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Increased glycation of hemoglobin and plasma

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 Article in press - uncorrected proof
Clin Chem Lab Med 2007;45(8):996–999
2007 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2007.248
Increased glycation of hemoglobin and plasma proteins in
normotensive, non-diabetic obese Indian subjects: putative
role of lipid peroxides
Viswanathan Sathiyapriya1,a, Nambiar
Selvaraj1,a, Hanumanthappa Nandeesha1,a,
Zachariah Bobby1,*, Aparna Agrawal2, Magadi
Gopalakrishna Sridhar1, Purushothaman
Pavithran3 and Nadaradjan Rattina Dasse1
1
Department of Biochemistry, Jawaharlal Institute of
Postgraduate Medical Education and Research,
Pondicherry, India
2
Department of Medicine, Jawaharlal Institute of
Postgraduate Medical Education and Research,
Pondicherry, India
3
Department of Physiology, Jawaharlal Institute of
Postgraduate Medical Education and Research,
Pondicherry, India
Abstract
Background: Glycation and lipid peroxidation are
spontaneous reactions believed to contribute to the
pathogenesis of many clinical disorders. The purpose
of the present study was to evaluate the levels of lipid
peroxides and glycated proteins in normotensive,
non-diabetic obese Indian subjects and to assess pos-
sible associations between them.
Methods: A total of 28 obese male subjects and 20
non-obese subjects were included in the present
study. Whole blood glycated hemoglobin, plasma lip-
id peroxides and fructosamine levels were estimated
in both groups.
Results: Lipid peroxides, glycated hemoglobin and
fructosamine levels were significantly higher in obese
subjects in comparison with non-obese subjects. We
also found a significant association between malon-
dialdehyde and body mass index (rs0.424, ps0.025).
Partial correlation analysis revealed that malondial-
dehyde was significantly correlated with glycated
hemoglobin (rs0.590, ps0.01) and fructosamine
(rs0.442, ps0.021) after controlling for glucose.
Conclusions: Increased glycation of proteins was
found in normotensive, non-diabetic obese Indian
subjects. These data also support the premise that lip-
id peroxides per se play a role in the glycation of
hemoglobin and plasma proteins.
Clin Chem Lab Med 2007;45:996–9.
a
These authors contributed equally to this work.
*Corresponding author: Dr. Zachariah Bobby, Assistant
Professor, Department of Biochemistry, Jawaharlal Institute
of Postgraduate Medical Education and Research,
Pondicherry-605 006, India
Phone: q91-413-2273078, Fax: q91-413-2372067,
E-mail: zacbobby@yahoo.com
2007/488
Keywords: fructosamine; glycated hemoglobin; gly-
cation; lipid peroxides; obesity.
Introduction
Obesity is a major public health problem in both
developed and developing countries. Besides being
associated with increased susceptibility to cardiovas-
cular diseases, obesity correlates with the occurrence
of type 2 diabetes mellitus (1). In animal models of
obesity, changes have been identified in insulin
release and cellular action, as well as in the meta-
bolism of many peripheral tissues (2).
Although obesity has a genetic etiology, the major
precipitating factor is environment, mostly related to
sedentary lifestyle (3). In recent years, oxidative stress
has been considered as a pivotal factor in various
complications associated with obesity (4).
There is now overwhelming evidence indicating
that oxidative stress, defined as an imbalance
between oxidants and antioxidants in favor of the for-
mer, leads to many biochemical changes and is an
important pathological mediator in a wide spectrum
of human disease (5). Oxygen free radicals are highly
reactive and attack almost all cell components, caus-
ing damage to surrounding tissues (5).
The most deleterious impact of oxidative stress is
lipid peroxidation, which has been implicated in the
pathogenesis of numerous diseases, including
atherosclerosis, diabetes, cancer, and aging (6–8).
Lipid peroxidation is widely measured by determina-
tion of its final breakdown product, malondialdehyde
(MDA). Lipid peroxides in vitro have been found to
enhance the glycation of proteins (9, 10), yet another
process known to have pathological consequences
(11).
Although body mass index (BMI) is the most fre-
quently used index of obesity, it does not reflect fat-
ness uniformly in all populations, and interethnic
extrapolations are not justified (12). For a given age,
sex and body fat level, Caucasians have higher BMI
than Asians. This has prompted the World Health
Organization to issue preliminary Asian obesity
guidelines (13), in which it is recommended to con-
sider Asians as obese if their BMI is G25 kg/m2.
The objectives of this study were: 1) to determine
whether glycated hemoglobin (HbA1C), fructosamine
and MDA concentrations were increased in non-dia-
betic, non-hypertensive obese individuals; and, if so,
2) to determine the relationship between MDA con-
centrations and fructosamine and HbA1C.
 Article in press - uncorrected proof
Sathiyapriya et al.: Increased glycated circulating proteins in obese subjects 997

