Hemorrhage-Adjusted Iron Requirements, Hematinics and Hepcidin Define Hereditary Hemorrhagic Telangiectasia as a Model of Hemorrhagic Iron Deficiency

Abstract

Background
Iron deficiency anemia remains a major global health problem. Higher iron demands provide the potential for a targeted preventative approach before anemia develops. The primary study objective was to develop and validate a metric that stratifies recommended dietary iron intake to compensate for patient-specific non-menstrual hemorrhagic losses. The secondary objective was to examine whether iron deficiency can be attributed to under-replacement of epistaxis (nosebleed) hemorrhagic iron losses in hereditary hemorrhagic telangiectasia (HHT).Methodology/principal findingsThe hemorrhage adjusted iron requirement (HAIR) sums the recommended dietary allowance, and iron required to replace additional quantified hemorrhagic losses, based on the pre-menopausal increment to compensate for menstrual losses (formula provided). In a study population of 50 HHT patients completing concurrent dietary and nosebleed questionnaires, 43/50 (86%) met their recommended dietary allowance, but only 10/50 (20%) met their HAIR. Higher HAIR was a powerful predictor of lower hemoglobin (p = 0.009), lower mean corpuscular hemoglobin content (p

Full text

Hemorrhage-Adjusted Iron Requirements, Hematinics
and Hepcidin Define Hereditary Hemorrhagic
Telangiectasia as a Model of Hemorrhagic Iron
Deficiency
Helen Finnamore
1,4,7
, James Le Couteur
2
, Mary Hickson
2
, Mark Busbridge
3
, Kevin Whelan
4
,
Claire L. Shovlin
5,6
*
1National Heart and Lung Institute, Cardiovascular Sciences, Imperial College London, London, United Kingdom, 2Nutrition and Dietetics, Imperial College Healthcare
NHS Trust, London, United Kingdom, 3Clinical Chemistry, Imperial College Healthcare NHS Trust, London, United Kingdom, 4Diabetes and Nutritional Sciences Division,
King’s College London, School of Medicine, London, United Kingdom, 5National Heart and Lung Institute, Cardiovascular Sciences, Imperial College London, London,
United Kingdom, 6Respiratory Medicine, Imperial College Healthcare NHS Trust, London, United Kingdom, 7University of Liverpool Medical School, Liverpool, United
Kingdom
Abstract
Background:
Iron deficiency anemia remains a major global health problem. Higher iron demands provide the potential for
a targeted preventative approach before anemia develops. The primary study objective was to develop and validate a
metric that stratifies recommended dietary iron intake to compensate for patient-specific non-menstrual hemorrhagic
losses. The secondary objective was to examine whether iron deficiency can be attributed to under-replacement of epistaxis
(nosebleed) hemorrhagic iron losses in hereditary hemorrhagic telangiectasia (HHT).
Methodology/Principal Findings:
The hemorrhage adjusted iron requirement (HAIR) sums the recommended dietary
allowance, and iron required to replace additional quantified hemorrhagic losses, based on the pre-menopausal increment
to compensate for menstrual losses (formula provided). In a study population of 50 HHT patients completing concurrent
dietary and nosebleed questionnaires, 43/50 (86%) met their recommended dietary allowance, but only 10/50 (20%) met
their HAIR. Higher HAIR was a powerful predictor of lower hemoglobin (p = 0.009), lower mean corpuscular hemoglobin
content (p,0.001), lower log-transformed serum iron (p = 0.009), and higher log-transformed red cell distribution width
(p,0.001). There was no evidence of generalised abnormalities in iron handling Ferritin and ferritin
2
explained 60% of the
hepcidin variance (p,0.001), and the mean hepcidinferritin ratio was similar to reported controls. Iron supplement use
increased the proportion of individuals meeting their HAIR, and blunted associations between HAIR and hematinic indices.
Once adjusted for supplement use however, reciprocal relationships between HAIR and hemoglobin/serum iron persisted.
Of 568 individuals using iron tablets, most reported problems completing the course. For patients with hereditary
hemorrhagic telangiectasia, persistent anemia was reported three-times more frequently if iron tablets caused diarrhea or
needed to be stopped.
Conclusions/significance:
HAIR values, providing an indication of individuals’ iron requirements, may be a useful tool in
prevention, assessment and management of iron deficiency. Iron deficiency in HHT can be explained by under-replacement
of nosebleed hemorrhagic iron losses.
Citation: Finnamore H, Le Couteur J, Hickson M, Busbridge M, Whelan K, et al. (2013) Hemorrhage-Adjusted Iron Requirements, Hematinics and Hepcidin Define
Hereditary Hemorrhagic Telangiectasia as a Model of Hemorrhagic Iron Deficiency. PLoS ONE 8(10): e76516. doi:10.1371/journal.pone.0076516
Editor: Dermot Cox, Royal College of Surgeons, Ireland
Received March 28, 2013; Accepted August 27, 2013; Published October 16, 2013
Copyright: ß2013 Finnamore et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study received support from donations by families and friends of British patients with hereditary haemorrhagic telangiectasia, and internal
Bachelor of Science project funding (for HF). EPIC-Norfolk is supported by programme grants from the Medical Research Council UK (G9502233, G0300128) and
Cancer Research UK (C865/A2883). CLS and MH acknowledge support from the National Institute of Health Research Biomedical Research Centre Funding Scheme
(Imperial BRC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: c.shovlin@imperial.ac.uk
Introduction
Iron deficiency is one of the most common nutritional
deficiencies, globally affecting more than one billion people [1]
including 12% of US females aged 12–49 years. [2] In 2002, the
World Health Organisation estimated that 0.8 million (1.5%) of
deaths worldwide and 35 million healthy life years lost (2.4% of
global DALYs) were attributable to iron deficiency. [3] Iron has
multiple essential roles, being present in oxygen binding-molecules
such as hemoglobin and myoglobin, cytochromes, and many
intracellular enzymes. Low serum iron concentrations provoke a
hepcidin-dependent homeostatic response to co-ordinately in-
crease intestinal iron absorption and release iron from intracellular
stores, [4] but the latter are depleted in chronic iron deficiency
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which develops when iron demands and losses are not met by iron
intake. Demands are highest during growth and pregnancy; losses
are usually due to bleeding of which the most prevalent worldwide
are menstruation, gastrointestinal bleeding due to hookworm, and
urinary blood loss from schistosomiasis. [2][3] Normal menstrual
losses reaching 60–80 mls per month [5][6] are why the
recommended dietary iron intake for pre-menopausal females is
higher than for post-menopausal females and males: recom-
mended dietary allowances (RDA) are 18 mg/day and 8 mg/day
respectively in the US. [2] Inadequate dietary iron intake is
common in populations whose diets are predominantly based on
starchy foods with low meat intake, [3] but is also common in
developed countries: In the UK, dietary surveys indicate that up to
50% of women consume less than the national recommended iron
intake for males. [7][8] Despite this, current guidelines for
management of iron deficiency anemia do not emphasise poor
dietary intake as an identifiable cause of iron deficiency precluding
the need for further investigations [9].
Transfusional requirements and circulatory demands that result
from iron deficiency anemia are the best recognised of the diverse
detrimental consequences from iron deficiency: [1][2][3][10][11]
Anemic patients unable to sustain normal hemoglobin concentra-
tions have hyperdynamic circulations, with high cardiac output
accompanied by lower systemic vascular resistance. [12] Addi-
tional sequelae of iron deficiency include suboptimal immune,
skeletal muscle and thyroid function; prematurity; poor maternal
and perinatal outcomes in pregnancy; and impaired motor and
cognitive development in children. [1][2][3][10] Recently, new
disease associations of iron deficiency have been recognised,
including accentuation of hypoxic pulmonary hypertension, [13]
and in hereditary hemorrhagic telangiectasia (HHT), association
with venous thromboemboli (VTE), attributable to elevated
plasma levels of coagulation factor VIII, a VTE risk factor in
the general population [14].
Individuals with HHT are commonly iron deficient, generally
ascribed to chronic blood loss from gastrointestinal and nasal
telangiectasia. [14][15][16][17][18] More than half have visceral
arteriovenous malformations (AVMs), when they need to generate
supra-normal cardiac outputs to compensate for blood flow
through hepatic [18] and/or pulmonary [19] AVMs. The
additional burden from anemia is emphasised by the largest
prospective hepatic AVM series: [18] seven of the eight cases of
high output cardiac failure were precipitated by iron deficiency
anemia. [18] This mirrors findings in patients from the general
population with impaired cardiac reserve due to ischemic heart
disease [20][21].
