka of ∼2–5 ×
108M−1) (125,132) so that these complexes are de facto not
dissociated once formed in the circulation. However, actin-DBP
is rapidly cleared from the circulation with a half-life of <1 h in
rabbits (74). The damage caused by actin polymerization has been
directly demonstrated in DBP null mice (133). Actin infusion
in such mice resulted in more severe acute lung inflammation
(vascular leakage, hemorrhage, and thickening of the vascular
wall) compared with normal mice. This was confirmed in
vitro as lung endothelial cells, exposed to DBP-actin complexes
generated enhanced cell death (133). Third, the 3-D structure
shows all three domains of DBP being in direct contact with
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actin and interacting through a variety of contact mechanisms.
The substantial surface areas from both DBP and actin that are
involved in intimate protein-protein contacts explains the high
affinity interaction between these two proteins. Moreover, the
DBP residues involved in actin binding are less well-conserved
between all species with known DBP structures than the vitamin
D-binding domain. The contact surface is greater than the
contact surface of actin with two other actin-binding proteins
(gelsolin and profilin) together (126–128). These data confirm
the actin-scavenger role of DBP in higher vertebrates. However,
DBP from some fish and amphibians are unable to bind to actin
and create an actin-DBP complex. The molecular reasons for
that absence of actin interaction are so far unknown. Fourth,
DBP-actin interaction can be used to isolate DBP from several
species, as actin can be used for affinity chromatography when no
species-specific antibodies against DBP are available. In clinical
or research situations, the presence of actin-DBP complexes in
serum or the (low) DBP concentration in serum can be used a
marker for the disease severity and has prognostic significance in
patients with severe illness (134). The greatest decrease in serum
DBP has been found in patients with acute/fulminant hepatic
necrosis (whereby serum DBP may fall to about 10% of normal
values) rhabdomyolysis, sepsis, or major traumas (135–137).
DBP Binds Fatty Acids
Triglycerides are bound in serum to several lipoproteins whereas
free fatty acids are mainly loosely bound to albumin. However,
all members of the albuminoid family are able to bind fatty acids.
DBP binds to fatty acids present in membrane phospholipids
(see DBP and inflammation) of leucocytes but also bind fatty
acids, and especially poly-unsaturated fatty acids (16:1, 18:1,
18:2, and 20:4) are associated with DBP/GC as measured by gas
chromatography (17). When [3H]arachidonic acid was added to
serum about 75% was found to be bound to DBP. The molar
ratio of fatty acids/DBP is about 0.4 compared to 1.8 for albumin.
Mono- and polyunsaturated fatty acids impair the binding of
25OHD and 1,25(OH)2D to DBP. Indeed, high concentrations
(36 µmolar) of linoleic or arachidonic acid decreased the
apparent affinity two to five-fold. Their effect was much greater
for inhibition of binding of 1,25(OH)2D than for 25OHD
(18). Saturated fatty acids and many other lipid soluble serum
substances do not interfere with the binding of DBP to vitamin
D metabolites. Physiologic concentrations of unsaturated fatty
acids may thus impair the binding of 1,25(OH)2D to DBP (18).
Whether this transport of fatty acids plays a real role in energy or
vitamin D transport is not known.
DBP and Inflammation
DBP has no direct effects on inflammation. Its ligand,
1,25(OH)2D has many immune and inflammatory effects
[reviewed in (34,35)] and DBP inhibits the cellular entry of
1,25(OH)2D. DBP, however, is able to bind to membranes and
chondroitin sulphated proteoglycans of leucocytes. In association
with annexin A2, this binding enhances complement C5a-
stimulated chemotactic activity. Quiescent neutrophils do not
bind DBP and binding is dependent on prior neutrophil
activation. As this activity is linked to activated leucocytes, it may
play a role in local inflammation in response to cell damage by
whatever mechanism (19,138,139). A more extensive discussion
on this action of DBP has been published (34,140) and no further
data have recently been published on that topic.
DBP and Macrophage Activating Factor
(MAF)
Several in vitro studies suggested that the deglycosylation of
DBP can activate DBP to become a macrophage activating factor
(MAF). A membrane bound β-galactosidase present on the
surface of immune B cells and a sialidase present on T cells
would remove two of the three sugar residues of DBP/GC1
protein (141). This DBP would then be able to facilitate the
differentiation of monocytes to become osteoclasts and even
correct the osteopetrosis phenotype of mice (142). In other
circumstances DBP-MAF would activate macrophages in their
battle against cancer cells or infections (143,144). A few clinical
trials tested the potential effects of DBP-MAF against HIV
infections or cancer. Most of the data on DBP-MAF comes from
Yamamoto’s laboratory but he had to retract several major papers
because of irregularities in the documentation of his data after
institutional review [retracted manuscripts included (145,146)].
Moreover, the availability of nagalase to act as endoglycosylase
(and thus to deglycosylate DBP) has been questioned (147).
Therefore, there is serious doubt on the existence and role of
DBP-MAF despite a large number of publications [reviewed in
(34,148)].
Polymorphisms of DBP and Disease
The polymorphisms of DBP have been associated with
susceptibility or resistance to a large number of chronic
conditions, such as osteoporosis (149–151), type 1 and type
2 diabetes (152), thyroid autoimmunity (153), inflammatory
bowel disease (154), and chronic obstructive lung disease (155).