Materials and methods

Subjects

The study population consisted of 28 obese male subjects

(BMI )25 kg/m2) and 20 non-obese age-matched subjects
(BMI -25 kg/m2). Subjects with a history of diabetes, hyper-
tension or any infection were excluded from the study.
Informed consent was obtained from all individuals after the
purpose and nature of the study had been explained. This
study was approved by the Ethics Committee of our institute.

Anthropometric measurements Body weight and height of

the participants were measured. BMI was calculated as
weight divided by height squared (kg/m2).

Sample collection After subjects had fasted overnight for

at least 8 h, blood (5 mL) was drawn and collected in EDTA
bottles. Whole blood was used for the estimation of HbA1C.
Plasma was collected from the remainder of the sample by
centrifugation at 2500=g for 5 min at 48C. Plasma glucose
levels were estimated immediately and the remainder of the
sample was stored at –708C for estimation of MDA and
fructosamine.
Biochemical analyses
Fasting plasma glucose was measured using a fully auto-
mated glucose oxidase method with a commercially availa-
ble kit (Agappe Diagnostics, Kerala, India) on a Ciba Corning
550 express plus autoanalyzer (Ciba Corning Diagnostics,
Oberlin, OH, USA). HbA1C was estimated by ion-exchange
chromatography (Biocon Diagnostik, VöhlMarienhagen,
Germany). Plasma fructosamine was measured by the p-
indonitrotetrazolium violet kinetic method using Raichem
kits (Hemagen Diagnostics, San Diego, CA, USA) adapted for
the Ciba Corning 550 express plus analyzer. MDA was meas-
ured using the thiobarbituric acid-reactive substances meth-
od (14).
Statistical analysis
All results are presented as mean"SD, except for HbA1C and
MDA, which are presented as median (interquartile range).
Student’s t-test and the Mann-Whitney U-test were used to
assess the significance of differences between the groups.
Correlation analysis was performed using the Pearson test.
A p-value of -0.05 was considered significant.
Results
Data for the obese and non-obese subjects are shown
in Table 1. There was no significant difference
Figure 1 Linear regression analysis of MDA and glycated
hemoglobin in obesity (rs0.588, ps0.001).
between the two groups with respect to age, waist-
to-hip ratio and fasting glucose concentrations. Of the
obese subjects, 11 had impaired fasting glucose val-
ues. Lipid peroxides and the extent of protein glyca-
tion, measured as HbA1C and fructosamine, were
significantly higher in obese subjects compared to
controls. Lipid peroxides were significantly indepen-
dently associated with HbA1C (rs0.59, ps0.01) when
the influence of fasting glucose was excluded during
partial correlation analysis. Regression analysis of
MDA and HbA1C is shown in Figure 1. Statistical anal-
ysis revealed a significant correlation between MDA
and fructosamine, even when the influence of plasma
glucose was subtracted by partial correlation analysis
(rs0.44, ps0.02). Linear regression analysis of fruc-
tosamine and MDA is depicted in Figure 2. A signifi-
cant positive correlation was also obtained between
BMI and MDA (rs0.42, ps0.03).
Discussion
Glycation of human proteins is currently of great
experimental and clinical interest (15). Glycation of
hemoglobin and plasma proteins affords the clinician
an index of diabetic control (15), while glycation of
other proteins is reportedly linked to late complica-
tions of diabetes mellitus and chronic renal failure
(11). Glycation can markedly affect protein function
(16).

Table 1 Mean"SD for age, BMI, MDA, glycated hemoglobin and fructosamine in obese subjects and non-obese controls.

