Comparison of the effects of cow’s milk, fortified soy milk, and calcium supplement on plasma adipocytokines in overweight or obese women

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Please cite this paper as:
Faghih s, Hedayati M, Abadi A, Kimiagar SM. Comparison of the effects of cow’s milk, fortified soy milk, and calcium supplement on
plasma adipocytokines in overweight or obese women. Int J Endocriol Metab. 2010; 8(4):188-93.
Comparison of the effects of cow’s milk, fortified soy milk, and calcium
supplement on plasma adipocytokines in overweight or obese women
Shiva Faghih ¹*, Mehdi Hedayati ², Alireza Abadi ³, Seyed Masoud Kimiagar 4
1 Department of Nutrition, School of Health and Nutrition, Shiraz University of Medical Sciences, shiraz, IR Iran
2 Obesity Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran
3 Department of Statistics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran
4 Department of clinical nutrition, Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran
Copyright c 2010, Iran Endocrine Society, Published by Kowsar M.P.Co. All rights reserved.
* Corresponding author at: Shiva Faghih, Department of Nutrition,
School of Health and Nutrition, Shiraz University of Medical Sciences,
Razi Blvd, Shiraz, IR Iran. Tel:+9126305829
E-mail: shivafaghih@gmail.com
Implication for health policy/practice/research/medical education:
Obesity and its complications are the major concerns of health care practitioners, and finding proper diet to reduce this problem is
critical. Results of this article are useful for nutritionists, epidemiologists and physicians.
ABSTRACT
Background: Obesity is an escalating public health problem. Adipose tissue synthesizes
and secretes a variety of biological molecules, termed adipcytokines, that may contrib-
ute to obesity-linked metabolic abnormalities including cardiovascular diseases.
Objectives: We compared the effects of cowʹs milk, calcium-fortified soy milk, and
a calcium supplement on adipo cytokines in premenopausal overweight and obese
women.
Materials and Methods: In this clinical trial, 100 healthy, overweight or obese premeno-
pausal women were randomly assigned to one of the following dietary regimens for 8
weeks: (a) a control diet (b), a calcium-supplemented diet containing 800 mg/day cal-
cium carbonate, (c) a high-milk diet containing three servings of low-fat milk, and (d) a
soy-milk diet containing three servings of calcium-fortified soy milk. All diets required
a 500-kcal/day reduction in energy. At baseline and after 8 weeks, anthropometric indi-
ces and plasma leptin, adiponectin, TNFα, CRP, and IL-6 were measured.
Results: Plasma CRP and leptin were significantly correlated with all anthropometric
indices except for WHR, and plasma adiponectin had a significant negative correlation
with WHR at baseline. Plasma leptin, CRP, and IL-6 decreased significantly in all groups
(P < 0.01; except for CRP in the control group), but there were no significant differences
among the four groups for these three measures.
Conclusions: We conclude that a dietary reduction of 500-kcal/day has beneficial effects
on plasma adipocytokines, but calcium intake either as calcium carbonate or as milk
leads to no differences. These results merit further research.
ARTICLE INFO
Article history:
Received: 13 May 2010
Revised: 10 Jun 2010
Accepted: 1 Jan 2011
Keywords:
Calcium
Dairy
Obese
Adipocytokines
Article Type:
Original Article
c 2010 Kowsar M.P.Co. All rights reserved.
1. Background
Obesity is an escalating public health problem (1-4) De-
fined as the accumulation of excess body fat (5, 6). obe-
sity is a principle causative factor in the development of
metabolic disorders such as insulin resistance, hyperten-
sion, hyperglycemia, dyslipidemia, and atherosclerosis
(6, 7). Adipose tissue is currently considered to be a hor-
monally active system in the control of metabolism and
not only as a store of excess energy (5, 8, 9). It synthesizes
and secretes a variety of biological molecules, including

Int J Endocrinol Metab. 2010;8(4):188-193
189
Cow’s milk, soy milk, and calcium and plasma adipocytokines Faghih S et al.
adiponectin, leptin, tumor necrosis factor alpha (TNFα),
and interlukin-6 (IL-6) (5, 9-11). Recent research has re-
vealed that substances such as C-reactive protein (CRP),
IL-6, and TNFα mirror oxidative stress and may play a role
in promoting adverse vascular outcomes in metabolic
syndrome and type 2 diabetes (12, 13). TNFα is overex-
pressed in white adipose tissue in obesity and decreases
with weight loss and improvement of insulin sensitiv-
ity (14). Subsequent studies have demonstrated that CRP
concentrations are significantly related to various mea-
sures of body fat, and weight loss may lead to a decrease
in CRP concentration (15, 16).
