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Circulating Concentrations of “Free” Leptin in Relation to Fat Mass
and Appetite in Gastrointestinal Cancer Patients
A. Michael Wallace, Anne Kelly, Naveed Sattar, Colin S. McArdle, and Donald C. McMillan
Abstract: Recent studies have suggested that circulating con
-
centrations of leptin might play a role in cancer cachexia. In
the first part of the study, we compared circulating concentra
-
tions of free and total leptin, percent fat mass, and the inflam
-
matory markers C-reactive protein (CRP) and interleukin-6
(IL-6), together with appetite score, in age- and gender-
matchedhealthy controls (n = 11) and advanced gastrointesti
-
nal cancer patients (n = 26). In the second part of the study, the
same measurements were repeated before and after megestrol
acetate treatment of weight-losing gastrointestinal cancer pa-
tients (n = 10). Body mass index and percent fat mass were sig-
nificantly lower (P < 0.05) and IL-6 and CRP were signifi-
cantly higher (P < 0.05) in cancer patients than in controls.
There was no difference in the percentage of leptin bound in
the circulation between controls and cancer patients. Circu-
lating “free” leptin concentrations correlated with percent fat
mass in controls(r = 0.745, P = 0.008) and cancer patients (r =
0.600, P = 0.001). In cancer patients, circulating leptin con-
centrations, either free or total, were not correlated with IL-6
or CRP concentrations. When adjusted for fat mass, the circu
-
lating concentrations of free and total leptin were signifi
-
cantly lower in the cancer patients (P < 0.01). Megestrol ace
-
tate treatment significantly increased circulating free and
total leptin concentrations in the cancer patients (P < 0.05).
There was a significant positive correlation between the
change in circulating concentrations of free and total leptin
and the change in percent fat mass (r = 0.685, P < 0.05 and r =
0.661, P < 0.05, respectively). The results of the present study
indicate that the proportions of freeand bound leptin in the cir
-
culation do not differ between normal subjects and patients
with gastrointestinal cancer and in both groups are related to
fat mass. Furthermore, the increase in circulating leptin con
-
centrations after megestrol acetate treatment is not associated
with any alteration in leptin binding.
Introduction
Weight loss in advanced cancer patients is a major clinical
problem that reduces the efficacy of anticancer treatment, re
-
sults in loss of independence, and reduces the quality of life
(1,2). Little is known about the mechanisms involved, but it is
characterized by the disproportionate reduction of lean body
tissue (3–5). Clearly, loss of appetite may contribute to such
weight loss, and there is increasing evidence that the systemic
inflammatory response also plays an important role (2,4).
Body weight homeostasis is maintained by a series of
complex interactions between the brain (particularly, the hy
-
pothalamus) and the periphery involving the hormone leptin,
which is synthesized in and secreted from adipose tissue
(6–8). It has been suggested that increased circulating leptin
concentration decreases appetite and food intake, increases
energy expenditure, and is important for the regulation of
metabolism and body weight in humans (9).
According to this paradigm, low circulating leptin con-
centrations increase appetite and decrease energy expen-
diture, resulting in gain of fat mass. This occurs in the rare
condition of congenital leptin deficiency (10); in other patho-
logical situations, the role of leptin is less clear. In healthy in-
dividuals, it has been shown that circulating leptin concentra-
tions reflect fat mass (11). Furthermore, this relationship
between fat mass and total leptin appears to be maintained in
gastrointestinal cancer, where total leptin concentrations are
low and correlate with the low fat mass (12–14).
It is known that specific binding components for leptin
are present in the circulation (11,15–17), and one explana
-
tion for the apparent inability of a circulating low total
leptin concentration to increase appetite in cancer patients
might be a decrease in the leptin-binding component, re
-
sulting in high circulating concentrations of free leptin,
which is the form of leptin in cerebrospinal fluid (18). In
addition to measuring free leptin concentrations in a cross-
sectional study, this hypothesis may be more rigorously
tested in the context of a longitudinal study of treatment
with an appetite stimulant such as megestrol acetate (19).
To our knowledge, this approach has not been examined in
cancer patients.
In the present study, we investigated the relationship be
-
tween circulating concentrations of free leptin, appetite, fat
mass, and the systemic inflammatory response in gastrointes
-
NUTRITION AND CANCER, 44(2), 156–160
Copyright © 2002, Lawrence Erlbaum Associates, Inc.
