Research Article

EFFECT OF 8 WEEKS LOW CALORIE HIGH PROTEIN AND STANDARD PROTEIN

DIET ON PLASMA MALONDIALDEHYDE AND GLUTATHIONE LEVEL IN OBESE
WITH WEIGHT CYCLING
Septian Ika Prasetya1, Fiastuti Witjaksono2, Ninik Mudjihartini3*
1Undegraduate Program of Medicine, Faculty of Medicine Universitas Indonesia
2Department of Nutrition, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
3Department of Biochemistry and Molecular Biology, Faculty of Medicine, Univeersitas

Indonesia
*corresponding author e-mail: ninikbiokim@gmail.com

ABSTRACT
Objective: This study is aimed at evaluating the effects of two kinds of low calorie diet, which are
high protein and standard protein proportion diet upon the change in plasma malondialdehyde (MDA)
and glutathione (GSH) level in obese people with weight cycling.
Methods: An open-randomized clinical trial was conducted in a province worker health center in
Jakarta, Indonesia. Participants were assigned to a 1000 kcal calorie reduction from usual daily caloric
intake diet with two different intervention groups, namely high protein/HP group (25-30 percent of
total caloric intake were from protein) and standard protein/SP group (15-20% protein). The diet
program was applied for 8 weeks with daily reminder by phone and weekly counseling. Venous blood
sample was taken in the first day as well as in the last day of the program. Plasma MDA level was
measured according to Wills method while GSH level was measured with Ellmans method.
Result: Twenty one participants completed the program, 9 in HP group and 12 in SP group. In HP
group, plasma MDA level increased after diet program from 0.444 + 0.107 to 0.488 + 0.166 nmol/ml
plasma (P=0.423) while plasma GSH level decreased from 3.520 + 0.2045 to 3.460 + 0.2473 g/ml
plasma (P=0.316). On the other hand, in SP group, both plasma MDA and GSH level decreased. In
this group, MDA level decreased from 0.571 + 0.185 to 0.536 + 0.145 (P=0.310) nmol/ml plasma
while GSH level decreased from 3.549 + 0.241 to 3.519 + 0.41 g/ml plasma (P=0.708). Mean
difference in change of MDA level between HP (0.0448+ 0.117) and SP (-0.0345 + 0.1174) group (P=
0.193) as well as mean change of GSH level (-0.059 + 0.1673 in HP group and -0.03 + 0.388 in SP
group, P=0.833) were not significant.
Conclusion: Low calorie diet for 8 weeks with high protein proportion did not result in significantly
different outcome with standard protein in terms of oxidative stress state which were measured in
plasma MDA and GSH level.
INTRODUCTION
According to WHO, in 2014, more than 1.9 (39%) billion adults aged 18 years and older were
overweight with 600 (13%) million among them were obese. Meanwhile in Indonesia, according to
basic health research (RISKESDAS) 2013, the prevalence of adult with overweight was 13.5% and
adult with obesity was 15.4% in 2013. Approximately, 2.8 million people die each year resulting from
being overweight or obese as well as causing an estimated 35.8 million (2.3%) of global disability-
adjusted life years. As body mass index (BMI) increases, the risk of developing coronary heart disease,
ischemic stroke, and type 2 diabetes mellitus increase accordingly since overweight and obesity bring
about adverse impacts on blood pressure, cholesterol, and triglycerides. The rise of BMI also
increasing the risk of developing various types of cancer such as cancer of the breast, colon, prostate
endometrium, kidney and gall bladder.1
Oxidative stress is regarded as the major underlying mechanism of chronic diseases development
resulted from overweight and obesity. Fat accumulation as well as BMI rise as occurred in obesity
were proven to be correlated with systemic oxidative stress. Accumulated fat showed a marked
increase in oxidative stress which induces metabolic syndrome through several ways, most probably
by causing impaired regulation in adipocytokines production and selective increase in reactive oxygen
species (ROS) generation due to elevation of systemic oxidative stress. Within accumulated fat, the
increase in NADPH oxidase and concurrent decrease in antioxidant enzymes result in oxidative stress.
Oxidative stress exerts its effect on adipocytokines production by increasing production of
plasminogen activator inhibitor-1 and TNF- which in turn participate in the formation of thrombosis
and insulin resistance, respectively. Oxidative stress also dampen the plasma adiponectin which main
functions are insulin-sensitizing and anti-atherogenic effects hence resulting in insulin resistance and
atherosclerosis. Systemic oxidative stress elicited by fat accumulation causes increase in ROS in
vascular wall which later initiating atherosclerosis formation and subsequent cardiovascular diseases.2
Weight loss attempts through various means are often suggested or self-intended as peoples awareness
on potential adverse impacts of obesity to health rises. Dietary program as one of mainstay in weight
reduction has proven to bring about improvement in pro-oxidant antioxidant balance by reducing
markers of oxidative stress such as F2 isoprostanes3 and lipid peroxidation as well as increase in
antioxidant activity for instance catalase, glutathione reductase,4 and glutathione peroxidase4.
However, many people who had succeeded in reducing their body weight failed to maintain it at the
desired level and then underwent subsequent body weight gain. A state of body weight gain after a
marked reduction from weight loss attempts which occurred alternately is called weight cycling.
Regaining body weight after previous weight loss is dominated by increase in fat mass rather than
increase in free fat mass thus increasing fat mass percentage. This condition is undesirable since free
fat mass which is predominantly muscle mass is an essential part of body for various activity while
greater fat mass percentage results in adverse health impacts. Therefore, an efficient weight loss dietary
program which results in greater fat mass reduction and muscle mass preservation must be formulized
to address issue of weight cycling. The objective of this study was to evaluate the effect of low calorie
high protein diet compared to low calorie standard protein based on oxidative stress and antioxidant
status which were measured by plasma malondialdehyde and glutathione level, respectively.
METHOD
Subjects
This study is part of a main research assessing the effects of low calorie high protein diet on body
composition, inflammation marker, and oxidative stress in obese people with weight cycling. The
research has been approved by health research ethical committee of Faculty of Medicine Universitas
Indonesia Cipto Mangunkusumo Hospital with letter approval number of 237/UN2.F1/ETIK/2017.
Potential participants were obtained from the list of provinces civil workers visiting workers health
service center of special capital region of Jakarta in 2016 (Pusat Pelayanan Kesehatan Pegawai
Provinsi DKI Jakarta). The inclusion criteria were men and women more than 20 years old with BMI
ranging from 25 35 kg/m2 and with history of weight cycling. In this study, weight cycling was
defined as history of weight loss 2 kg and regaining weight into or exceeding its initial body weight
at least twice in last five years. Weight cycling history of each potential participant was firstly self-
reported for screening purpose based on history of body weight changes yet in the latter recruitment it
was confirmed through interview by nutritionist. Exclusion criteria were diabetes mellitus, history of
gastrointestinal tract resection, hormonal disorders such as abnormal thyroid function, hormonal
contraception user, menopause, and abnormal kidney function which was assessed from serum urea
and creatinine levels. Out of 712 potential participants screened by questionnaire, only 33 participated
initially and 22 subjects completed the 8-weeks diet intervention.