Leptin is a hormone primarily secreted by the adipose
tissue and represents several physiological functions
(17). Although the principal effects of leptin in the central
nervous system are the control of food intake and energy
expenditure, there is a significant relationship between
leptinaemia and the chronic subinflammatory state that
accompanies obesity which suggests that other possible
peripheral biological effects are associated with its cyto-
kine-like structure (18).
In sharp contrast to most adipokines, adiponectin ex-
pression and serum concentrations do not increase and
actually decrease in a variety of obese and insulin-resis-
tant states (14). Adiponectin is produced in fat cells (19),
and in addition to its effects on insulin sensitivity, it has
anti atherogenic properties (14, 18).
2.Objectives
Various studies have demonstrated that weight loss
results in significant increases in serum levels of adipo-
nectin and significant reduction in plasma CRP (20, 21).
Sun and Zemel’s findings indicated that dietary calcium
suppresses obesity-associated inflammatory status by
modulating proinflammatory and anti-inflammatory
factor expression in mice (22). A few studies have specifi-
cally focused on the effects of calcium or dairy consump-
tion on plasma adipocytokines (16, 22-25). One of these
studies was conducted on mice (22), and 2 of them evalu-
ated the effect of calcium on CRP (16, 23). In the present
study, we examined the effects of cowʹs milk, fortified soy
milk, and calcium supplement on serum adipocytokines
in overweight or obese women.
3. Material and Methods
3.1 Study sample
Using an effect size of 0.55 for our sample size equa-
tions, 20 people for each group were estimated. Then we
added 25% to this number, so 25 people for each group
were included in the analyses. One hundred healthy, pre-
menopausal, overweight or obese women ranging in age
from 20 to 50 years volunteered to participate in this ran-
domized clinical trial, and 85 completed the study. Fif-
teen subjects dropped out for various reasons, including
thyroid disease, not wanting or being able to consume
soy milk or a calcium supplement, and choosing not to
continue participation. There were no significant differ-
ences between those who completed the study and those
who did not for any of the main variables. Inclusion crite-
ria were as follows: body mass index (BMI) more than 25
kg/m²; taking no medications or supplements that might
affect metabolism of calcium, vitamin D, or weight loss;
absence of menopause; stable body weight (body weight
changed less than 3kg for the last 2 months); and absence
of diabetes, hypertension, coronary-artery, thyroid, and
kidney diseases. The subjects were nonpregnant and non-
lactating with no allergy to milk or soy milk and lactose
intolerance. We also made sure that the subjects had not
participated in any other studies in the 6 months preced-
ing the screening. The study was approved by the ethics
committee of the National Nutrition and Food Technol-
ogy Research Institute. Each potential participant was in-
formed of the possible risks and benefits associated with
this study and provided written signed consent.
3.2 Study interventions
For the first 2 weeks of the study, we collected the sub-
jects’ baseline dietary data and physical activity assess-
ments. Then, we randomized the subjects to one of the
following dietary regimens for 8 weeks:
1. A control diet that required a 500-kcal/day reduction
in total intake (based on the Harris-Benedict equa-
tion), with 500 to 600 mg/day of dietary calcium
(about 1.5 to 2 servings of dairy products);
2. A calcium-supplemented diet identical to the control
diet except for the addition of 800 mg/day of calcium
( as calcium carbonate);
3. A milk diet that required a 500 kcal/day reduction in
total intake and containing three servings (220 ml
each) of low fat milk (1.5%); and
4. A soy-milk diet that required a 500-kcal/day reduc-
tion in total intake and three servings of calcium
fortified soy milk per day. The total calcium intake
for the milk diet and the soy-milk diet were between
1,200 and 1,300 mg/day.
Daily caloric requirements for all diets were calculated
by using the Harris-Benedict equation (15). After adjust-
ing the caloric intakes for each participant’s physical
activity level, individualized meal plans were given to
each participant based on a 500 kcal/day reduction from
their estimated caloric requirements (26). The diets for
all groups were designed to provide comparable levels
of macro nutrients as follows: 55% carbohydrate, 18%
protein, and 27% fat. At baseline and at 2-week intervals,
weight, waist circumference, and hip circumference
were measured, and 24-hr dietary records (27) as well as
physical-activity records (2 weekdays and 1 weekend day)
were taken. Body fat was measured at the beginning of
the study and at Week 8 using a Bodystat bioelectric im-
pedance analysis (Quadscan4000 model).