A. M. Wallace, A. Kelly, and N. Sattar are affiliated with the University Department of Pathological Biochemistry and C. S. McArdle and D. C. McMillan
with the Department of Surgery, Royal Infirmary, Glasgow G4 0SF, UK.
tinal cancer patients. In addition, we examined the effect of the
appetite stimulant megestrol acetate on these relationships.
Materials and Methods
Subjects
Patients with histologically proven locally advanced or
metastatic gastrointestinal cancer were included in the study.
Cancer patients were weight stable (<5% gain or loss in body
weight, n = 13) or weight losing (>5% body weight loss, n =
13) over the previous 6 mo. No patient complained of moder
-
ate or severe dysphagia, and none had an obvious functional
obstruction to food intake.
After an overnightfast, height, weight, and percent fat mass
were measured, and an appetite score was recorded. A venous
blood sample was taken for the measurement of circulating
concentrations of leptin, leptin binding, interleukin-6 (IL-6),
and C-reactive protein (CRP). Similar measurements were
performed in healthy weight-stable subjects for comparison.
In addition, 10 weight-losing cancer patients (>5% body
weight loss overtheprevious6mo) were studied before and af
-
ter 6–12 wk of treatment with megestrol acetate BP (480
mg/day; Megace, Bristol-Myers Pharmaceuticals).
The study was approved by the local ethical committee.
All patients were informed of the purpose of the study, and all
gave written consent.
Methods
Total human leptin was measured by a validated “in-
house” radioimmunoassay (11). Briefly, test serum or leptin
standard was incubated with sheep antileptin antiserum (anti-
bodies generated against recombinant human leptin) and
radioiodinated (
125
I) leptin at 4°C for 16 h. Sepharose-don
-
key anti-sheep globulin was added to the tubes after incuba
-
tion, and the samples were reincubated for1hatambient
temperature. Free and bound fractions were then separated
by centrifugation and washed three times. The bound frac
-
tion was counted on a gamma counter. The intra- and inter
-
assay coefficients of variation were <7% and <10%, respec
-
tively, over the concentration range, and limit of detection
was 0.5 ng/ml.
Percent leptin binding in the circulation was determined
after separation of bound and free fractions by a validated
Sephadex G-100 gel filtration procedure (11). Briefly, serum
and radioiodinated (
125
I) leptin were incubated for 20–24 h at
4°C. Bound and free analyte were separated by gel filtration
chromatography, also at 4°C, on a Sephadex G-100 column
(1.5 × 40 cm). Eighty 1-ml fractions were collected, and each
was counted on a gamma counter. The free fraction peak was
eluted later and well separated from the bound fraction, and
the concentration of free leptin was calculated as follows:
free leptin (ng/ml) = total leptin (ng/ml, measured by radio
-
immunoassay) × %free leptin. The interassay coefficient of
variation was 4.7% for multiple (n = 18) analyses of a serum
sample in which bound fraction averaged 21.2% of total
leptin.
Human IL-6 was measured by an enzyme-linked immu
-
nosorbent assay kit (Diaclone Research, Cedex, France, as
supplied from IDS, Tyne and Weir, UK). A monoclonal an
-
tibody specific for IL-6 was provided coated onto the wells
of microtiter strips. During the first incubation, standards,
samples, and quality controls were incubated with the IL-6
antigen and a biotinylated monoclonal antibody specific for
IL-6. After the samples were washed, the enzyme
(streptavidin peroxidase) was added. After the samples
were incubated and washed to remove unbound enzyme, a
substrate solution that acts on the bound enzyme was added
to produce a colored reaction product. The intra- and
interassay coefficients of variation were <5% and <7%, re
-
spectively, over the sample concentration range. The limit
of detection of the assay was 2 pg/ml.
CRP was measured on an Olympus analyzer (model
AU5200, Olympus Diagnostic Systems, Eastleigh, UK) by
turbimetry after binding to a specific antibody. The limit of
sensitivity was 5 mg/l, and the intra- and interassay coeffi
-
cients of variation were 5% and 7%, respectively, over the
sample concentration range.
Appetite was measured using a 10-cm linear analog scale,
ranging from poor to good appetite (20).
Total body water was determined by bioelectrical imped-
ance (model 4000B, Xitron Technologies, San Diego, CA).
