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OJHAS Vol. 10, Issue 2:
(Apr-Jun 2011) |
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| Correlation
of Lipid Peroxidation with Glycated Haemoglobin Levels in Diabetes Mellitus |
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Varashree BS, Assistant Professor,
Gopalakrishna Bhat P, Professor,
Department of
Biochemistry, Kasturba Medical College, Madhav Nagar,
Manipal- 576104 |
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Address for Correspondence |
Department of
Biochemistry,
Kasturba Medical College,
Madhav Nagar,
Manipal- 576104, India
E-mail:
varasuhas@yahoo.com |
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Varashree BS, Bhat GP. Correlation
of Lipid Peroxidation with Glycated Haemoglobin Levels in Diabetes Mellitus. Online J Health Allied Scs.
2011;10(2):11 |
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Submitted: Apr 28,
2011; Accepted: Jul 16, 2011; Published: Jul 30, 2011 |
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| Abstract: |
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Reactive oxygen species are
crucial to normal biological processes; they are potentially dangerous
and are commonly referred to as prooxidants. The reactive oxygen intermediates
can cause direct cellular injury by including lipid and protein peroxidation
and damage to nucleic acid. The polyunsaturated fatty acids present
in the cells are vulnerable to free radicals causing lipid peroxidation.
Determination of Malondialdehyde (MDA) by using thiobarbituric acid
is used as an index of the extent of lipid peroxidation. This study
was done to know if lipid peroxidation correlated with the glycated
haemoglobin levels. Diabetic status was assessed by estimating fasting
blood sugar and glycated haemoglobin level while oxidant stress was
evaluated by estimating erythrocyte MDA levels. The lipid peroxidation
in erythrocyte lysates was significantly increased in diabetic individuals
compared to controls (p<0.001). The result of this study indicates
that in diabetic individuals are more prone to oxidative stress
and glycated haemoglobin is a marker in evaluating the long term glycemic
status in diabetic individuals.
Key Words:
Oxidative stress; Glycated haemoglobin; Lipid peroxidation; Malondialdehyde
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Cells can tolerate mild oxidative
stress, which often results in up regulation of the synthesis of antioxidant
defence systems in an attempt to restore the balance, but when severe,
cause derangement in all metabolism causing cell injury and death. In
most human diseases, oxidative stress is secondary phenomenon, a consequence
of the disease activity. There is a growing awareness that oxidative
stress plays a role in various clinical conditions e.g. malignant diseases,
diabetes, atherosclerosis etc.
Diabetes mellitus, a common
metabolic disorder resulting from defects in insulin secretion or action
or both, is characterized by hyperglycemia often accompanied by glycosuria,
polydipsia, and polyuria.(1) During diabetes, persistent hyperglycemia
causes increased production of free radicals especially reactive oxygen
species (ROS), for all tissues from glucose auto-oxidation and protein
glycosylation.(1) In addition, superoxide is generated by the process
of glucose autoxidation that is associated with the formation of glycated
proteins in the plasma of diabetic patients. Many factors are
responsible for this like polyol pathway, prostanoid synthesis and protein
glycation which disturbs the antioxidant defence system thereby increasing
the amount of reactive oxygen species.(2) The increase in ROS production
contributes to the development of diabetic complications.
Monitoring blood glucose control
as performed by patients and health care providers is considered as
the cornerstone of diabetes care. Carbohydrates such as glucose can
bind non enzymatically to proteins such as hemoglobin in a process known
as glycation. The human erythrocytes are freely permeable to glucose
and within each erythrocyte glycated hemoglobin is formed continuously
from hemoglobin. The formation of glycated hemoglobin is dependent on
the ambient glucose concentration. Individuals with higher levels of
blood glucose will have higher levels of glycated hemoglobin.(3) The
glycation process is slow and continuous that occurs over days to 3-4
months in vivo. In a normal person about 3-6% of HbA is glycated; in
a diabetic patient the percentage of HbA may double or triple depending
on the degree of hyperglycemia.(4-6) Glycated hemoglobin is the
best surrogate marker for setting the treatment goals. Nonenzymatic
glycation is a spontaneous chemical reaction between glucose and the
amino groups of proteins in which reversible Shiff bases and more stable
Amadori products are formed.(7) Advanced glycation end products (AGEs)
are then formed through oxidative reactions and cause irreversible chemical
modifications of proteins(7).