Study design and intervention

Initially, 33 participating subjects were distributed by block randomization into one of two groups,
high protein (HP) group and standard protein (SP) group. Two weeks before diet intervention were
given, subjects were interviewed using 24-hours food recall to determine baseline daily caloric intake
and their anthropometric data as well as 1.5 ml of vein blood samples prior to 8 hour-fasting were
taken. Subjects were instructed to stop all previous diet program and to keep their daily physical
activity at their usual level.
Nutritional consultation was given to each participant of both group addressing the diet plan of
reducing the daily caloric intake by 1000 kcal from usual daily caloric intake before joining the study.
Amount of previous daily caloric intake were obtained from 24 hours food recall by asking type,
cooking method, and estimation of amount of food consumed by using household size based on food
photo books issued by Tim Survey Konsumsi Makanan Individu, Ministry of Health Republic of
Indonesia. Subjects in high protein group were instructed to decrease their daily caloric intake by 1000
kcal and the remaining amount of daily caloric intake were divided based on the macronutrient source
with composition of 25-30% protein, 50-55% carbohydrate, and 20-25% fat. On the other hand,
standard protein (SP) group received a 1000 kcal caloric restriction diet plan with energy source
comprised of 15-20% protein, 55-60% carbohydrate, and 20-30% fat. Subjects were taught about
appropriate kind and amount of foods for daily consumption to comply the assigned amount and source
of caloric intake along with eligible cooking methods. Subjects were also provided a menu book
explaining details about it. Diet program were conducted in 8 weeks. Subjects were asked to make a
self-reported daily food consumptions which were noted in a logbook. Subjects were also followed up
on a daily basis by phone and weekly counseling to ensure adherence to the diet plan.