The error of themethod is ~10%(20). Percent body fatwascal-
culated as follows: fat-free mass (kg) = total body water ÷ 0.73
and body fat (%) = [(weight – fat-free mass)/weight] × 100.
Statistics
Data are presented as the median and range; where ap-
proriate, control and group differences were examined us
-
ing the Mann-Whitney U test. The Wilcoxon signed rank
test was used to investigate changes after megestrol acetate
treatment. Measures of association were performed using
Spearman’s rankcorrelationtest (Minitab, State College, PA).
Results
The characteristics of healthy controls (n = 11) and gastro
-
intestinal cancer patients [n = 26 (20 colorectal, 3 gastric, 2
gastroesophageal, 1 pancreatic)] are shown in Table 1. Body
mass index and percent fat mass were significantly lower (P
< 0.05) and IL-6 and CRP were significantly higher (P <
0.05) in the cancer patients than in the controls. The circulat
-
ing concentrations of free and total leptin were significantly
lower in the cancer patients (P < 0.01). When free and total
leptin concentrations were adjusted for percent fat mass, they
remained significantly lower in the cancer patients (P <
0.05). There was no significant difference in percent leptin
binding between cancer patients and controls.
In the controls and cancer patients, the free and total leptin
concentration correlated with the measured percent fat mass
Vol. 44, No. 2 157
(r = 0.745, P = 0.008 [controls] and r = 0.600, P = 0.001 [pa
-
tients] for free leptin concentration and r = 0.745, P = 0.008
[controls] and r = 0.558, P = 0.003 [patients] for total leptin;
Figs. 1 and 2).
In the cancer patients, there was a positive correlation be
-
tween the circulating free and total leptin concentrations and
appetite scores (r = 0.575, P = 0.003 and r = 0.605, P = 0.001,
respectively). When free and total leptin concentrations were
adjusted by dividing by percent fat mass, there remained a
significant positive correlation with appetite scores (r =
0.720, P < 0.0001 and r = 0.720, P < 0.0001, respectively). In
the cancer patients, circulating leptin concentrations, either
free or total, were not correlated with IL-6 or CRP
concentrations.
Treatment with megestrol acetate significantly increased
circulating free and total leptin concentrations (P < 0.05; Ta-
ble 2). There was a significant positive correlation between
the change in circulating concentrations of free and total
leptin and the change in percent fat mass (r = 0.685, P < 0.05
and r = 0.661, P < 0.05, respectively; Fig. 3). However, there
was no correlation between the change in free and total leptin
concentrations and appetite when adjusted for percent fat
mass.
Discussion
Body weight homeostasis appears to be maintained by a
normal, dynamic equilibrium between anabolism and catab
-
olism controlled by orexigenic and anorexigenic neuropep
-
tides. In cancer cachexia, this normal mechanism is dis
-
rupted, and it is not surprising that leptin has been implicated,
because it has been reported to control the balance between
these neuropeptides (9). Furthermore, recent research indi
-
cates that advanced cancer is associated with the systemic in
-
flammatory response and increased concentrations of rele
-
vant cytokines such as IL-6 (4,22,23). In addition, circulating
leptin has been shown to increase as part of the acute-phase
158 Nutrition and Cancer 2002
Table 1. Characteristics of Healthy Controls and
Gastrointestinal Cancer Patients
a,b
Healthy
Controls Cancer Patients P
Gender, M/F 7/4 18/8
Age, yr 66 (46–74) 62 (47–88) NS
BMI, kg/m
2
25.2 (22.0–28.0) 23.4 (16.6–29.2) 0.01
% Fat mass 35 (14.5–47.1) 31.1 (15.2–41.2) <0.05
Free leptin, ng/ml 8.9 (0.9–31.6) 2.5 (0.9–6.6) <0.01
Total leptin, ng/ml 12.9 (0.9–42.3) 3.9 (0.9–9.3) <0.01
Leptin-to-% fat mass ratio 0.40 (0.1–0.26) 0.14 (0.04–0.9) <0.05
% Bound leptin 36.3 (21.1–41.9) 34.3 (9.2–47) NS
IL-6, pg/ml <2 (<2 to <2) 2 (<2 to 178) <0.05
CRP, mg/l <5 (<5 to 30) 21 (<5 to 127) <0.01
a: Values are medians, with range in parentheses.
b: Abbreviations are as follows: M, male; F, female; BMI, body mass in
-
dex; IL-6, interleukin-6; CRP, C-reactive protein; NS, not significant.