The concepts that iron deficiency is insufficiently assessed and
managed, [22] and not addressed by all therapies used to
improve the oxygen carrying capacity in blood (such as
erythropoeisis stimulating agents [23]), have been emphasised
recently for the general population. [22][23] If anemia develops,
diet alone is considered inadequate as a source of iron; [22]
treatment is with oral iron supplements, and if insufficient or
poorly tolerated, iron infusions and/or blood transfusions.
[24][25][26] To supplement dietary intake, the World Health
Organisation recommends 60 mg/day of elemental iron to
prevent iron deficiency anemia in at-risk individuals, and
120 mg per day for iron deficient individuals in 2–3 divided
doses. [3] There are recognised dose-dependent side effects of
iron tablets, which were best demonstrated in a 1966 study of
1,496 subjects receiving iron tablets at conventional doses (180–
222 mg elemental iron/day) or placebo. [27] Calculations
performed for the current study indicate that side effects were
reported by 26.2% [95% CI 23.5,28.8] of iron users compared
to 13.2% [10.1, 16.3] in placebo groups (p,0.0001), with tablet
discontinuation due to side effects in 7.6% [6.0, 9.3] and 2.2%
[0.8, 3.5] respectively (p,0.0001) [27].
Strategies to optimise iron intake focus on achieving the RDA,
and treatment of existing anemia, when the iron deficit to be
replaced can be formally calculated utilising iron stores and
hemoglobin. [22] Specific groups at-risk of iron deficiency
(individual disease cohorts and pregnant women), have iron levels
and or hematinic indices monitored, but guidance from the Center
for Disease Control (CDC) and elsewhere is that no routine
screening for iron deficiency is recommended for men or
postmenopausal women [28][29].
Many individuals have higher iron demands than normal, and
this provides the potential for a targeted, informed approach for
prevention and/or treatment of iron deficiency at earlier stages,
even before anemia has developed. This may also be helpful for
individuals where ferritin, hemoglobin, and/or blood volume are
affected by concurrent pathologies such as inflammation [30][31]
or hypoxia. [19][32][33] The use of pre-emptive iron supplements
without proven iron deficiency is controversial, as evident from
discussions on management of blood donation (which lowers iron
stores [34][35]), with concern expressed because of the potential
risk of iron overload. [36] Except for menstruation and pregnancy,
recommendations on additional dietary iron intake are vague, and
not stratified according to those with the greatest iron losses.
[1][3][9][10][11]. As emphasised by Hallberg however, [36] iron
store kinetics imply it is difficult to very develop iron overload by
consuming diets with high dietary iron content, unless there is a
concurrent iron storage disorder. Additionally, enhanced dietary
intake is not reported to cause similar side effects to iron tablets.
We hypothesised that a stratified metric which defined dietary
iron requirements according to quantified hemorrhagic losses,
may be helpful in assessment, prevention and treatment of iron
deficiency anemia.
The HHT population potentially offered an ideal group in
which to evaluate such a metric against concurrent dietary iron
intakes, since nosebleeds, being both evident and usually external,
can be measured to quantify hemorrhagic iron losses.
[14][15][16][17][18][37] It was therefore important to formally
establish why HHT patients are iron deficient. Epistaxis (nose-
bleeds) is not mentioned in current guidance on evaluating iron
deficient individuals, [9] although 60% of the general population
experience nosebleeds, [38][39] reflecting the superficial and easily
traumatised multidirectional arterial anastomotic system in the
nose. [40] Significant gastrointestinal bleeding affects relatively
small proportions of individuals with HHT. [15][16][17][18] We
therefore also tested the hypothesis that iron deficiency may be
aggravated in HHT patients due to perturbed regulation of the
iron regulatory hormone hepcidin. [4] Plasma hepcidin concen-
trations should be lower in the setting of iron depletion, but are
inappropriately elevated in several disease states including
pulmonary arterial hypertension [41] which affects a small
subgroup of HHT patients with mutations in ACVRL1/ALK1
[16][17][42].
The study aims, to develop a metric that stratifies recommended
dietary iron intake to compensate for non-menstrual hemorrhagic
losses, and evaluate whether iron deficiency can be attributed to
under-replacement of hemorrhagic iron losses in HHT, were
achieved: Here we present the hemorrhage-adjusted iron require-
ment (HAIR), that, together with hepcidin/ferritin relationships
and hematinic validations, establish HHT as a model of
hemorrhagic iron deficiency.
Iron Deficiency, HHT and HAIR
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Materials and Methods
Ethics Statement
Named institutional review boards or ethics committees
specifically approved this study: The dietary and nosebleed study
was given a favorable Ethics opinion by the South West London
REC3 Research Ethics Committee (11/H0803/8). All participants
provided written informed consent. The survey study, named
‘‘HHT and other medical conditions’’ (NRES 12/EM/0073), was
given a favorable Ethics opinion by the NRES Committee East
Midlands- Derby 1 Research Ethics Committee on 2nd February
2012. All participants provided written or online informed
consent.
Literature Searches
At the outset of this study, it had been anticipated that an
existing method to stratify iron intake according to quantified
hemorrhagic losses would be available, but Pubmed searches for
the terms {‘‘iron’’ ‘‘intake’’ ‘‘hemorrhage’’ and either ‘‘adjust’’ or
‘‘stratify’’} repeated most recently on 26.06.2013, identified no
relevant articles other than a Cochrane review of iron intake in
pregnancy. More general searches were performed using the terms
‘‘iron’’ and ‘‘deficiency,’’ initially filtered by ‘‘Practice Guidelines’’
or by ‘‘Systematic Review’’. These identified 47 English language
articles that had been published since 1992. All were reviewed for
potential relevance to the current study. Three were not relevant.
25 articles referred to the management of iron deficiency in
specific disease settings, characterised by non-hemorrhagic states
of renal failure (n = 11); childhood (n = 5); helico pyloridus
infection (n = 4); Crohn’s disease (n = 1), bariatric surgery (n = 1);
thalassemia (n = 1); anemia of chronic disease (n = 1); and cancer
(n = 1). An additional five articles referred to evidence for cancer
screening (n = 2) or diagnostic yields from enteroscopy; colonos-
copy; or gastrointestinal bleed investigations (1 each). Pubmed
searches were expanded to ‘‘iron’’ ‘‘deficiency’’ ‘‘guidelines.’’ This
retrieved 330 English language articles published since 1976, and
131 in the last 5 years. Again, most referred to management in
pregnancy, renal disease, pediatric populations, cancer and
guideline development/compliance. It was concluded that there
were no appropriate existing indices, and the HAIR metric was
devised.
Key data and opinion articles indicate that in hemodynamically
tolerated iron deficiency anemia, optimal management with oral
iron is generally favoured to avoid parenteral therapy. [22][25] A
Pubmed search for iron tablets side effects identified 180 articles,
most referring to pregnancy or poisoning, but we were aware from
our clinical practice of the difficulties non-pregnant patients
experience when using iron tablets as prescribed. Published
Table 1. Demographics and personal HAIR calculations for the 50 dietary study participants.
Binary demographic variables N % (95% CI)
Gender, male 28 56 (41.8,70.3)%
Post-menopausal females 17 34 (20.4, 47.6)%
Pre-menopausal females 5 10 (1.4, 18.6)%
Achieving RDA for iron, overall 40 80 (68.5, 91.5)%
Males/Post-menopausal females (n of 45) 40 88.9 (79.3, 98.4)%
Pre-menopausal females (n of 5) 0 0% (0)
ESS3: Nosebleed quality gushing or pouring 17 34 (20.4, 47.6)%
ESS4: Medical attention for nosebleeds 23 46 (31.7, 60.3)%
ESS5: Currently anemic 13 26 (13.4, 38.6)%
ESS6: Transfused for nosebleeds 12 24 (11.7, 36.3)%
Continuous demographic variables Median Range [interquartile range]
Age (years) 55 20–80 [48.8, 67.3]
Iron intake overall (mg/day) 11 6.3–20.6 [9.6, 13.5]
ESS1: Number of nosebleeds (per month) 17.7 0–70 [3.5, 70]
ESS2: Nosebleed duration (mins) 2.5 0.5–23 [2.5, 10]
ESS Final score* (units) 4.4 0–9.1 [2.6, 5.4]
Calculated losses and requirements Median Range [interquartile range]
Nosebleed losses (ml per month), median, range (IQR) 276.7 0–12,719 [21.0, 1,398]
Extra dietary iron requirements (mgs/day), median, range (IQR) 21.1 0–970 [1.6, 107]
HAIR (mg/day), median, range (IQR) 29.1 8.0–978 [13.4, 115]
Dietary iron shortfall (mg/day), median, range (IQR) 17.0 210.3–967 [0.008, 103]
General, dietary intake and nosebleed demographics and derivatives, calculated from the FFQ (Table S1) and raw data for nosebleeds (see Figure 2). RDA, recommended
dietary allowance (8 mg for males and post-menopausal females, 18 mg for non pregnant pre-menopausal females); 95% CI, 95% confidence intervals; ESS, epistaxis
severity score, where number refers to ESS question number. [50] *The final ESS score ranges from 0–10, where a higher score equates to greater blood losses. Dietary
iron intake was no higher in pre-menopausal females (Kruskal Wallis pvalue 0.22), none of the pre-menopausal females achieved their recommended iron intake, and
the difference in the proportion of pre-menopausal females and males/postmenopausal females meeting their RDA was statistically significant (p,0.001, Mann
Whitney). Male gender weakly correlated with a higher dietary iron intake, but once corrected for gender, individuals with longer nosebleeds tended to have higher
dietary iron intakes (data not shown).