At baseline and after 8 weeks, fasting blood samples
(10 ml) were obtained in the morning. The plasma
was separated and frozen at -80ºC for later analy-

Int J Endocrinol Metab. 2010;8(4):188-193
190 Cow’s milk, soy milk, and calcium and plasma adipocytokines
Faghih S et al.
sis. Adiponectin (Mercodia, Uppsala, Sweden), leptin
(Mercodia,Uppsala,Sweden), TNFα (Diaclon, France), IL-6
(Diaclon, Besancon, France), and CRP (Diagnostics Bio-
chem Canada) were measured using a sandwich Elisa
method.
3.3 Statistical analysis
Dietary records were analyzed using Nutritionist 4 (N
IV). Statistical analyses were performed with SPSS soft-
ware, version 15. One-way ANOVAs were used to compare
the characteristics of the subjects in the four groups at
baseline and also to compare the mean differences of
plasma adipocytokines among the four groups. The ef-
fect of the intervention in each group was tested using
paired-samples t-tests, and the relationships between
plasma adipocytokines and anthropometric indices
were estimated using Pearson correlations. P < 0.05 was
considered significant.
4. Results
Of the 100 women meeting the general eligibility cri-
teria, 15 dropped out before completing the weight-loss
period (5, 3, 3, and 4 persons in control, calcium supple-
ment, high milk, and soy milk groups respectively). Plas-
ma CRP, IL-6, and TNFα were not normally distributed, so
we used logs of them in the analysis.
The baseline characteristics of subjects are shown in
Table 1. It is apparent that there were no significant dif-
ferences between the age, weight, BMI, or energy and
calcium intake of subjects across the four groups.
The relationship among plasma adipocytokines and an-
thropometric indices at baseline are shown in Table 2. It
is clear that plasma CRP and leptin were significantly cor-
related with all anthropometric indices except for waist
to hip ratio WHR, that plasma adiponectin had a signifi-
cant negative correlation with WHR, and that there were
no significant correlations among TNFα and IL-6 and the
anthropometric indices.
Table 3 shows that after 8 weeks, the correlations among
plasma adipocytokines and anthropometric indices were
the same as they were at baseline, except for plasma
leptin, which showed no significant correlation. The an-
thropometric indices of all groups decreased significant-
ly after 8 weeks of weight-loss intervention (P < 0.001for
all), but the amount of these reductions was significantly
different among the four groups for only waist circum-
ference (P = 0.029) and WHR (P = 0.015). After adjusting
for baseline values with analyses of covariance, however,
changes in weight and BMI were significantly different
among the four groups (P = 0.017 and P = 0.019, respec-
tively). Post hoc tests showed that the changes in waist
circumference and WHR were significantly higher in the
high-milk (6.32 ± 2.50 cm and 0.048 ± 0.019) and soy-milk
(5.84 ± 1.47 cm and 0.044 ± 0.019) groups than in the con-
trol group (3.98 ± 2.77 cm and 0.021 ± 0.016; P < 0.01 and
P < 0.05, respectively). Also, the reductions in weight and
BMI were significantly greater in the high-milk group
(4.43 ± 1.93 kg and 1.74 ± 0.73 kg/m²) than in the control
group (2.87 ± 1.55 kg and 1.15 ± 0.62 kg/m²; P < 0.01for all).
Variables Control
(n = 20)
Ca supplement
(n = 22)
High milk
(n = 22)
Soy milk
(n = 21)
P b
Age, y38.25 ± 9.49 a35.77 ± 8.70 38.27 ± 10.43 37.54 ± 9.27 0.78
Weight, kg 76.78 ± 9.6 78.16 ± 11.43 76.24 ± 10.57 80.05 ± 13.32 0.69
BMI, kg/m² 30.78 ± 3.13 31.54 ± 4.12 30.01 ± 3.55 31.09 ± 4.13 0.58
Energy, kcal/d 1839 ± 169 1870 ± 201 1937 ± 177 1901 ± 148 0.32
Calcium, mg/d 512 ± 172 532 ± 149 484 ± 131 509± 101 0.73
a mean ± SD
b One way ANOVA
Table 1. Baseline characteristics of subjects under study
a NS = not significant
Table 2. Correlation of plasma adipocytokines and anthropometric indices at baseline
Anthropometric
indices
adipocytokines
Weight, kg Waist circumference, cm BMI, kg/m² Body fat mass (%) WHR
CRP, ng/ml r = 0.34
p = 0. 001
r = 0.41
p = 0. 0001
r = 0.42
p = 0. 0001
r = 0.41
p = 0. 0001
NS
TNFα, pg/ml NS aNS NS NS NS
IL-6, pg/ml
NS NS NS NS NS
Leptin, ng/ml r = 0.49
p = 0. 0001
r = 0.52
p = 0. 0001
r = 0.56
p = 0. 0001
r = 0.47
p = 0. 0001
NS
Adiponectin, ng/ml NS NS NS NS r = - 0.27
p = 0. 013

Int J Endocrinol Metab. 2010;8(4):188-193
191
Cow’s milk, soy milk, and calcium and plasma adipocytokines Faghih S et al.