Free radicals are very reactive
chemical species, which can cause oxidation injury to the living beings
by attacking the macromolecules like lipids, carbohydrates, proteins
and nucleic acids.(8) The significant
targets for injury are mainly proteins and DNA than lipids. Lipid peroxidation
occurs late in the injury process. An increased concentration of end
products of lipid peroxidation is the evidence most frequently quoted
for the involvement of free radicals in human disease. It is likely
that increased oxidative damage occurs in most, if not all human diseases
and plays an significant pathological role in them.(9) Lipid peroxidation
end-products very commonly detected by the measurement of thiobarbituric
acid reactive substances (TBARS).(10)
Free radicals are produced
as a result of glycosylation of several proteins including hemoglobin
(Hb) by non-enzymatic mechanisms.(11) Subsequently free radicals change
lipid/protein ratio of membranes by affecting polyunsaturated fatty
acids and lipid peroxidation causes functional irregularities of several
cellular organelles.(11) Lipid peroxidation is a free radical-related
process, which is potentially harmful because its uncontrolled, self-enhancing
process causes disruption of membranes, lipids and other cell components.
(12) Lipid peroxidation is the oxidative deterioration of polyunsaturated
fatty acids. The free radicals steal electrons from the lipids in the
cell membrane, resulting in cell damage. Lipid peroxidation is a late
event accompanying rather than causing final cell death. The end products
of lipid peroxidation process are aldehydes, hydrocarbon gases and chemical
residues including malondialdehyde. MDA is an important reactive carbon
compound which is used commonly as an indicator of lipid peroxidation
(11). Abnormally high levels of lipid peroxidation and the simultaneous
decline of antioxidant defence mechanisms can lead to damage of cellular
organelles and lead to oxidative stress.(12) Diabetes mellitus is characterised
by hyperglycaemia together with biochemical alterations of glucose and
lipid peroxidation.(12) Significantly higher values of thiobarbituric
acid-reactive substances (TBARs) in serum, which provide an indirect
measurement of lipid peroxidation and decreased erythrocyte antioxidant
enzyme activities, have been observed in adult diabetic patients.(7)
Some complications of diabetes mellitus are associated with increased
activity of free radical-induced lipid peroxidation and accumulation
of lipid peroxidation products.(12) Diabetic red blood cells (RBC)
s were shown to be more susceptible to lipid peroxidation as measured
by TBARS in rats and humans.(10) In erythrocytes from diabetic patients
increased membrane lipid peroxidation may lead to abnormalities in composition
and function.(13) Diabetes erythrocytes have higher malondialdehyde
levels.(13) Diabetes produces disturbances of lipid profiles, especially
an increased susceptibility to lipid peroxidation.(1) An enhanced oxidative stress has been observed in these patients
as indicated by increased free radical production, lipid peroxidation
and diminished antioxidant status.(1)
The objective of the present
study is to evaluate the oxidant stress in diabetes mellitus and its
association with glycated hemoglobin levels in diabetes mellitus. The
diabetic status was assessed by estimating the fasting blood sugar and
glycated hemoglobin while the oxidant stress was evaluated by estimating
erythrocyte malondialdehyde in terms of thiobarbituric acid reacting
substance.
Sample collection:
The study group comprised of
nondiabetic individuals and diabetic patients attending the Kasturba
hospital, Manipal. Informed consent from the patients was obtained for
the study. Patients were selected at random and no distinction was made
between those with insulin dependent or non- insulin dependent diabetes.
The diabetic status was assessed by estimating the fasting blood sugar
(FBS) using glucose oxidase method. Test group consisted of fifty diabetic
individuals; whose fasting glucose level was more than 126mg%. Blood
(2ml) was collected by venepuncture into tubes containing 3.6mg EDTA
and stored at 4°C. The mean age of controls was 54±12.1 and that of
cases was 52 ± 12.1.
Erythrocyte malondialdehyde
was estimated within 24 hours of blood collection. The hemolysates prepared from the
above blood samples were stored at -25°C.
Estimation of glycated haemoglobin
by affinity chromatography: (14)
Affinity gel columns (Glycogel
B)were used to separate bound, glycosylated haemoglobin from the non-glycosylated
fraction. The gel contains immobilized m- amino-phenylboronic acid and
cross linked beaded agarose. The boronic acid reacts with the cis- diol
groups of glucose bound haemoglobin to form a reversible 5- membered
ring complex, thus relatively holding the glycosylated haemoglobin on
the column. The non- glycosylated haemoglobin is eluted. The complex
is next dissociated by sorbitol, which permits elution of glycosylated
haemoglobin. Absorbances of the bound and unbound fractions, measured
at 415nm are used to calculate the percent of glycosylated haemoglobin.