Anthropometric data and plasma sample

Anthropometric data of the participants were collected in the first encounter. Subjects blood sample
was taken in the first day as well as in the last day of 8 weeks diet program. Venous blood samples
were taken as much as 1.5 ml and then were stored in heparin vacutainer tube. Next, samples were
centrifuged and stored frozen until further use.
Statistical analysis
Mean differences significance in baseline characteristics of age, BMI, and number of weight cycling
cycles between HP and SP groups were assessed by independent samples t-test. Meanwhile, Fisher
exact test were used to determine proportion difference significance of gender between both groups.
Significance of intragroup mean difference of MDA and GSH before and after 8-weeks diet were
evaluated by paired samples t-test if the data were normally distributed as indicated by P>0.05 in
Shapiro-Wilk test. If the distribution of the data is not normal, Wilcoxon test as the non-parametric
test was used instead. On the other hand, independent samples t-test was used to determine intergroup
statistical significance of mean difference of change rate in MDA and GSH between HP and SP group
if the data distribution was normal. If the data was not normally distributed, Mann-Whitney test was
used for this purpose.
Measuring plasma malondialdehyde (MDA) and glutathione level
The measurement of MDA level was performed using Wills method6 while plasma GSH measurement
was conducted according to Ellman method7, both with slight modification briefly described as
follows.
Determination of malondyaldehyde concentration
The basic principle of MDA measurement is reacting MDA with thiobarbituric acid that will produce
a colorized compound which has maximum absorbance at 530 nm in spectrophotometry. A set of
mixture comprised of tetraethoxypropane, tricholoroacetic acid (TCA) 20% and thiobarbituric acid
(TBA) 0.67% with the varying concentration of tetraethoxypropane was used as a standard solution.
Standard curve was arranged by plotting tetraethoxypropane concentration in x-axis to solutions
absorbance at 580 nm in y-axis. Using Microsoft Excel software, the value of curves slope and
intercept were derived from arrays of standard solutions mean absorbance and concentration. TCA
20% solution and standard TEP solution with varying concentrations were added to plasma
homogenates. The mixtures were then centrifuged and the precipitates were removed. TBA 0,67%
solution were added. The final mixture were then heated and centrifuged to obtain the supernatant
which later were read in duplicates at 530 nm. The plasma MDA level was calculated as follows: mean
absorbance value minus standard curves intercept and then divide by standard curves slope.

Determination of glutathione concentration

The principle of Ellman method is reacting a compound which contains SH group with
dithiobisnitrobenzoic acid (DNTB) which have S-S- bond to produce thionitrobenzene which have
yellow color and absorbs light maximally at 412 nm. Standard glutathione solution with varying
concentration and phosphate buffer 0.1 M pH 8 both were mixed with TCA 5% 0.8 ml and DNTB
0.001 mL and then their absorbance were read at 412 nm. GSH standard solutions absorbance were
plotted against its concentration to generate GSH standard curve. Plasma sample as much as 50 l
were reacted with phosphate buffer solution 0.1 M pH 8.0 1.78 ml and 0.2 ml TCA 5%. The mixture
were then centrifuged to remove the precipitate and 0.8 ml of supernatant was taken as the sample.
Finally, samples absorbance were read in duplicate at wavelength 412 nm. To convert absorbance
value into GSH level, mean sample absorbance was subtracted by the value of standard curves
intercept and then divide by standard curves slope.
RESULT
As it was shown in table 1, mean age of subjects in standard protein group were higher while
proportion of male subjects were higher in high protein group. Mean body mass index as well as
frequency of weight cycling of both group were identical. Baseline characteristics of subjects from
both group of intervention did not show any significance in mean or proportion difference. Hence, it
could be assumed that characteristics of subjects in high protein low calorie diet as well as in standard
protein low calorie diet before diet intervention commenced were similar thus comparing of both group
after the intervention was eligible.

Table 1. Subjects characteristic prior to low calorie-diet intervention with high protein and
standard protein
High protein Standard protein
Variable P*
(n: 9) (n: 13)
Age (y) 33.67 + 7.22 35.31 + 7.70 0.62t
Gender
Male 2 (22.8%) 1 (7,7%)
0.54f
Female 7 (77.8%) 12 (92.3%)
Body mass index (kg/m2) 30.65 + 4.03 30.32 + 2.81 0.82t
Number of weight cycle history 31 3 (2 4) 0.404t
*
significant value was set at P<0.05
t
independent samples t-test
f
Fisher exact test