doi:10.1371/journal.pone.0076516.t001
Iron Deficiency, HHT and HAIR
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strategies to reduce gastrointestinal side effects include calculating
the iron deficit to be replaced, [22] and use of different
preparations, [22][25][27][43][44][45][46][47] lower dosages,
[22][45] or wider dose-spacing. [22][45] However, the best
primary data on iron tablet side effects were found in the 1966
Hallberg paper, [27] for which no online Abstract or data were
available. This manuscript was obtained from the British Library
archives, and the published data re-analysed for presentation in a
format more accessible for a modern readership. The article was
also used for earlier source references.
UK service costings for investigations were obtained from
national costing reports. [48][49] In the UK, colonoscopies cost
£577, an average computerised tomography scan £100–172, [48]
and out-patient appointment costs averaged £147 (IQR £101,
£171) [49].
Developing the Hemorrhage Adjusted Iron Requirement
(HAIR)
The hemorrhage-adjusted iron requirement (HAIR) was
calculated as the sum of the normal recommended dietary iron
intake, and requirements to compensate for non-menstrual blood
losses. To replace normal menstrual losses, [5][6][46] premeno-
pausal females are advised to consume substantially more iron
than post-menopausal females or men: In the US, the Recom-
mended Dietary Allowance (RDA) for premenopausal women is
18 mg/day, increasing to 27 mg/day when pregnant, compared
to 8 mg/day for men and post-menopausal women. [2] Menstrual
losses average 35–50 ml/month, but may reach 60–80 ml/month
in the normal setting. [5][6] Abnormal menstrual flow (menor-
rhagia) is defined at flows exceeding 80 ml/month. The additional
increment of daily dietary iron recommended for non-pregnant,
pre-menopausal women was therefore taken to represent that
required for 80 ml of blood losses per month. In the US, this ‘‘pre-
menopausal increment’’ equates to (18–8)mg/day = 10 mg/day,
[2] though the principle can be extrapolated to any set of
recommended nutrient allowances.
Blood losses are often acute, but dietary compensation takes
place over many weeks. Using the paradigm of blood donation
recommendations, where blood is usually donated no more than
once every 3 months, and the recently validated epistaxis severity
score, [50] the HAIR formula covers an equivalent 3 month
period for rectification of losses. If there was a single blood loss in a
3 month period, for example following surgery or blood donation,
then blood losses for months 2 and 3 would be zero:
HAIR~Usual diet recommendation
zEstimated blood loss volumes in 3 months
80 (ml=month) x 3

x Pre-menopausal increment
e:g:Using RDA :
HAIR ~RDA (8 or 18 mg=day)
z(
Estimated extra blood losses (ml) in
month 1zmonth 2zmonth 3
240ml (f or 3 months)
0
B
B
B
@
1
C
C
C
C
A
x10mg=day)
The usual dietary recommendation is determined by whether
the individual is a premenstrual female with the higher RDA (or
country-specific equivalent). For personalised HAIR calculations
for the 50 participants in the validation survey, nose bleed volume
losses per month were calculated from minutes of bleeding per
month, which were converted to volumes per month according to
reported intensity, and a survey of timed nose bleeds of known
volume (see below).
Figure 1. Actual and recommended dietary iron intakes for the
50 study participants. A) Current recommendations using US
recommended dietary allowance (RDA [2]) values for iron (blue dropped
line circles, at 8 or 18 mg/day): the left hand five datasets, with higher
RDA values, represent the five premenopausal women, the remaining
45 datasets with lower RDA values represent males and post
menopausal females. Red columns indicate each individual’s iron intake
per day from their personalised food frequency questionnaire (FFQ,
intake of 130 food items presented in Table S1). Note that the RDA was
not met by any of the pre-menopausal females. B) HAIR recommen-
dations: The same intake data as in A) are now illustrated on a natural
logarithmic scale to allow presentation of each individual’s personalised
HAIR value, calculated according to their personalised iron losses, and
US based recommended dietary allowance (RDA) for iron, presented in
Table 1. Note that a log(HAIR) of 3 corresponds to a HAIR of 20 mg/day
(approximate needs of a male blood donor); a log(HAIR) of 4 to a HAIR
of 55 mg/day (approximate needs over 3 months to replace a 3–4 g/dl
drop in hemoglobin), and a log(HAIR) of 5 to a HAIR of 148 mg/day.
Generally short (0.5–2.5 min) nosebleeds less than once per month
resulted in log(HAIR) of approximately 2; several nosebleeds per week
of 5 minutes or more in a log(HAIR) of ,3; daily 10 min nosebleeds a
log(HAIR) of ,4, and several nosebleeds per day, each lasting 2.5–10
minutes, in a log(HAIR) of 5.
doi:10.1371/journal.pone.0076516.g001
Iron Deficiency, HHT and HAIR
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The calculations were then repeated for published average
volume losses in common clinical settings, [51][52][53] based on
the clinical rule of thumb that one unit of blood raises the
hemoglobin by 1 g/dl in the non-bleeding adult patient [51].
HHT Diet and Nosebleed Questionnaire Study
Study design. A study to formally capture dietary intake in
relation to hemorrhagic losses in hereditary hemorrhagic telangi-
ectasia (HHT) was designed in late 2010, incorporating the newly
published Epistaxis Severity Score. [50] Inclusion criteria were a
definite diagnosis of HHT [54] and ability to give written,
informed consent. Only one person per household could
participate.
Population recruitment. Potential participants were recruit-
ed between 18
th
April and 3
rd
September 2011, primarily through
the Imperial College London HHTIC London Clinical Service,
either during attendance at clinic (N = 25), or by post, targeting the
44% of individuals in the clinic database described by Livesey et al
[14] with a recorded ferritin ,20 mg/L, or serum iron of less than
7mmol/L (N = 24). One individual was also recruited by
advertisement through the UK Telangiectasia Self Help website
on the same page (http://www.telangiectasia.co.uk/) as the
dietary iron advice sheet available since 2002. Ethical approval
permitted recruitment of international HHT patients through the
Kemer HHT Meeting in April 2011, but dietary intakes proved
too different to food items in the EPIC FFQ to permit analyses,
and the three Turkish dietary/epistaxis data sets were not
included. 62 consenting British individuals provided basic
demographic information and were given study questionnaires:
all 50 (81%) who completed and returned their questionnaires
were included in this study.
Quantification of nosebleed losses. Nosebleed losses were
quantified using Epistaxis Severity Score (ESS) methodology. [50]
Participants provide characteristics of their typical nosebleeds
within the previous three months stratified by typical frequency,
duration and intensity. Tick box options provided for typical
frequency were less than monthly, once per month, once per week,
several per week, once per day, or several per day. Options
provided for typical duration (in minutes) were less than 1, 1–5, 6–
15, 16–30 or more than 30. Options provided for intensity were
‘‘typically pouring or gushing’’ or ‘‘typically not pouring or
gushing’’. The ESS score incorporates three further tick box
questions regarding medical attention for nose bleeds, current
anemia, and transfusional requirements, and has been validated as
an objective measure of nosebleed severity. [50] The current study
utilised raw ESS data of typical frequency per month, typical
duration, and typical intensity. To calculate blood volume lost per
month, average nosebleed duration (in minutes) was multiplied by
Figure 2. Raw data on nosebleed frequency and duration.