The exact role of DBP in the pathophysiology of these diseases
is however not completely understood [see (156,157) for an
extensive discussion].
In summary, GC/DBP has two major functions:
1) The transport of all vitamin D metabolites (at a single binding
cleft of the A domain of the protein). Due to the combination
of high affinity for vitamin D metabolites and a high protein
concentration, free concentrations of all vitamin D metabolites
are extremely low.
2) Tight binding of actin, creating a DPB-Actin complex that
avoids actin polymerization in serum after tissue damage.
3) Other possible functions of GC/DBP need further validation.
SUMMARY AND PERSPECTIVES
Vitamin D and all its metabolites are bound to a specific vitamin
D binding protein, DBP. This discovery was made independently
by studying the electrophoretic mobility of antirachitic activity
or radiolabeled vitamin D (metabolites) and by studying the
polymorphism of a major serum protein called Group-specific
Component (GC). We now know that DBP and GC are the
same protein and appeared early in the evolution of vertebrates.
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TABLE 4 | Major remaining research problems and questions.
1 International standard for (gene-specific) DBP and reference method
for the measurement of DBP
2 Validation of measurement of free 25OHD and its potential clinical value
in comparison with total 25OHD
3 Validation of measurement or calculation of free 1,25(OH)2D and its
potential physiologic and clinical implications
4 Understanding of the environmental drive (and health implications) for
the world-wide gene polymorphism of DBP/GC
5 Full understanding of the implications of actin-DBP binding in health
and diseases
6 Understanding of the role of DBP in inflammation
7 Role of megalin and DBP-25OHD uptake, if any, in non-renal cells
DBP is genetically the oldest member of the albuminoid family
(including albumin, α-fetoprotein, and afamin, all involved in
transport of fatty acids or hormones). Some fish use lipoproteins
as a sole transport protein but many other fish and (nearly) all
amphibia, reptiles, birds, and mammals use DBP. This protein
has a single binding site for all vitamin D metabolites and has a
high affinity for 25OHD and 1,25(OH)2D. This allows creating a
large pool of circulating 25OHD, which prevents rapid vitamin D
deficiency when the supply of new vitamin D is compromised.
DBP also regulates the access of all vitamin D metabolites
to cells and tissues. In birds and mammals, DBP combines a
high affinity with high concentration, whereas its concentration
in early vertebrates is much lower. DBP of higher vertebrates
(not amphibians or reptiles) binds with very high affinity actin,
creating a 1:1 DBP:actin complex, and thereby preventing the
formation of polymeric actin fibrils in the circulation after
tissue damage. Megalin is a cargo receptor and is expressed in
many epithelial cells, including the luminal border of the renal
tubuli. Megalin and cubilin are needed to reabsorb DBP or
the DBP-25OHD complex (and many other proteins and their
ligands), filtered in the renal glomerulus, thereby preventing
the urinary loss of these proteins and 25OHD. Absence of one
of these receptors results in rickets, unless the daily supply
of vitamin D is able to replace its excessive urinary loss. The
total concentrations of 25OHD and 1,25(OH)2D in DBP null
mice or humans are extremely low (at the limit of detection)
but their free concentrations in serum and tissues are probably
normal. This does not impair the action of the vitamin D
endocrine system on gene expression or tissue action, and
therefore is the strongest argument for claiming that the “free
hormone hypothesis” also applies to the vitamin D hormone,
1,25(OH)2D. The relative importance of free 25OHD compared
to total 25OHD is not yet settled. The ongoing standardization
of DBP assays and the validation of a direct ELISA for free
25OHD should allow generating the data needed to answer this
question in the near future. DBP also transports fatty acids,
and unsaturated fatty acids compete with vitamin D metabolites,
thereby decreasing the apparent affinity of DBP for 25OHD and
especially 1,25(OH)2D. DBP can bind to proteoglycans present in
the membrane of immune cells and thereby enhance complement
C5a-stimulated chemotactic activity of activated neutrophils.
This role in inflammation is not fully understood. DBP null mice
and a single woman with total absence of DBP are healthy and
do not have a clinical phenotype related to vitamin D transport
or actin, megalin, or complement binding. DBP is genetically
very polymorphic with three frequent alleles (DBP/GC 1f, 1s,
and 2) but in total more than 120 different variants. There is a
clear North-South gradient of this polymorphism but its health
consequences, if any, are not understood. There is a need for
standardization of assays for serum DBP as to allow the studies
needed to further explore the role of DBP in physiology and
diseases. Despite the enormous progress in the deciphering of the
structure of DBP and its function, there remains an impressive list
of major research questions (Table 4).
AUTHOR CONTRIBUTIONS
RB: design and overall writing of the manuscript. FS: genetic
origin of DBP and writing of several chapters of manuscript. LA:
polymophism of DBP and overall correction of manuscript and
references. FR: structure-function analysis of DBP and overall
correction of manuscript.
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
The reviewers JS and JSS declared a past co-authorship with one of the authors RB
to the handling editor.
Copyright © 2020 Bouillon, Schuit, Antonio and Rastinejad. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these
terms.
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