a NS = not significant
Anthropometric indices
Adipocytokines
Weight, kg Waist circumference, cm BMI, kg/m² Body fat mass (%) WHR
CRP, ng/ml r = 0.31
p = 0.003
r = 0.37
p = 0.0001
r = 0.41
p = 0.0001
r = 0.40
p = 0.0001
r = 0.24
0.026
TNFα, pg/ml NS aNS NS NS NS
IL-6, pg/ml
NS NS NS NS NS
Leptin, ng/ml NS NS NS NS NS
Adiponectin, ng/ml NS NS NS NS r = - 0.23
p = 0.03
Table 3. Correlation of plasma adipocytokines and anthropometric indices after intervention
The data in Table 4 indicate that plasma IL-6 and leptin
decreased significantly in all four groups, but there were
no significant reduction in plasma TNFα or adiponectin.
The reduction of plasma CRP was significant in the three
experimental groups but not the control group. Mean
differences of plasma adipocytokines among the four
groups were not significant.
5. Discussion
Results of this study showed that plasma CRP and
leptin were significantly correlated with body weight,
waist circumference, body fat, and BMI. Inflammatory
markers, such as CRP and IL-6, are higher in obese indi-
viduals than in lean subjects (12). The insulin resistance
and atherosclerosis study showed that CRP values corre-
lated with BMI, waist circumference, and fasting hyper-
insulinemia (13). There are several studies that indicate
that serum leptin concentration is proportional to body-
fat mass (17, 28, 29).
Additionally, in this study there was a significant cor-
relation between plasma adiponectin and WHR. It seems
a All values are Mean ± SD
b No significant differences were seen among groups (One way ANOVA)
c Significant difference between before and after P < 0.001
d Significant difference between before and after P < 0.01
e Significant difference between before and after P < 0.05
Variables aGroups b Time Changes
Before After
TNFα, pg/ml Control (19)
Ca supplement (21)
low fat milk (21)
Soy milk (20)
6.60 ± 4.57
6.48 ± 1.94
8.30 ± 6.72
4.96 ± 2.51
6.26 ± 4.17
5.91 ± 1.78
7.91 ± 5.91
4.94 ± 2.84
-0.34 ± 0.44
-0.82 ±0.26
-0.38 ± 0.50
-0.12 ± 0.40
CRP, ng/ml Control (19)
Ca supplement (21)
low fat milk (21)
Soy milk (20)
2.92 ± 1.99
2.60 ± 2.24
1.92 ± 2.06
3.29 ± 2.35
2.60 ± 2.63
2.05 ± 1.88
1.17 ± 1.32
2.27 ± 2.11
-0.31 ± .043
0.54 ± 0.26 e
0.75 ± 0.19 c
0.97 ± 0.38 e
IL-6, pg/ml Control (19)
Ca supplement (21)
low fat milk (21)
Soy milk (20)
1.56 ± 1.20
1.54 ± 0.64
1.18 ± 0.57
1.02 ± 0.48
1.15 ± 0.86
1.03 ± 0.51
0.89 ± 0.37
0.76 ± 0.42
-0.40 ± 0.11 d
-0.55 ± 0.11 d
-0.29 ± 0.08 c
-0.24 ± 0.10 d
leptin, ng/ml Control (19)
Ca supplement (21)
low fat milk (21)
Soy milk (20)
21.54 ± 9.42
21.96 ± 8.65
20.37 ± 12.3
20.75 ± 9.85
14.88 ± 7.56
18.56 ± 9.31
11.75 ± 9.05
14.29 ± 5.85
-6.65 ± 1.40 c
-5.10 ± 1.22 e
-8.61 ± 1.27 c
-5.78 ± 1.68 d
Adiponektin, ng/ml Control (19)
Ca supplement (21)
low fat milk (21)
Soy milk (20)
8.37 ± 1.99
8.61 ± 2.69
10.32 ± 3.65
8.33 ± 2.62
7.96 ± 2.55
8.36 ± 2.04
10.08 ± 3.41
7. ± 2.1063
-0.40 ± 0.61
-0.55 ± 0.46
-0.24 ± 0.56
-0.53 ± 0.31
Table 4. Within and between groups comparison of the effects of 8-week weight-loss intervention on plasma adipocytokines

Int J Endocrinol Metab. 2010;8(4):188-193
192 Cow’s milk, soy milk, and calcium and plasma adipocytokines
Faghih S et al.