Typical number of nosebleeds per month (blue symbols/lines, data from
ESS question 1), and typical duration of nosebleeds per month (red
symbols/lines, data from ESS question 2) reported by the 50 study
participants, ordered by increasing value of HAIR. Nosebleeds reported
as ‘‘typically gushing or pouring’’ were significantly longer than
nosebleeds reported as ‘‘typically not gushing or pouring’’ (mean
[standard deviation] 8.9 [6.4], versus 4.5 [4.9] minutes, Mann Whitney
p = 0.0038).
doi:10.1371/journal.pone.0076516.g002
Figure 3. Details of the nosebleeds reported by the online survey respondents. A) Reported volume (mls) of individual nosebleeds,
converted where appropriate from original units of measurement to mls as described in the methods. B) Reported duration (minutes) of individual
nosebleeds. Corroborating evidence for specified major bleeds was provided by 16 individuals, and included acute hemodynamic consequences
(faints, collapses, n = 5); hematocrit/hemoglobin falls (n = 4 including 3.2 g/dl hemoglobin fall in 8 hours; 8 units of hematocrit over 3 days); and
unspecified acute transfusions or hospital admission (n = 8). There was no corroboratory evidence for the two indicated outliers (red crosses) whose
values were excluded from calculations for the median, 20
th
and 5
th
percentile values used in nosebleed rate conversions.
doi:10.1371/journal.pone.0076516.g003
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Figure 4. Iron intakes from diet and iron supplements. The nine individuals using ferrous sulphate, ferrous gluconate, ferrous fumarate or
other iron supplements, are illustrated with pairwise comparisons of their dietary iron intake from the FFQ (left red bar) and dietary FFQ intake plus
their supplement iron intake (right red bar) in addition to each individual’s log(RDA) and personalised log(HAIR), which were calculated according to
their personalised iron losses and RDA for iron. As in Figure 1, note that generally short (0.5–2.5 min) nosebleeds less than once per month resulted in
log(HAIR) of approximately 2; several nosebleeds per week of 5 minutes or more in a log(HAIR) of ,3; daily 10 min nosebleeds a log(HAIR) of ,4, and
several nosebleeds per day, each lasting 2.5–10 minutes, in a log(HAIR) of 5. The two highest intakes were seen in individuals using ferrous sulphate
325 mg bd; the next six in users of once daily ferrous sulphate or ferrous fumarate.
doi:10.1371/journal.pone.0076516.g004
Figure 5. Stratification of blood hematinic and iron indices according to HAIR values and iron supplement use. A–E: Distributions of
all participants, either by conventional recommended dietary allowance (RDA, left two box plots (mens., menses distinguishing males and post
menopausal women from pre-menopausal women), or by approximate quintile (Qu) determined by HAIR (right five box plots, exact figures provided
in methods). A) HAIR values (mg of iron per day). B) Serum iron (mmol/L). C) Hemoglobin (Hb, g/dL)). D) Mean corpuscular hemoglobin
concentration (MCHC, g/dl). E) Red cell distribution width (RDW). F) Quadratic regression plots for the distribution of hemoglobin according to HAIR,
in study participants using oral iron supplements (continuous line with 95% confidence interval indicated), and those who did not (dotted line).
doi:10.1371/journal.pone.0076516.g005
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average number of bleeds per month, and by the rate appropriate
to the reported intensity of bleeding. The ESS nosebleed intensity
descriptions of ‘‘typically pouring or gushing’’ or ‘‘typically not
pouring or gushing’’ effectively distinguish arterial and non arterial
bleeding. There were no published data that formally captured the
rate of nasal blood loss in these settings, although two of the
dietary study participants reported nosebleeds of 270 ml in 30
minutes (9 mls/minute), and 1500 ml in approximately 2 hours
(12.5 mls/minute) respectively. A larger dataset of timed nose-
bleeds of known volume was obtained through the Imperial
2012 HHT Survey described below. For conversions, the rate
assigned for ‘‘typically pouring or gushing’’ nosebleeds was 7.9 mls
per minute, and the rate for ‘‘typically not pouring or gushing’’
was 2.3 mls per minute (for derivations of these values, see below).
Dietary iron intake. Dietary iron intake was quantified
using the European Prospective Investigation into Cancer (EPIC)
food frequency questionnaire. [55] Questions are asked about the
frequency of consumption of 130 food items over the previous
year, methods of cooking, and use of dietary supplements. Raw
data were entered into a validated EPIC software programme, the
Compositional Analysis of Frequency Estimates (CAFE), to give
estimated dietary iron intake. [56] This robust dietary intake
assessment method has good intra-rater reliability [57].
Blood samples. Blood indices were measured concurrently
(n =39) or at the closest timepoints measured in the clinical service
(n = 9). These measured the serum hemoglobin (Hb), hematocrit,
mean corpuscular volume (MCV), mean corpuscular hemoglobin
(MCH), mean corpuscular hemoglobin concentration (MCHC),
red cell distribution width (RDW), serum iron, serum transferrin
saturation index (TfSI), and serum ferritin. In addition to the
above clinic assessments, 21 individuals (12 males and 9 females)
consented to have research blood sampling which measured
bioactive hepcidin peptide [4] by competitive radioimmunoassay
[58].
Imperial 2012 HHT Survey
To capture large numbers of nosebleed volume/time relation-
ships in HHT, an online survey was conceived by CLS and HF
during the summer of 2011. Recognising the overlapping
demographic questions for same group of respondents, method-
ologies available through SurveyMonkey, and potential benefits of
pooling questionnaires to reduce bias, CLS incorporated the
nosebleed questions, and iron tablet tolerance questions relevant to
the current study, into a wider survey. Following ethical approvals,
the study went live on line on 6th April 2012, accessed through a
specially designed Imperial College entry page at www.imperial.
ac.uk/medicine/HHTsurvey2012. Potential participants were
recruited through the Imperial College London HHTIC London
Clinical Service databases by post; during attendance at the HHT
clinics; and by advertisement by the HHT Foundation Interna-
tional. [37] As recently demonstrated for nosebleed precipitants
[37] and antiplatelet/anticoagulant therapies, [59] subsections of
the survey addressed different aspects of health and treatments for
people with HHT and general population controls. Answer and
question logic was applied so that individuals were only directed to
questions relevant to them. Overall, of 172 questions, most
respondents were directed to between 52 and 107 questions.
To obtain the data to determine the rates of blood loss in HHT
nosebleeds, respondents with HHT were asked ‘‘If you have ever
measured how much blood you lost in a timed bleed (‘‘when you
knew how long it lasted) please tell us here:’’ Reported Imperial
and US cup measurements for nosebleeds of known duration were
converted to mls via metricconversions.org; specific filled contain-
ers such as proprietary cups or cans via known volumes, and where
hemoglobin drops over a matter of hours were available, a drop of
of 1 g/dl was estimated as 750 mls of blood by back calculation
from cross match requirements for surgical blood losses [51].
Additional general and HHT demographic question survey
responses which were included in the current study were age,
gender, and for HHT-affected respondents, whether they had
required any specialised treatment for HHT, with tickboxes for
specialised treatments for nosebleeds, skin telangiectasia, gastro-
Table 2. Quadratic regression of hematinic and iron indices with HAIR.
Age and gender-adjusted Age, gender and iron supplement-adjusted
Variable Coefficient (95% CI) Pseudo R
2
p value p value
Hemoglobin 20.0038 (20.0066, 20.001) 0.12 0.009 0.009
Hematocrit 20.000062 (20.0005, 0.000022) 0.08 0.15 0.15
MCV 0.0023 (20.0074, 0.012) 0.002 0.64 0.73
MCH 20.0060 (20.012, 20.00055) 0.03 0.032 0.74
MCHC 20.0049 (20.0074, 20.0025) 0.1 ,0.001 0.032
ln(RDW) 20.00022 (20.0000355, 28.15e-06) 0.09 0.002 0.0971
ln(iron) 20.00081 (20.0014, 20.00021) 0.09 0.009 0.009
ln (TfSI) 20.00103 (20.0016, 20.00046) 0.1 0.001 0.001
ln(ferritin) 20.0021 (20.0014, 0.00095) 0.01 0.72 0.72
Age and gender-adjusted HAIR regression coefficients with the designated variable were calculated using the designated variable as the dependent variable
(distributional graphs are provided in Figure S1) for quadratic regression, and simultaneously examining the relationships with age, gender, and HAIR. MCV, mean
corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; ln(RDW), log-transformed red cell distribution width;
ln(iron), log-transformed serum iron; ln(TfSI), log-transformed transferrin saturation index; ln(ferritin), log-transformed serum ferritin. 48 datasets were available except
for RDW (n = 46), iron and TfSI (n = 45), and ferritin (n = 41). Excluding outliers in the MCV, MCH and MCHC regressions did not materially alter the results (p = 0.76 for
MCV, n = 44; p = 0.035 for MCH, n = 43 and p = 0.001 for MCHC, n = 36). Age, gender and iron supplement-adjusted HAIR regression coefficients with the designated
variable were calculated by quadratic regression, using the designated variable as the dependent variable, and simultaneously examining the relationships with age,
gender, iron supplement use, and HAIR. Iron supplements contributed significantly to the final models of MCHC and ln(RDW), reducing the respective HAIR p values in
age, gender and iron supplement adjusted regression.
doi:10.1371/journal.pone.0076516.t002
Iron Deficiency, HHT and HAIR
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intestinal/gut bleeding, pulmonary AVMs, cerebral AVMs, or
hepatic AVMs. Those stating they had received specialised
nosebleed treatments were directed to a question with tickboxes
for the most common specialised treatments for HHT nosebleeds
[37].