Estimation of
malondialdehyde (MDA):
Erythrocyte MDA concentration
was determined using the method described by Jain et al.(15)
At low pH and elevated temperature,
MDA readily participates in nucleophilic addition reaction with 2- TBA
generating a red fluorescent 1:2 MDA- TBA adduct. The absorbance was
read at 532 and 600nm using a spectrophotometer. Butylated hydroxyl
toluene is added to the assay mixture in order to prevent lipid peroxidation
during heating. A standard graph was prepared by taking concentration
of standard in moles/ ml along the x- axis and absorbance (532-600nm)
along the y- axis. TBARS values were calculated from the standard graph
and expressed as nanomoles/ gram of haemoglobin. Estimation of haemoglobin
was done by the method of Drabkin.(16)
The erythrocyte malondialdehyde
levels was determined in the erythrocytes taken from both individuals
with diabetes mellitus (test group) and normal healthy individuals (control
group). Erythrocyte malondialdehyde levels was higher in cases (4.7±1.7
nmoles/gHb) than the controls (3.3±2.2 nmoles/gHb). The glycated hemoglobin
level was higher in cases (8±2.9) than the controls (6.1± 2.2). The
fasting blood sugar did not correlate with the erythrocyte malondialdehyde
levels but did correlate with glycated hemoglobin i.e. p<0.05. Among
the cases the erythrocyte MDA did not correlate with glycated hemoglobin.
Thus the lipid peroxidation in the diabetic erythrocytes were significantly
higher when compared to the control group (p=0.001) (Table 1, Figure
1-4).
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Table 1:
FBS, MDA and Glycosyltaed Hemoglobin levels |
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Parameters
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Mann-
Whitney ‘u’ test |
‘p’value |
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Fasting blood sugar
(mg %) |
8.61 |
0.001 |
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MDA (nmoles/gHb) |
4.20 |
0.001 |
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Glycated hemoglobin
(% of Hb) |
8.0 |
0.001 |
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Figure 1: Age
Distribution |
Figure 2: Comparison
of Mean FBS Between Cases and Controls |
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Figure 3: Comparison
of Mean Glycated Hb Between Cases and Controls |
Figure 4: Comparison
of Mean MDA Between Cases and Controls |
Oxidative stress depicts the
existence of products called free radicals and reactive oxygen species
(ROS) which are formed under normal physiological conditions but become
deleterious when not being quenched by the antioxidant system. There
are convincing experimental and clinical evidence (1) that generation
of reactive oxygen species is increased in both types of diabetes mellitus
and that the onset of diabetes is closely associated with oxidative
stress. Free radical mediated cytotoxic process of lipid peroxidation
appears to have a role in the development of multifactorial disease,
diabetes mellitus. Possible sources of elevated free radicals
in type 2diabetes include increased production of radical oxygen species,
especially from glycation or lipoxidation processes, auto-oxidation
of glucose and oxidizing of glucose and decreased antioxidant defense
systems.(17)
In the present study the individuals
with diabetes mellitus showed statistically significant levels of lipid
peroxidation as indicated by the levels of erythrocyte MDA. The increased
levels of thiobarbituric acid-reactive substances (TBARS) suggest a
net increase in the levels of oxygen free radicals which could be due
to their increased production and/or decreased destruction. This increased
level of MDA could be because of increased glycation of proteins in
diabetes mellitus. The glycated protein may themselves act as a source
of free radicals. There is a clear association between lipid peroxide
and glucoses concentration which also could be thought to play a role
in increased lipid peroxidation in diabetes mellitus. The exact mechanism
by which the elevated blood glucose leads to membrane lipid peroxidation
is not known. Some studies have shown that glucose can enolise and then
reduce molecular oxygen to give α- keto aldehydes, hydrogen peroxide
and ROS. Hydrogen peroxide formed by superoxide dismutation regenerates
the catalytic metal oxidation state and produces hydroxyl radicals.
The ROS formed causes peroxidative breakdown of phospholipid fatty acids
and accumulation of MDA.(15) Elevated levels of MDA could also be due
to alteration in the function of erythrocyte membrane.
The present study was carried
out to know the relation of fasting sugar with glycemic control i.e.
by determining the glycated haemoglobin levels. In the present study
there was an increase in the level of glycation of haemoglobin in diabetic
patients. Results of the present study suggest that increased production
of high levels of free oxygen species is linked to glucose oxidation.
Several studies have reported similar results. The glycated haemoglobin
as a marker of glycemic status over last 2-3months and malondialdehyde
was taken as an oxidative marker of diabetes mellitus. The glycation
induced structural modification of hemoglobin.
The present study results show
that lipid peroxidation might not contribute to glycation of haemoglobin.
Martin and others (18-21) have reported similar results, whereas
Jain et al have found a positive correlation.
In conclusion, the estimation
of lipid peroxide along with lipid profiles in diabetes mellitus would
serve as a useful monitor to judge the prognosis of the patient. Improvement
of glycemic control appears to be a beneficial factor in decreasing
lipid peroxidation in patients with diabetes. Prevention of lipid peroxidation
may help to delay the development of diabetic complications. The detection
of the risk factor in the early stage of the disease helps to improve
and reduce the mortality rate.
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