Figure 1. Relationship between percent fat mass and circulating free leptin
concentration in controls (open squares) and gastrointestinal cancer patients
(filled squares).
Figure 2. Relationship between percent fat mass and circulating total leptin
concentration in controls (open squares) and gastrointestinal cancer patients
(filled squares).
Table 2. Characteristics of Weight-Losing Gastrointestinal
Cancer Patients Before and After Treatment With
Megestrol Acetate
a
Before Treatment After Treatment P
BMI, kg/m
2
17.9 (15.9–21.4) 18.0 (15.4–22) NS
Appetite score 1.3 (0–10) 6.9 (0–10) NS (<0.10)
% Fat mass 21.5 (10.4–34.7) 21.7 (11–34.8) NS
Total leptin, ng/ml 1.8 (0.8–7.7) 2.3 (0.9–9.6) <0.05
Free leptin, ng/ml 1.1 (0.6–5.1) 1.4 (0.6–6.8) <0.05
% Bound leptin 35.5 (26–49) 35.4 (29–51) NS
IL-6, pg/ml <2 (<2 to 37.4) 7.6 (<2 to 52.3) NS (<0.10)
CRP, mg/l 6 (<5 to 160) 10 (<5 to 165) NS
a: Values are medians, with range in parentheses; n = 10.
response during surgical stress (24,25) and acute sepsis (26)
and after cytokine administration (27,28).
In the present study, we confirm earlier findings of low to
-
tal leptin concentrations in gastrointestinal cancer patients
(12,14) and the correlation between these low concentrations
and percent fat mass. We have extended these findings to
show that circulating free leptin concentrations parallel total
leptin concentrations. This observation is of considerable
significance, because the presumed bioavailable component
of total leptin is the free fraction, which is the only form
found in cerebrospinal fluid (18). In addition, we have shown
that there is no difference in the percentage of leptin bound
between cancer patients and controls, suggesting that there is
no alteration in the overall concentration of leptin-binding
proteins in the circulation. These novel findings in cancer pa-
tients are consistent with accumulating evidence that, in
healthy subjects, circulating free leptin parallels the total
leptin concentration (11,16,29) and also that circulating
leptin-binding proteins are directly related to the amount of
bound leptin (30).
A significant inverse relationship between circulating
IL-6 and total leptin concentrations in advanced cancer has
previously been reported (13). In contrast, after adjustment
for fat mass, Moses and co-workers (14) reported a signifi
-
cant positive correlation between total leptin and IL-6 con
-
centrations. In the present study, despite higher circulating
IL-6 and CRP concentrations in the cancer patients, there
were no correlations with free or total leptin concentrations
with or without adjustment for percent fat mass. The basis of
the different relationship between leptin and IL-6 reported in
these three studies remains unclear, and such relationships
merit further investigation in a larger group of cancer patients
to clarify this issue. Nevertheless, the low leptin concentra
-
tions in the cancer patients do not suggest a systemic role for
increased leptin in the anorexia/cachexia of cancer patients.
In addition, even when corrected for percent fat mass, the
cancer patients produce significantly lower circulating leptin
concentrations than controls. The reason for this suppression
is not known, but it is known that leptin-to-fat mass ratios are
reduced in periods of extended fasting or starvation (31), and
it is possible that a similar mechanism might occur here. It
could also be that a leptin-suppressing substance is produced
in advanced cancer. Obviously, further research is required to
investigate this in more detail.
In the present study, there was a positive, rather than, as
might be expected, a negative, relationship between free
leptin and appetite and total leptin and appetite in cancer pa
-
tients. Although results from self-reporting appetite scores
have their limitations (32), we believe that this is a reliable
approach in a study of this type. Our results may simply re
-
flect the fact that the circulating leptin concentration is an in
-
dicator of fat mass and that decreasing fat mass is a conse
-
quence of poor appetite, rather than a direct association
between leptin and appetite. Indeed, this is supported by our
finding that, on megestrol acetate treatment, the change in
free and total leptin concentrations was correlated with the
change in percent fat mass, but not appetite.