Any survey respondent who stated they had used iron tablets
(general population controls or HHT) was directed to non-biased
questions about tolerability, commencing with the question ‘‘If you
have used iron tablets, we want to know if these were easy to take,
or if they seemed to cause any problems. Please tick all that apply.
When I took iron tablets………’’ Participants were provided with
tick box options for (in order) i) ‘‘They were always fine to take and
caused no problems’’; ii) ‘‘They were sometimes fine to take, but
sometimes seemed to cause problems’’; iii) ‘‘I was able to complete
the course/continue with the iron as recommended’’; iv) ‘‘I had to
switch to a different type of iron supplement’’; v) ‘‘I had to stop
taking them’’; vi) ‘‘I felt sick (nauseous)’’; vii) ‘‘I had pains in my
stomach’’; viii) ‘‘I was constipated’’; ix) ‘‘I had diarrhoea’’. A
subsequent question asked ‘‘Have you still been anaemic despite
using iron supplements?’’ Participants were provided with tick box
options for (in order) i) ‘‘Yes’’; ii) ‘‘No’’; iii) ‘‘I’m not sure’’, and iv )
‘‘None of these options are applicable to me.’’
For the purposes of the current study, 90 day survey data were
downloaded on 12.6.2012. At the time of data download, of 913
individuals starting the survey, 756 (82.8%) had completed all of
their questions. Responses for respondents who declared that they
did not personally have HHT but had blood relatives with HHT
were only used as general population controls if there was no
declaration of HHT-related symptoms elsewhere in their survey
answers: 42 self-declared controls were reclassified as being of
unknown status, though none of these had used iron tablets.
Overall, 569 respondents stated they had used iron tablets, and
568 (521 with HHT and 47 general population controls) provided
responses to the subsequent questions on tolerability and, for HHT
respondents, persistent anemia.
Statistical Analyses
Power calculations. For the dietary study, pre-study power
calculations using blood indices for which standard deviations
were available (haemoglobin, [60] iron, [61] and transferrin
saturation index [61]) indicated that 48 recruits would provide
90% power to detect clinically significant differences in these
parameters between 24 participants stratified by higher and lower
iron intakes, using a two group t test with a 0.05 two sided
significance level. For the HHT survey, pre study power
calculations were based on the most difficult endpoints to
differentiate (differences in specific medical pathologies between
HHT patients and controls), resulting in the aim for 1000 HHT
responses, of whom only a proportion would be iron users.
Additional statistical details. All analyses were performed
using STATA IC version 11 (Statacorp, Texas), including
distributions of participant-specific variables, and two way
comparisons between groups by Spearman rank correlation and
Mann Whitney. Two way data plots presented individuals’ data
ordered by HAIR requirements, with relevant plots superimposed.
Multivariate adjusted regression coefficients and odds ratios were
calculated using stepwise linear, logistic or quadratic regression:
Logarithmic and inverse transformations were used to identify the
most appropriate dependent variables for regression.
For the approximate quintile distributions of HAIR, stratifica-
tion was made blinded to all other study parameters, and utilised
naturally occurring breaks between clusters to determine the
appropriate cut-offs. This resulted in four groups being larger or
smaller than the true quintile size of n = 10: Quintile 1:8.7–
10.4 mg/day (n = 12); Quintile 2:14.0–24.2 mg/day (n = 13);
Quintile 3:35.3–56 mg/day (n = 9); Quintile 4:114–121.5 mg/
day (n = 10) and Quintile 5:195–979 mg/day (n = 6).
For the survey data, variables entered into the logistic regression
model for persistent anemia if iron tablets were taken were: age;
gender; indications of hemorrhagic severity (need for any
nosebleed specialised treatment, that is nosebleed packs, cauter-
isation, laser therapy, septal dermoplasty, Youngs procedure, nasal
Table 3. Distribution of variables in 21 patients (12 males, 9 females) participating in iron regulatory study.
Demographic Normal range Median Range IQR
P value with (ln)
hepcidin
{
Age (years) – 41.0 20–69 23, 58 0.39
Iron (mmol/L) M 9–29; F 7–27 17.0 9–30 14,19 0.76
Transferrin saturation index (%) 20–45 29.0 12–46 26, 29.8 0.86
Ferritin (mg/L) M 20–300; F 10–120 54.0 6–203 33,88 –
Hepcidin (ng/mL) 2–55 24.0 3.1–87 9.2, 46 –
Hepcidin:Ferritin ratio
`
0.5 0.1–1.2 0.3, 0.6 n/a
Soluble transferrin receptor (sTfR),
nmol/L)
8.7–28.1 21.5 13.2–44.1 19.1, 24.5 0.59
sTfR/log Ferritin (Thomas ratio) 1.0 0.5–4.8 0.8, 1.5 0.32
Hemoglobin(g/dL) M 12.5–17; F 11.4–15 14.3 12.2–16.7 13.9, 15.9 0.51
Mean corpuscular volume, MCV (fL) 83–101 87.8 77.8–97.8 84.6, 90.3 0.69
Creatinine (mmol/L) M 60–125; F 60–110 74.0 59.-99 66, 81 0.61
eGFR (mL/min/1.73 msq) .59 87.0 9–95 76, 82.7 0.93
Albumin(g/L) 33–47 41.0 33–49 40, 43 0.24
M, males; F, female;eGFR, estimated glomerular filtration rate; n/a, not appropriate.
{indicates p value on separate addition to final model for hepcidin, once adjusted for ferritin and ferritin
2
.
`For the control group reported in [63] the geometric mean was 0.68 (95% confidence intervals 0.41, 0.96). In contrast, Piperno et al reported upper 95%
confidence intervals of the means for hemachromatosis heterozygotes and homozygotes as ,0.4 and ,0.2 respective ly [63].
doi:10.1371/journal.pone.0076516.t003
Iron Deficiency, HHT and HAIR
PLOS ONE | www.plosone.org 8 October 2013 | Volume 8 | Issue 10 | e76516

arterial ligation, or nasal arterial embolisation; or treatments for
gastrointestinal hemorrhage); other aspects of HHT status
(previous treatment of skin telangiectasia, pulmonary AVMs,
hepatic AVMs, or cerebral AVMs); need to stop iron tablets; need
to switch iron tablets; and iron tablet generation of diarrhoea,
abdominal pain, constipation or nausea. The least significant
variable was removed stepwise using the p values generated by
logistic regression until p = 0.10: subsequently, p values were
calculated post estimation, both by likelihood ratio tests (which
assume independence of observations within a cluster, an
assumption that was not met with these variables), and the non
parametric Wald test which does not make such assumptions.
Results
HAIR Calculations Reveal Substantial Dietary Iron
Shortfalls in HHT Study Participants
Dietary iron intake in relation to normal dietary
recommendations. The 50 dietary study participants com-
prised 28 males, and 22 females (17 post-menopausal, 5 pre-
menopausal), aged 20–80 (median 55) years. Detailed intake
distributions for more than 130 specified food items over the
previous year are reported in Table S1. Food items included meat
and fish products (17 types); bread and savory biscuits (5 types);
Table 4. Multiple regression of log transformed (ln) hepcidin.