In graph 1, it was shown that before diet intervention, mean concentration of plasma malondialdehyde
of high protein group (0.444 + 0.107) was lower than that of standard protein group (0.571 + 0.185)
but this difference was not statistically significant (P=0.08). Mean difference of plasma MDA after
intervention was also lower in high protein group (0.488 + 0.166) than in standard protein group (0.536
+ 0.145) and again this difference was not significant (0.481). Interestingly, in this study we found that
after completing the 8-weeks diet program, plasma MDA level in high protein low-calorie group
underwent a slight increase while in standard protein-low calorie group plasma MDA level decreased
very slightly. In high protein-group, mean plasma MDA level increased from 0.444 + 0.107 before
intervention into 0.488 + 0.166 after completing diet program. This slight increment was not significant
statistically (P=0.423). The opposite condition happened in standard-protein group where plasma
MDA level showed a slight reduction although this finding was statistically insignificant (P=0.48).
Post-intervention MDA level in standard protein group was 0.536 + 0.145, down from 0.571 + 0.185
before intervention (P=0.310).
 Mean plasma concentration of MDA (nmol/ml plasma)

t
independent samples t-test
p
paired samples t-test
significance level was set at P<0.05
Graph 1. Mean plasma concentration of malondialdehyde (MDA) in high protein low
calorie and standard protein low calorie diet before and after 8 weeks of intervention

For plasma concentration of glutathione (GSH), intergroup mean difference of either before and after
intervention were not significant, with P=0.774 for intergroup mean difference of GSH level before
intervention and P=0.708 for difference after intervention. Both high protein and standard protein low-
calorie diet brought about reduction in plasma GSH level yet these decrement were not significant. In
the high protein group, plasma GSH level decreased from 3.520 + 0.2045 before intervention to 3.460
+ 0.2473 after completing 8-weeks of diet (P=0.316). Whereas in standard protein group, GSH level
decreased very slightly from 3.549 + 0.241 to 3.519 + 0.41 with P=0.708.
 t
independent samples t-test
M
Mann-Whitney test
p
paired samples t-test
*
significance level was set at P<0.05
Graph 2. Mean plasma concentration of glutathione (GSH) in high protein low
calorie and standard protein low calorie diet before and after 8 weeks of intervention

As it was depicted in graph 3, in high protein group, MDA level increased by 0.0448+ 0.117 while
GSH level decreased by -0.059 + 0.1673 after diet intervention. On the contrary, in standard protein
group, both MDA and GSH decreased after completing the diet with rate of decrement of -0.0345 +
0.1174 and -0.03 + 0.388 for MDA and GSH, respectively. Intergroup comparison analysis showed
that rate of changes between two groups were not significant in both MDA (P= 0.193) and GSH level
(P=0.833).
 t
independent samples t-test
significance level was set at P<0.05