that visceral obesity is predominantly associated with
reduced levels of adiponectin; obese women who have
normal upper-body fat have normal adiponectin levels
as well (13).
In this study, a dietary reduction of 500 kcal/day for
8 weeks led to significant decreases in plasma IL-6 and
leptin and no significant changes in plasma TNFα in any
group. Plasma CRP decreased significantly in all three
experimental groups but not the control group. Leptin
is related to the risk of diabetes (30) and cardiovascular
disease (31) in the elderly. It has been reported that an
imposed energy deficit causes decreases in circulating
leptin (29, 32). Copolla et al. reported that after 12 months
of a multidisciplinary program of weight reduction,
obese women lost at least 10% of their original weight,
and their CRP levels decreased significantly (P < 0.01)
(20). Additionally, Madsen et al. showed that weight loss
was associated with a significant decrease in CRP (21). Sun
and Zemel found that a high-calcium diet significantly
inhibited the expression of TNFα and IL-6 and stimulated
the expression of adiponectin in visceral fat (P < 0.001)
in male aP2-agoti transgenic mice (6). They also found
similar effects in a retrospective analysis of archival clini-
cal samples from obese subjects, with high dairy intake
reducing CRP and increasing adiponectin under both
eucaloric and hypocaloric conditions. Sun and Zemel
suggested that although the reduction of CRP levels was
independent of changes in body weight, it is not possible
to fully ignore the effect of reduced adiposity, as subjects
exhibited reduced body fat. Circulating 1α, 25-dihydroxy-
cholecalciferol was reduced with increased Ca intake,
concurrent with reductions in pro-inflammatory indi-
ces, suggesting a role for dietary Ca in attenuating the
cytokine dysregulation related to diet-induced obesity
(25). Also, research has suggested that parathyroid hor-
mone regulates the circulating levels of the inflamma-
tory cytokines interleukin-6 and tumor necrosis factor-α,
which in turn stimulate production of CRP and calcium
supplementation, by suppressing parathyroid hormone
production, might also decrease serum CRP (23).
In contrast, Pittas et al. found that daily consumption of
either 500 mg of calcium citrate and 700 IU of vitamin D
or placebo for 3 years had no significant effects on plas-
ma IL-6 and CRP (26). Also Grey et al. reported that after 1
year of calcium supplementation (1g calcium citrate per
day), there was no difference between the CRP levels of
the supplement and placebo groups (25).
Despite the 5-cm reduction in waist circumference and
approximately 5% reduction in body weight, there was no
significant change in plasma adiponectin in this study.
Maestu et al. reported that after a 10-week intervention,
no change in adiponectin concentration was observed,
despite significant weight and fat mass loss (6.5% reduc-
tion) in male competitive bodybuilders (17). Xydakis et
al. observed no change in plasma adiponectin after 4 to
6 weeks of weight loss (11). On the other hand, Madsen
et al. found that a mean weight loss of about 12% after 8
weeks of VLCD resulted in a significant 22% increase in se-
rum levels of adiponectin. They also found that relatively
large weight losses (more that 10-11%) were necessary to
obtain a significant increase in plasma adiponectin lev-
els (21).
Despite the reductions in waist circumference, WHR,
body weight, and BMI were significantly different across
all four groups, but plasma adipocytokine changes were
not significantly different in any group. We assume that,
although the reductions in anthropometric indices were
significantly different among the four groups, they were
not high enough to exert different effects on the subjects’
adipocytokines expressions.
For this study, we conclude that calcium intake as cal-
cium carbonate, calcium-fortified soy milk, or low-fat
milk leads to no differences in plasma adipocytokines.
Still, given the short duration of this study, future studies
with longer experimental periods are warranted.
Financial support
This article was funded by Institute of Nutrition and
Food Technology, Shahid Beheshti University of Medical
Sciences.
Conflict of interest
There is no conflict of interest.
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