Variable
Regression
coefficient (95% CI)
Standard
error p value
A)
Ferritin restricted
Ferritin 0.04 (0.023, 0.058) 0.0083 ,0.0001
Ferritin
2
20.00015
(20.00028, 20.000066)
0.000039 0.001
B)
Complete model
Ferritin 0.036 (0.028, 0.044) 0.0037 ,0.001
Ferritin
2
20.00011
(20.00015, 20.000076)
0.000017 ,0.001
Hepcidin:ferritin 1.88 (1.43, 2.34) 0.22 ,0.001
A) Model using ferritin alone. 95% CI, 95% confidence intervals. Overall model
parameters: sum of squares 17.2; 20 degrees of freedom; mean square 0.86;
variance ratio (F) 11.0; adjusted r
2
= 60.0; overall p value for model = 0.0001. B)
The final model for ln(hepcidin), generated by stepwise multiple regression in
data from 21 HHT patients. Overall model parameters: sum of squares 17.2, 20
degrees of freedom, mean square 0.86, variance ratio (F) 81.5, adjusted r
2
= 92.4,
overall p value for model ,0.0001. No other captured variable contributed to
the model.
doi:10.1371/journal.pone.0076516.t004
Figure 6. Relationships between hepcidin and iron indices. A and B) Hepcidin levels according to approximate tertile groupings of A) serum
iron, and B) serum ferritin. Note that the reference range for hepcidin using this radioimmunoassay is 1.1–55 ng/mL. [58] Details of individual
participants are provided in Table 4. C) and D) Best fit quadratic regression relationships (with shaded areas indicating the 95% confidence intervals)
for hepcidin with C) serum iron, and D) ferritin.
doi:10.1371/journal.pone.0076516.g006
Iron Deficiency, HHT and HAIR
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breakfast cereals (porridge or other cereals, for which the names of
the two most frequently ingested were provided); potatoes, rice,
and pasta (10 types); dairy products or fats (19 types, with details
about cooking methods); sweets and snacks (18 types); soups,
sauces and spreads (8 types); drinks (15 types); fruits (11 types); and
vegetables (26 types).
The diets provided a total iron intake ranging from 6.3–20.6
(median 11.3, interquartile range 9.6–13.3) mg of iron per day
(Table 1). With these diets, 43 of the 50 (86%) study participants
met their personal recommended intake (RDA) of 8 or 18 mg of
iron per day, based on their menstrual status (Figure 1A).
However, iron intake was no higher in pre-menopausal females;
none of the pre-menopausal females reached their RDA of 18 mg
of iron per day; and overall, only four of the study participants met
the higher RDA for pre-menopausal females.
Quantification of additional nosebleed hemorrhagic iron
losses. In addition to usual iron demands, the HHT study
participants needed to replace iron lost through HHT bleeds.
They reported an average of 17.7 nosebleeds per month
(interquartile range 3.5–7), with an average duration of 2.5
minutes (interquartile range 2.5–10; Table 1, Figure 2.) 17 of the
50 (34%) stated their nosebleeds were usually gushing or pouring
in nature, indicative of a higher rate of blood loss.
To derive blood loss rates in HHT, a larger dataset was
obtained through the online HHT Survey. Of the 756 survey
respondents at the time of data download, 141 reported either
duration (n = 112) or volume (n = 67) for specific HHT nosebleeds,
and 38 (5%) reported both indices for the same nosebleed
(Figure 3) The median volume lost was 473 mls (interquartile
range 100, 560 mls). The survey nosebleeds were of longer
duration (median 40 minutes [interquartile range 20, 90 minutes])
Table 5. Multivariate logistic regression of the risk of persistent anemia, if using iron tablets.
Variable Odds ratio (95% CI) Standard error z test LR test p value Wald test p value
Had to stop iron tablets 3.70 (1.68, 8.18) 1.50 3.25 0.0005 0.0012
Iron tablets cause diarrhoea 3.09 (1.45, 6.62) 1.20 2.91 0.0020 0.0036
Had to switch iron tablets 2.24 (1.25, 4.02) 0.67 2.70 0.0059 0.0070
Nasal septal dermoplasty performed 2.35 (1.30, 4.27) 0.71 2.82 0.0038 0.0048
Treatment for gastrointestinal hemorrhage 3.06 (1.61, 5.81) 1.00 3.42 0.0003 0.0006
Treatment for skin telangiectasia 1.70 (1.06, 2.72) 0.41 2.21 0.0266 0.0273
The final model presenting all variables making a significant contribution to the risk of persistent anemia, once adjusted for the presence of other variables, in HHT
online survey respondents using iron tablets. The model details 424 observations providing a pseudo r
2
of 0.14, and overall model pvalue of ,0.0001. P values were
calculated post estimation, both by likelihood ratio (LR) tests which assume independence of observations within a cluster (an assumption that was not met with these
data), and the non parametric Wald test which does not make such assumptions. There was no clear relationship between persistent anemia and iron tablet-induced
nausea, constipation, or abdominal pain (likelihood ratio test pvalues 0.14, 0.11, and 0.09 respectively). There was also no relationship with age, gender, other
otorhinolaryngologic treatments [37] either combined or individually, or other reported HHT treatments for pulmonary, cerebral or hepatic arteriovenous malformations
(data not shown).
doi:10.1371/journal.pone.0076516.t005
Table 6. HAIR values for typical clinical settings.
US values based on RDA, mg/day UK values based on RNI, mg/day
Setting
Approximate
volume loss
(ml)
Males or post
menopausal females
Pre menopausal
non pregnant
females
Males or post
menopausal
females
Pre menopausal
females
No additional losses
0 8 18 8.7 14.8
Blood donation every 3 months
470 27.6 37.5 20.6 26.7
Hemorrhagic hemoglobin fall* of
1 g/dl, e.g normal peri-partum loss [52] 470 27.6 37.6 20.6 26.7
2 g/dl e.g. TURP; colectomy, minor postpartum
hemorrhage [52]
940 47.2 57.2 32.6 38.7
3 g/dl e.g. moderate post partum hemorrhage [52] 1410 66.8 76.8 44.5 50.6
4 g/dl e.g. nephrectomy [51] 1880 86.3 96.3 56.5 62.6
Femoral head fracture
[
53
] 611 33.5 43.5 24.2 30.3
HAIR values were calculated by the formula given in the Methods, correcting over 3 months. *HAIR values can be extended to incorporate existing deficits manifest by
hemoglobin falls, although other guidance exists in this setting; [22] these figures exclude any hemorrhagic losses and hemoglobin falls that are corrected by
transfusions, since the transfused red cell iron content remains available for body stores. TURP, transurethral resection of the prostate. The reasons for the differences
between columns are that the recommended dietary iron intake for premenopausal females is substantially higher than for post-menopausal females and for men, and
because normal dietary intake recommendations vary between countries: In the UK, the respective iron Reference Nutrient Intake (RNI) values are 14.8 mg/day and
8.7 mg/day [64]. In the US, premenopausal women are advised to consume 18 mg/day, increasing to 27 mg/day when pregnant, compared to 8 mg/day for men and
post-menopausal women (recommended dietary allowance, RDA, as used in calculations presented in the current study). [2] UK HAIR values are therefore lower than
US-based HAIR values because of the lower recommendations for premenopausal women, and because the UK increment of 6.1 mg/day applies instead of the US
increment of 10 mg/day for premenopausal women. The principles and calculation can be extended to any other set of dietary allowances.
doi:10.1371/journal.pone.0076516.t006
Iron Deficiency, HHT and HAIR
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than the typical nosebleeds reported by the 50 dietary study
participants (p,0.0001), suggesting individuals in the online
survey were more likely to record and report timed volumes for
their more severe nosebleeds. The median rate of blood loss
(excluding the two outliers indicated in Figure 3) was 7.9 mls/
minute (interquartile range 4.7, 16.7 mls).
To convert dietary study nosebleeds to volume loss per month,
the median survey rate of 7.9 mls/min was used for nosebleeds
described as ‘‘typically gushing,’’ and 20th percentile value of
2.3 ml/minute for nosebleeds described as ‘‘typically not pouring/
gushing.’’ Using these conversion calculations, the 50 dietary study
participants reported median losses of 277 mls of blood per month
from their nose bleeds (interquartile range 21–1398 mls per
month). Further details of these demographics are presented in
Table 1.
Quantification of additional iron required to replace
nosebleed hemorrhagic iron losses. The hemorrhage-ad-
justed iron requirement was calculated for each individual by
summing the RDA determined by their menstrual status (8 or
18 mg/day), and additional requirements for their nosebleed
losses in mls per month. Their resultant hemorrhage adjusted iron
requirements (HAIR) averaged 29.1 mg of iron per day (inter-
quartile range 13.4, 115 mg/day).