It was also of interest that in the cancer patients the im
-
provement of appetite with megestrol acetate treatment was
associated with a significant increase in circulating free and
total leptin. There was, however, no overall change in body
mass index or fat mass. The small, but significant, increase in
leptin may suggest that it is more sensitive to an improve
-
ment in energy balance than either weight or fat mass mea-
surements. Indeed, a similar situation has recently been de-
scribed in growth hormone-deficient adults treated with
growth hormone, where a decrease in the circulating leptin
concentration occurred before a reduction in fat mass (33).
The results of the present study indicate that the propor-
tions of free and bound leptin in the circulation do not differ
between controls and cancer patients and are related to fat
mass in both groups. Furthermore, the increase in circulating
leptin concentrations after megestrol acetate treatment is not
associated with any alteration in leptin binding.
Acknowledgments and Notes
This research was funded from the Glasgow Royal Infirmary Research
Endowment Fund. Address correspondence to A. M. Wallace, Dept. of Clin
-
ical Biochemistry, Macewen Bldg., Royal Infirmary, Glasgow G4 0SF, UK.
Phone: 0141-211-4490. FAX: 0141-553-1703. E-mail:
awallace@clinmed.gla.ac.uk.
Submitted 13 March 2002; accepted in final form 22 July 2002.
References
1. Jatoi A Jr and Loprinzi CL: Current management of cancer-associated
anorexia and weight loss. Oncology (Huntingt) 15, 497–502, 508,
2001.
2. O’Gorman P, McMillan DC, and McArdle CS: Impact of weight loss,
appetite, and the inflammatory response on quality of life in gastroin
-
testinal cancer patients. Nutr Cancer 32, 76–80, 1998.
3. McMillan DC, Preston T, Watson WS, Simpson JM, Rearom KC, et
al.: Relationship between weight loss, reduction of body cell mass and
Vol. 44, No. 2 159
Figure 3. Relationship between change in percent fat mass and free (open
squares) and total (filled squares) leptin concentrations in gastrointestinal
cancer patients.
160 Nutrition and Cancer 2002
inflammatory response in patients with cancer. Br J Surg 81,
1011–1014, 1994.
4. Kotler DP: Cachexia. Ann Intern Med 133, 622–634, 2000.
5. McMillan DC, Scott HR, Watson WS, Preston T, Milroy R, et al.: Lon
-
gitudinal study of body cell mass depletion and the inflammatory re
-
sponse in cancer patients. Nutr Cancer 31, 101–105, 1998.
6. Campfield LA, Smith FJ, Guisez Y, Devos R, and Burn P: Recombi
-
nant mouse OB protein: evidence for a peripheral signal linking adi
-
posity and central neural networks. Science 269, 546–549, 1995.
7. Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, et al.:
Physiological response to long-term peripheral and central leptin infu
-
sion in lean and obese mice. Proc Natl Acad Sci USA 94, 8878–8883,
1997.
8. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, et al.:
Effects of the obese gene product on body weight regulation in ob/ob
mice. Science 269, 540–543, 1995.
9. Jeanrenaud B and Rohner-Jeanrenaud F: Effects of neuropeptides and
leptin on nutrient partitioning: dysregulations in obesity. Annu Rev
Med 52, 339–351, 2001.
10. Montague CT, Farooqi IS, Whitehead JP, Soos MA, Rau H, et al.: Con
-
genital leptin deficiency is associated with severe early-onset obesity
in humans. Nature 387, 903–908, 1997.
11. McConway MG, Johnson J, Kelly A, Griffin D, Smith J, et al.: Differ
-
ences in circulating concentrations of total, free and bound leptin relate
to gender and body composition in adult humans. Ann Clin Biochem
37, 717–723, 2000.
12. Wallace AM, Sattar N, and McMillan DC: Effect of weight loss and
the inflammatory response on leptin concentrations in gastrointestinal
cancer patients. Clin Cancer Res 4, 2977–2979, 1998.
13. Mantovani G, Maccio A, Mura L, Massa E, Mudu MC, et al.: Serum
levels of leptin and proinflammatory cytokines in patients with ad-
vanced-stage cancer at different sites. J Mol Med 78, 554–561, 2000.
14. Moses AG, Dowidar N, Holloway B, Waddell I, Fearon KC, et al.:
Leptin and its relation to weight loss, ob gene expression and the
acute-phase response in surgical patients. Br J Surg 88, 588–593,
2001.