Graph 3. Mean rate of change (decrease or increase) of MDA and GSH level in high protein
low calorie and standard protein low calorie diet before and after 8 weeks of intervention
DISCUSSION
Eight weeks of low calorie diet intervention to obese people with weight cycling resulted in an increase
of plasma malondialdehyde (MDA) level for group of subjects which received high protein yet it
brought about MDA level reduction in the standard protein group. The rise of plasma MDA level in
high protein group indicated that probably there was still an ongoing oxidative stress process within
subjects belonging to this group. On the other hand, the measurement of glutathione (GSH) level after
subjects had completed the diet program showed that the GSH level decreased in both high protein
and standard protein group. However, it should be noted that the rate of reduction of GSH level in
standard protein group was lower than that of in high protein group. A possible explanation for this is
that since in high protein group a state of oxidative stress was still taking place, GSH as an endogenous
non-enzymatic antioxidant was consumed to balance increasing formation of pro-oxidants. Generation
of superoxide will be attenuated by superoxide dismutase that catalyze the partitioning of superoxide
radical and result in hydrogen peroxide (H2O2) which undergo further reaction that generate water
molecule by the work of GSH and the other antioxidants. Hence, rate of GSH depletion was higher in
high protein group as it was utilized to counter the increase of pro-oxidants while standard protein
group underwent lower GSH reduction since the oxidative stress condition seemed to be less profound
as it was indicated by depletion of MDA level.
Oxidative stress implies that there is a disruption in the balance of pro-oxidants antioxidants system
in which the rate of pro-oxidant generation exceeds the capability of endogenous and exogenous
antioxidants to remove it. We assumed that the excess of protein intake in high protein group was
responsible for generating more pro-oxidants thus causing a state of oxidative stress. An intake of
protein exceeding the needed amount with concurrent deficit in carbohydrate caused shift in catabolism
where protein were utilized to harvest energy. There would be a state of imbalance in macromolecule
composition in which amino acids supply was excessive yet carbohydrate derivate were lacking. In a
body which trying to fulfill its energy need due to caloric restriction, consumption of amino acids will
increase as it is available in high amount. However, high protein supply gives rise to increase in
thermogenic response which is accompanied by a lower efficiency of food energy utilization, an
increase in oxygen consumption, and impaired oxidative phosphorylation capacities. Combination of
excessive amino acids availability and utilization with consequence of increased body thermogenic
response result in an increase in electron flow along the mitochondrial respiratory chain that eventually
lead to oxidative stress.8 Oxidized amino acids from the breakdown of protein will enter the Krebs
cycle, then advances to mitochondrial respiratory chain to generate energy in the form of ATP.
Oxidation of amino acids as substrates is accompanied by the generation of reducing equivalents that
will be re-oxidized in mitochondrial electron transport chain. This will enhance electron flow in
respiratory chain that eventually produces reactive oxygen species in mitochondria such as superoxide
in coenzyme Q. Rate of ROS formation is so high that endogenous as well as exogenous antioxidants
could no longer counteract thus ROS begins to make deleterious effect through oxidative stress. One
of possible mechanism of cell injury by the oxidative stress is by cell membranes lipid peroxidation
with MDA as the end product of the reaction.
The result of this study was coherent with several experimental studies in rats suggesting that high
protein diet enhances oxidative stress especially when measured in a specific organ. Study by
Camiletti-Moirn showed that treating rat with 45% high protein intake for 12 weeks significantly
increased brain thiobarbituric acid-reactive substances (TBARs, P=0.042), brain protein carbonyl
content (PCC, p=0.006) as well as brain antioxidant enzymes activity (P<0.01) which were measured
by total SOD, manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (CuZn-
SOD), and catalase activity when compared to the normal 10% protein group.9 Another study by
Sophia D et al suggested that high protein diet (100% raw soy flour) for 30 days resulted in
significantly higher level of lipid peroxidation and decrease in antioxidant enzymes including SOD,
catalase, and glutathione peroxidase in pancreatic tissue.10 Another experiment of high protein intake
in rats for 2 weeks showed that the diet significantly increased malondialdehyde and decreased T-AOC
and activities of SOD and GSH-Px in rats pancreas compared to standard potein control (all P<0.05).11
However, there is a study by Petzke KJ et al which having slightly contradictory result suggesting that
high protein intake given for rats in a long term basis (15 weeks) did not result in consistently
significant difference between different amount of protein group at different organs. In that study,
adequate protein (13,8%) and high protein without RRR-a-tocopherol acetate (HP-toc) resulted in
higher plasma protein carbonyl concentration and liver lipid peroxides (TBARs) levels compared with
medium protein (25,7%) and high protein (51,3%) diets (P<0.05). Besides, no significance was found
in the difference of plasma GSH concentration among various protein intake yet total blood GSH
concentrations were found to be significantly lower in the HP-toc diet compared with the remainder
diet (P<0.05). Furthermore, GSH concentration in liver was significantly lower in adequate protein
diet compared with the other diet (P<0.05).12
A randomized controlled trial conducted by Kitabchi AE et al which gave 6-months diet intervention
of high protein composition similar to this study (30% protein, 40% carbohydrates, and 30% fats) with
500 kcal of caloric restriction resulted in a somewhat contradictory conclusions with this study. The
former study suggested that the subjects on high protein diet underwent improvement in markers of
oxidative stress and lipid peroxidation. Dichlorofluoreschein (DCF) level as a marker of oxidative
stress underwent reduction from 3.2 + 0.1 mol/l in the baseline to 2.4 + 0.1 mol/l after 6 month of
intervention. Additionally, MDA level also decreased from 1.1 + 0.06 to 0.7 + 0.05 mol/l.13
Presumably this discrepancy was mainly caused by different duration of diet intervention where that
study was conducted in 6 month and restricted a smaller amount of daily caloric intake which was only
500 kcal/day based on resting energy expenditure.
CONCLUSION
Low calorie diet with high protein proportion for 8 weeks resulted in increase in plasma MDA and
GSH level while standard protein diet brought about decrement in both plasma MDA and GSH yet
mean difference of change in MDA and GSH level of both group were not statistically significant. An
increase of MDA as a marker of lipid peroxidation with concurrent decrease in GSH as endogenous
antioxidant after high protein diet indicated that probably high protein diet enhance oxidative stress.
ACKNOWLEDGEMENT
We would like to thank Directorate of Research and Community Services (DRPM) Universitas
Indonesia for funding this research by PITTA grant 2017.
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