Of the 50 study participants, only 10 (20%) met their HAIR by
diet alone. To illustrate graphically, individual HAIR values were
plotted for each study participant. As shown in Figure 1B, this
emphasised shortfalls in dietary intake for individuals who could
realistically meet their HAIR through diet (generally those with
typical nosebleeds occurring at most, several times per week). The
graphical representation also emphasises those with extreme
shortfalls due to nosebleeds once or several times per day, when
iron supplements would be essential to replace hemorrhagic iron
losses.
Nine study participants were using pharmaceutical iron
supplements (with elemental iron contents ranging from 20 to
65 mg) once or twice daily. The respective elemental iron content
was added to dietary intakes for pairwise comparisons. Figure 4
illustrates the resultant substantial increases in daily iron intake,
and that six of these nine individuals now met their HAIR.
HAIR Validation: Prediction of Hematinic Indices in HHT
In the HHT study group, there was no difference in HAIR
values according to the menstrual status determining the
recommended dietary allowance, or RDA (Figure 5A). There
was also no relationship between RDA, age, or gender with
hematinic or iron indices (data not shown).
For HAIR assessments, in order to illustrate trends, participants
were stratified into approximate quintiles of iron requirements (see
methods). HAIR values in the first quintile were similar to the
RDA for males and post menopausal females; HAIR values in the
second quintile similar to the RDA for premenopausal females. In
contrast to the relationships with the standard recommended
dietary allowance (RDA), stratifying participants by HAIR
quintiles revealed clear trends in serum iron indices (Figure 5B).
Trends were also apparent for hematinic indices such as
hemoglobin (Figure 5C), mean corpuscular hemoglobin concen-
tration (Figure 5D), and red cell distribution width (Figure 5E).
To more formally evaluate the relationships between HAIR and
iron/hematinic indices, stepwise quadratic regression analyses
were performed using log-transformation where appropriate to
generate more normally distributed dependent variables (Figure
S1). In age and gender-adjusted quadratic regression, higher
HAIR was a powerful predictor of lower log-transformed serum
iron (p = 0.009), lower hemoglobin (p = 0.009), higher red cell
distribution width (p,0.001), and other hematinic parameters
(Table 2).
There was a trend for hematinic and iron indices in participants
with higher HAIR values using oral iron supplements to more
closely resemble indices from participants who bled less but did not
use iron supplements (Figure 5F). However, relationships between
higher HAIR and hemoglobin/iron persisted after adjustment for
iron supplement use (Figure 5F; Table 2). This was despite the fact
that previous blood transfusions had been received by 12
participants (four of the oral iron supplement users, and eight of
the study participants not using iron supplements).
Hepcidin Levels are Appropriate for Iron Stores in HHT
21 of the dietary study participants consented to additional
research blood sampling for hepcidin analyses. These 12 males
and 9 females aged 20–69 (median 41) years displayed a wide
range of iron indices and hematinics (Table 3). Normal iron
metabolism handling relationships predict that hepcidin should be
lower in the setting of lower iron stores, [4] and in these 21
individuals, hepcidin levels appeared to be lower in patients with
lower serum iron and ferritin concentrations (Figure 6).
To evaluate formally, stepwise multiple regression were
performed, using log transformed (ln)hepcidin as the dependent
variable because this displayed a more normal distribution than
hepcidin. As shown in Table 4A, 60% of the variance in
ln(hepcidin) was explained by a model incorporating only ferritin
(p,0.0001) and its higher order variable, ferritin
2
(p = 0.001). This
proportion of 60% is almost identical to the proportion of hepcidin
variance explained by ferritin levels in the control population
reported by Busbridge et al. [58] The bulk of the remaining
variance (32.4% of the total variance in hepcidin levels) was
explained by the hepcidin:ferritin ratio (Table 4B). This ratio is
reduced in patients with liver disease [62] (hepcidin is synthesised
in the liver), [4] and modified in iron storage disorders such as
hemachromatosis. [63] In the HHT cohort, there was a wide
range of hepcidin:ferritin ratios (Table 2). Importantly, the
geometric mean and 95% confidence intervals (0.49 [95%
confidence intervals 0.35, 0.62]) corresponded to the hepcidin:-
ferritin ratio values reported for the control group studied by
Piperno et al. [63] Since the relationships between ferritin and
hepcidin in the HHT patients were very similar to those reported
for general population controls, [58][63] we concluded there was
no evidence to suggest a generalised abnormality of iron handling
in the HHT cohort.
A Role for HAIR Even if Iron Supplements are Prescribed
Eight study participants who had previously been transfused
were not using iron supplements, 46/50 (92%) study partici-
pants could have met their HAIR using twice daily oral iron
supplements (Figure 1),and HHT patients frequently reported
poor tolerance of iron tablets in clinic. These data prompted a
formal assessment of iron tablet tolerability which was
performed as part of the online survey used to obtain timed
nosebleed data. Of the 568 respondents who stated that they
had used iron tablets, barely half reported that iron tablets were
always or sometimes fine to take (280/521 HHT patients
(53.7%), 28/47 healthy controls (59.6%), p = 0.44). Very high
proportions reported constipation (218/568, 38.3%); nausea
(131/568, 23.1%) or diarrhea (60/568, 10.6%), with no
difference between the healthy controls and HHT patients.
Once adjusted for indices of HHT hemorrhage, persistent
anemia was reported three times as frequently by HHT patients
reporting diarrhoea, and almost four times more frequently if
iron tablets needed to be stopped (Table 5). We concluded that
Iron Deficiency, HHT and HAIR
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there was a role for guidance of dietary iron intake, even if iron
tablets were prescribed.
Application of HAIR to Common Clinical Settings in the
General Population
Having validated HAIR as a predictor of hematinic indices in
the HHT population, and demonstrated that iron handling
appeared to be normal in this specific hemorrhagic condition,
HAIR values were calculated for blood loss volumes relevant to
common clinical settings in the general population, such as post
partum bleeding, [52] surgery, [51] fractures, [53] and blood
donation. Table 6 illustrates the HAIR-based recommendations
for iron intake for the general population, based on two different
national sets of recommended dietary intakes: In the US,
premenopausal women are advised to consume 18 mg/day,
compared to 8 mg/day for men and post-menopausal women
(RDA, as used in calculations presented in the current study). [2]
In the UK, the respective iron Reference Nutrient Intake (RNI)
values are 14.8 mg/day for premenopausal women, and 8.7 mg/
day for men and post-menopausal women [64].
Discussion
In this study, an innovative approach and new metric revealed
extreme shortfalls in dietary iron intake that were not apparent if
dietary iron intake was only assessed using usual iron dietary
requirements. In the study population with HHT, iron-deficient
hematinics were predicted by the hemorrhage-adjusted iron
requirement (HAIR). Since serum hepcidin concentrations were
appropriate for ferritin levels, a generalised derangement in iron
handling seems highly unlikely, suggesting that iron deficiency in
HHT can be attributable purely to under-replacement of
hemorrhagic losses. Because the HAIR validations were per-
formed in a rare disease population, this may suggest that HAIR is
relevant only to a relatively narrow audience of clinicians caring
for HHT patients, but this is not the case. The concepts are
generic, and we simply exploited the quantifiable hemorrhagic
losses of nosebleeds to provide data that has been undeveloped for
many decades in internal medicine [5][6][46].
The strengths of the overall study included selection of a single
patient cohort with evident and quantifiable chronic hemorrhagic
iron losses; use of the currently accepted differential for dietary
iron intake to compensate for menstrual blood losses; participant
awareness of the need for iron-rich diets (evidenced by the higher
proportion meeting their recommended dietary intake than other
UK studies [7][8]); and the study participants’ marked hemor-
rhagic iron losses which allowed a clear differential between their
ability to meet usual recommended dietary intakes, and HAIR.
The HHT survey benefited from targeting a group with high
proportions requiring long term iron therapy, and highly
motivated to participate in research studies as evidenced by the
rapid response rate.
The survey of self-reported tolerance or iron tablets and anemia
is the weakest component of the current study, particularly as it did
not include a placebo group for medications where Kerr and
Davidson elegantly demonstrated suggestibility of side effects. [65]
The data are included because they provide a further large series
that suggests high proportions of patients have to limit or modify
their iron treatments, and that poor toleration of iron tablets is
associated with persistent anemia. Since actual blood loss from
nosebleeds may be greater than stated due to hemorrhaged blood
that is swallowed, the nosebleed-based estimation may be an
underestimation for some patients. A minor weakness of the study
was that the rate of each individual bleeding event was not
available, and instead calculated from patient reports of timed
nosebleed volumes, applied according to reported intensity which
effectively distinguishes arterial and non arterial bleeding.