15. Lammert A, Kiess W, Bottner A, Glasow A, and Kratzsch J: Soluble
leptin receptor represents the main leptin binding activity in human
blood. Biochem Biophys Res Commun 283, 982–988, 2001.
16. Houseknecht KL, Mantzoros CS, Kuliawat R, Hadro E, Flier JS, et al.:
Evidence for leptin binding to proteins in serum of rodents and hu
-
mans: modulation with obesity. Diabetes 45, 1638–1643, 1996.
17. Landt M: Leptin binding and binding capacity in serum. Clin Chem 46,
379–384, 2000.
18. Landt M, Parvin CA, and Wong M: Leptin in cerebrospinal fluid from
children: correlation with plasma leptin, sexual dimorphism, and lack
of protein binding. Clin Chem 46, 854–858, 2000.
19. Loprinzi CL, Schaid DJ, Dose AM, Burnham NL, and Jensen MD:
Body-composition changes in patients who gain weight while receiv
-
ing megestrol acetate. J Clin Oncol 11, 152–154, 1993.
20. Raben A, Tagliabue A, and Astrup A: The reproducibility of subjective
appetite scores. Br J Nutr 73, 517–530, 1995.
21. Hannan WJ, Cowen SJ, Plester CE, Fearon KC, and deBeau A: Com
-
parison of bioimpedance spectroscopy and multi-frequency
bioimpedance analysis for the assessment of extracellular and total
body water in surgical patients. Clin Sci (Colch) 89, 651–658, 1995.
22. McMillan DC, Elahi M, Sattar N, Anderson WJ, Johnstone J, et al.:
Measurement of the systemic inflammatory response predicts cancer
specific and non-cancer survival in patients with cancer. Nutr Cancer
41, 64–69, 2001.
23. Mantovani G, Maccio A, Lai P, Massa E, Ghiani M, et al.: Cytokine ac
-
tivity in cancer-related anorexia/cachexia: role of megestrol acetate
and medroxyprogesterone acetate. Semin Oncol 25, 45–52, 1998.
24. Wallace AM, Sattar N, and McMillan DC: The co-ordinated
cytokine/hormone response to acute injury incorporates leptin.
Cytokine 12, 1042–1045, 2000.
25. Stratton RJ, Dewit O, Crowe E, Jennings G, Villar RN, et al.: Plasma
leptin, energy intake, and hunger following total hip replacement sur
-
gery. Clin Sci (Colch) 93, 113–117, 1997.
26. Bornstein SR, Licinio J, Tauchnitz R, Engelmann L, Negrao AB, et al.:
Plasma leptin levels are increased in survivors of acute sepsis: associ
-
ated loss of diurnal rhythm in cortisol and leptin secretion. J Clin
Endocrinol Metab 83, 280–283, 1998.
27. Zumbach MS, Boehme MW, Wahl P, Stremmel W, Ziegler R, et al.:
Tumor necrosis factor increases serum leptin levels in humans. J Clin
Endocrinol Metab 82, 4080–4082, 1997.
28. Janik JE, Curti BD, Considine RV, Rager HC, Powers GC, et al.:
Interleukin 1" increases serum leptin concentrations in humans. J Clin
Endocrinol Metab 82, 3084–3086, 1997.
29. Sinha MK, Opentanova I, Ohannesian JP, Kolaczynski JW, Heiman
ML, et al.: Evidence of free and bound leptin in human circulation.
Studies in lean and obese subjects and during short-term fasting. J Clin
Invest 98, 1277–1282, 1996.
30. Landt M, Horowitz JF, Coppack SW, and Klein S: Effect of short-term
fasting on free and bound leptin concentrations in lean and obese
women. J Clin Endocrinol Metab 86, 3768–3771, 2001.
31. Bodden G, Chen X, Mozzoli M, and Ryan I: Effect of fasting on serum
leptin in normal human subjects. J Clin Endocrinol Metab 81,
3419–3423, 1996.
32. Hill AJ, Rogers PJ, and Blundell JE: Techniques for the experimental
measurement of eating behaviour and food intake: a practical guide.
Int J Obes 19, 361–375, 1995.
33. Ahmad AM, Guzder R, Wallace AM, Thomas J, Fraser WD, et al.: Cir
-
cadian and ultradian rhythm and leptin pulsatility in adult GH defi
-
ciency: effects of GH replacement. J Clin Endocrinol Metab 86,
3499–3506, 2001.