Parameter Controls (ns20) Obese subjects (ns28)

Age, years 34.40"10.51 39.04"8.32

BMI, kg/m2 21.54"1.13 27.08"1.55*
Waist-to-hip ratio 0.91"0.07 0.93"0.03
MDA, mmol/L 2.64 (1.33–3.47) 3.08 (2.17–4.9)*
Glycated hemoglobin, % 4.9 (4.26–5.43) 5.87 (5.37–6.83)*
Plasma glucose, mmol/L 4.90"1.00 5.19"0.91
Fructosamine, nmol/mL 2.16"0.43 2.39"0.30*
Glycated hemoglobin and MDA are reported as median (interquartile range). *p-0.05 compared to controls.
 Article in press - uncorrected proof
998 Sathiyapriya et al.: Increased glycated circulating proteins in obese subjects
Figure 2 Linear regression analysis of MDA and fructos-
amine in obesity (rs0.442, ps0.019).
The two traditional factors found to influence the
degree of glycation of proteins are the prevailing
glucose concentration and the half-life of the protein
(15). However, evidence in the literature indicates
increased glycated protein levels in some non-diabet-
ic pathological states (16–19).
In the present study, we found an increase in both
HbA1C and fructosamine concentrations in obese Indi-
an subjects. To the best of our knowledge, no study
to date has attempted to determine the concentra-
tions of HbA1C and fructosamine in non-diabetic, nor-
motensive obese subjects.
Apart from the presence of increased glycation
reaction, we also found increased MDA levels in plas-
ma from obese individuals. We observed a parallel
increase in HbA1C and MDA, with significant correla-
tion between their respective increments. Previous
studies carried out in other ethnic groups have also
found that levels of plasma MDA are increased in
obese subjects compared to age-matched controls
(20–22). Olusi et al. reported that obesity is an inde-
pendent risk factor for increased lipid peroxides in
obese individuals (20). Similarly, increased plasma
MDA levels have been reported in non-diabetic, nor-
motensive obese subjects (21).
The present study showed a significant positive
association between HbA1C and MDA, even when the
effect of fasting glucose was nullified by partial cor-
relation analysis. Similarly, a significant positive asso-
ciation was found between fructosamine and MDA.
Previous in vitro studies, including ours, have report-
ed higher HbA1C levels in erythrocytes co-incubated
with MDA and glucose than in erythrocytes incubated
with glucose alone (9, 10). We have previously dem-
onstrated that fructosamine levels are closely asso-
ciated with MDA levels in patients with nephrotic
syndrome, chronic renal failure, rheumatoid arthritis
and asthma (23–26). We have also reported a signif-
icant correlation between HbA1C and MDA in patients
with asthma, chronic renal failure and hyperthyroid-
ism (26–28). The mechanism by which MDA enhances
the glycation process has not been clearly elucidated.
It is thought that MDA enhances protein glycation by
acting as an anchor between sugar and hemoglobin
moieties (29). It has also been suggested that oxida-
tive stress can facilitate the autoxidation of glucose to
dicarbonyl intermediates, an early step in the Maillard
reaction (9). Obese individuals are known to be more
insulin-resistant (30, 31). Occasional transient higher
glucose levels in these subjects could also be a con-
tributing factor to their higher protein glycation.
Glycation and lipid peroxidation are known to play
a key role in complications of many pathophysiolo-
gical process (6, 11). Recent studies have uncovered
a myriad of pathological events induced by glycated
albumin, which includes increased inflammatory
responses (32, 33). Studies have indicated that obe-
sity is a chronic inflammatory state (34, 35). This
inflammatory state has been reported to contribute to
the major obesity co-morbidities, including type 2
diabetes, cardiovascular disease and the metabolic
syndrome (34). It has been demonstrated that obese
animals and humans have significantly higher levels
of circulating proinflammatory cytokines such as
interleukin-6 and tumor necrosis factor a (35).
In conclusion, results from the present study pro-
vide evidence of an increase in both glycated protein
and MDA levels in non-diabetic, normotensive obese
individuals and indicate that these processes are
closely associated. Given the detrimental role of gly-
cation in pathological conditions, strategies to avert
protein glycation in obese subjects would be of great
help in controlling the problems associated with
obesity.
Acknowledgements
This work was supported by a grant from the Department of
Science, Technology and Environment, Pondicherry, India
and by the Council of Scientific and Industrial Research, New
Delhi, India, in the form of a Senior Research Fellowship to
Ms. V. Sathiyapriya.
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Received November 23, 2006, accepted April 10, 2007