Importantly, using even the 5th percentile value (0.3 mls per
minute) in conversion of all nosebleed volumes, still resulted in
only 22 individuals meeting the resultant partially-adjusted iron
requirements. The proportion of dietary iron absorbed differs in
unpredictable ways according to factors such as genetic variability
in iron handling genes, iron status/hepcidin levels, [4] and co-
existent dietary intake of iron absorption modifiers such as
phytates, oxylates and polyphenols. [36] By basing the HAIR
calculation on the currently accepted differential for iron intake to
compensate for menstrual blood losses, we were able go some way
towards bypassing the uncertainties of individual iron absorption
rates, but this would be useful to address in future studies.
We suggest that the HAIR metric will be particularly helpful for
predictive recommendations, for patients in whom ferritin and
hemoglobin are affected by pathologies other than iron deficiency,
and as an accessible tool to use in clinic. It is very difficult to
consume diets containing 20 mg of iron per day without careful
dietary planning: in the current study the greatest dietary iron
intake achieved was 20.6 mg/day. Figure 1 suggests patients with
nosebleeds occurring less than daily (HAIR values ,20 mg/day;
log (HAIR) ,3) may be reasonably expected to achieve their extra
iron intake through diet alone, but daily nosebleeds are likely to
require formal iron supplements, whether or not there are any
additional hemorrhagic losses. This is important for clinicians as
epistaxis is not considered an important clinical question when
evaluating iron deficient individuals. The rich nasal plexi of vessels
required for the physiological warming and humidification of
inspired air run superficially within the nasal mucosa, and are
easily traumatised by dry, cold inflow, finger damage, and
constituents of nasal sprays. Nosebleeds are common [38] and
are more frequent and prolonged for individuals with hyperten-
sion, coagulopathies or using anti-platelet [39] and anti-coagulant
therapies. There is a tendency to assume that chronic anemia-
precipitating hemorrhage must be from occult gastrointestinal
bleeding. But the superficial blood supply of the nose is a rich
multidirectional arterial anastomotic system, [40] thus represent-
ing a significant potential site of bleeding, not captured by current
medical histories.
Within the HHT population, clearly there will be some
individuals where HAIR will offer lesser corrective opportunities
because of chance co-inheritance of concurrent iron handling
defects. There have also been theoretical HHT-specific concerns
regarding aberrant hepcidin regulation, but it is intriguing to note
that hepcidin perturbations would operate in different directions:
Inappropriately high hepcidin concentrations are found in
pulmonary arterial hypertension [41] which is usually caused by
mutations in BMPR2, but also affects a small subgroup of HHT
patients with mutations in ACVRL1/ALK1 [42]. Furthermore, the
proteins mutated in HHT patients can associate with bone
morphogenetic protein receptors (including BMPR2), that bind
BMP6, a master regulator of hepcidin. [4] However hepatic
arteriovenous malformations are also more common in the
ACVRL1/ALK1 HHT patient subgroup. [16][17][18] While
liver synthetic function is generally preserved, in late stage hepatic
AVM disease, abnormalities in synthetic function and frank
cirrhosis may occur, [16][17][18] and would be predicted to
reduce the hepcidin:ferritin ratio as seen in other forms of liver
disease/cirrhosis [62].
For the general population, we suggest HAIR could also play
important roles in the prevention and management of iron
deficiency as it does not require blood test results for calculation:
Iron Deficiency, HHT and HAIR
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The CDC does not recommend routine iron screening blood tests
for men or postmenopausal women [28][29], but symptomatic
presentations and incidental discovery carry attendant health care
burdens - for example, iron deficient patients are more likely to
require unplanned transfusions following elective surgery. [24]
Prospective application of the HAIR concept may therefore be
helpful for both clinicians and patients to reduce later morbidity.
HAIR may also have a retrospective role, in the assessment of iron
deficiency of unknown cause: In current guidance, poor dietary
intake is not always emphasised as an identifiable cause of iron
deficiency precluding the need for further gastrointestinal inves-
tigations to identify sources of occult blood loss. [31] This means
that patients may be referred for expensive investigations when a
simple assessment of iron intake and subsequent advice may
resolve both the cause and the deficiency. To address whether
adoption of HAIR will reduce iron deficiency rates and/or
invasive investigation costs, we suggest three randomised trials. In
the first, large post surgical cohorts could be prospectively
randomised to receive standard management, standard dietetic
advice, or dietetic advice incorporating HAIR values relevant to
their quantified surgical losses. The second could use a similar
approach to determine if HAIR calculations are useful for
prevention or treatment of iron deficiency due to menorrhagia,
which remains an important cause of iron deficiency in young
women. This would require estimation of excessive blood losses
beyond normal menses, but given there is little rigorous guidance
for iron replacement estimates in this clinical setting, the HAIR
formula may be helpful. Additionally, comparison of gastroenter-
ological referral rates and outcomes could be made in iron
deficient patients randomised to either standard referral practices,
or an initial HAIR-based dietary assessment.
In summary, hereditary hemorrhagic telangiectasia is confirmed
as a model of hemorrhagic iron deficiency, where nosebleed/iron
intake comparisons may be used to predict the severity of iron
deficient hematinic indices. There is a role for dietary guidance,
even if iron tablets are prescribed. We anticipate that wider
application of the HAIR metric, allowing clinicians to prospec-
tively adjust recommended iron intake to compensate for
hemorrhagic losses, may improve understanding, and reduce the
wider clinical and financial burdens of this very common
nutritional deficiency.
Supporting Information
Table S1 Portions of food items per day. Breakdown of
dietary intake in preceding year for 130 food items, as reported by
the 50 dietary study participants using the European Prospective
Investigation into Cancer (EPIC) food frequency questionnaire
(FFQ). (main article reference [55]) Questions are asked about the
frequency of consumption of 130 food items over the previous
year, methods of cooking, and use of dietary supplements, and are
presented in food groups: meat and fish products (17 types); bread
and savory biscuits (5 types); breakfast cereals (porridge or other
cereals); potatoes, rice, and pasta (10 types); dairy products or fats
(19 types, and details about cooking methods); sweets and snacks
(18 types); soups, sauces and spreads (8 types); drinks (15 types);
fruits (11 types); and vegetables (26 types). N: Number of
participants (of 50) reporting item; sd, standard deviation; min,
minimum; max, maximum.
(DOCX)
Figure S1 Normal quantile plots of dependent variables
used in regression analyses. In contrast to distributions for
(A) hemoglobin, and (B) hematocrit, distributions of other indices
were skewed. Normalisation was achieved by logarithmic
transformation of (C) iron, (D) transferrin saturation index (TfSI),
(E) ferritin, and (F) hepcidin. Distributions of mean corpuscular
volume (MCV), mean corpuscular hemoglobin (MCH), mean
corpuscular hemoglobin concentration (MCHC), and red cell
distribution width (RDW) remained skewed after logarithmic and/
or inverse transformation: The variables with distributions most
approximating to normality (G) MCV, (H) MCHC (and MCH,
distribution comparable, data not shown), and (I) lnRDW were
used for regression analyses, and regression results confirmed by
re-testing after exclusion of outliers (data not shown).
(TIF)
Acknowledgments
The authors are grateful to the NRES Committee London-West, East
Midlands-Derby 1 Research Ethics Committee and Imperial AHSC Joint
Research Compliance Office for efficient ethical reviews; the Telangiec-
tasia Self Help Group for advertising the dietary questionnaire study; and
Angela Mulligan and Amit Bhaniani of the EPIC-Norfolk Study team for
the use of FETA (Weta) FFQ EPIC Tool for Analysis. Dr Shovlin also
thanks members of the HHT Survey Research Team for sending out postal
questionnaires and entering data; and the HHT Foundation International
for advertising the online survey to their database.
Author Contributions
Conceived and designed the experiments: HF JLC MH MB KW CLS.
Performed the experiments: HF JLC MB CLS. Analyzed the data: HF MB
KW CLS. Contributed reagents/materials/analysis tools: HF MH MB
KW CLS. Wrote the paper: HF CLS. Performed the dietary study: HF.
Performed initial calculations: HF. Wrote the first manuscript draft: HF.
Assisted with patient recruitment: JLC. Assisted with dietary data
collection: JLC. Advised on dietary concepts and data analysis: MH
KW. Supervised the study: KW CLS. Performed hepcidin measurements:
MB. Advised on biochemical concepts: MB. Performed all statistical
analyses: CLS. Generated the figures: CLS. Reviewed and approved the
final article: HF JLC MH MB KW CLS. Guarantor of the data: CLS.
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