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Biomedical Research

The role of Methyl Glyoxal in relation to patho-physiological complications in diabetes mellitus

Author(s): Mukhopadhyay S. Gachhui R., Kar M.

Vol. 17, No. 2 (2006-05 - 2006-08)

Biomedical Research 2006; 17 (2): 111-116

Mukhopadhyay S. ٭٭Gachhui R., ٭Kar M.

٭Department of Biochemistry, NRS Medical College and Hospital, Kolkata, India
٭٭Department of Life Science and Biotechnology, Jadavpur University, Kolkata, India

Key words: Diabetes mellitus, Methyl Glyoxal, Reduced Glutathione, Free radical, Antioxidant, Malondialdehyde

Accepted February 17, 2006

Abstract

Methyl glyoxal (MG) is an endogenous reactive dicarbonyl compound, elevated in diabetes mellitus. It is produced primarily from triosphosphate by nonenzymatic and enzymatic pathways and detoxified to D-lactate by reduced glutathione-dependent glyoxalase enzyme system. We have investigated the serum levels of MG and its correlation with lipid peroxidation in type-II diabetes mellitus.

We studied 59 type- II diabetic patients and age- and sex- matched 43 normal con-trol. We collected the fasting blood samples of all the subjects and estimated blood glucose. According to the hyperglycaemic status, we subdivided the diabetic patients into 3 groups; Group I showed blood glucose of 140 – 200 mg/ dL, Group II: 201- 300mg /dL and Group III had > 301 mg / dL. The serum levels of catalase and superoxide dismutase (SOD) enzyme activities, total antioxidant status (TAS), reduced glutathione levels as well as MG and the cellular damage marker, malondialdehyde (MDA) of the patients and normal controls were estimated.

The cellular damage in diabetes mellitus as reflected by increased MDA levels was found to be correlated with the elevation of MG levels but not with the hyperglycaemic status of the patients. The reduced level of glutathione is thought to be responsi-ble for MG accumulation in diabetes mellitus.

We, therefore, proposed that the serum MG level may possibly be used as a marker to understand the free radical-mediated cellular insults in diabetes mellitus.

Introduction

In diabetes mellitus, oxidative [1-3] and carbonyl stress [4] are associated with increased production of reactive oxygen species(ROS) and supposed to act as major causal factors in the development of chronic complications like cataract, retinopathy, nephropathy, neuropathy and mi-croangiopathy [5-9]. Prevailing hypothesis suggests that poor antioxidant defense mechanism stimulates free radical formation [10,11]. Moreover, hyperglycaemia is thought to mediate cellular damage through mechanism involving nonenzymatic glycation of proteins. We previously reported that glycation of haemoglobin (Hb) re-leases free reactive iron from protoporphyrin cage due to its conformational alterations and further generate dreadly damaging hydroxyl radical by Fenton reaction [12,13].

Substantial recent data indicate that in diabetes mellitus, glucose toxicity is mediated through increased production of highly reactive dicarbonyl compound, methylglyoxal, glyoxal and 3 deoxyglucosone [14]. Methyl glyoxal (MG) is an endogenous metabolite, primarily produced from tri-osephosphate by enzymatic [15] and nonenzymatic pathways [16]. There are, however, other important sources of MG formation in vivo. It can be formed in lipid peroxidation, catabolism of ketone bodies, threonine [17] etc.

This compound is detoxified to nontoxic D-lactate by glu-tathione dependent glyoxalase enzyme system [18]. This small ketoaldehyde compound is more potent than glu-cose even 36 times faster 18] and it attacks basic amino acids (like histidine, arginine, lysine) of proteins by Mail-lad reaction [19,20] and modified proteins undergo re-ceptor mediated endocytosis followed by lysosomal degradation as well as cytokine synthesis and secretion [18]. Rapid post translational modifications of such pro-teins finally form Advanced Glycation Endproducts (AGE) which by further oxidations produce free radicals [21]. MG is extremely reactive glycating agent for collagen [19], enzymes and other important cellular constituents [22] and also reported to be toxic to cultured cells [23]. It has also been reported that MG induces altered chaperone activity of α crystallin protein in diabetes and aggregates early cataract formation [24].

Reduced glutathione plays a dual function in the cell as an antioxidant as well as a detoxifying agent. It is commonly considered as an antioxidant co-enzyme in glutathione peroxidase which reduces peroxides or superoxides, yielding oxidized glutathione. Moreover, reduced glu-tathione has a discrete detoxification function in glyox-alase pathway where it facilitates toxic MG to D-lactate formation.

However, no study has so far been correlated among the elevated levels of MG, antioxidant status, lipid peroxidation and the reduced levels of glutathione in diabetes mellitus. The present study was, therefore, undertaken to evaluate the role of MG in the development of complications in diabetes mellitus.

Materials and Methods

The study included 59 randomly selected type II diabetic patients from the out patient and inpatient Dept. of Diabetology, NRS Medical College & Hospital, with the history of common diabetic complications. The patients were divided into 3 subgroups according to the fasting blood glucose level at the time of presentation.

The new patients who were diagnosed for the first time as diabetic with the fasting blood glucose levels ranging from 140mg/dL to 200mg/dL and not under medication were considered as Group I. Patients having the fasting blood glucose levels below 140mg/dL were excluded from the study and the old patients who were under medication but showed the fasting blood glucose levels within the range of 140 – 200mg/dL were also excluded from the study.

Patients belonged to Group II were having the fasting blood glucose level above 201mg/dL but below 300mg/ dL at the time of presentation.

Table 1: Shows the design of study indicating categories, distribution of age and sex and the fasting blood glucose levels of the control and diabetic patients at the time of presentation.

Table 1

The patients who presented with the fasting blood glucose level above 301mg/dL were included in Group III.

Patients of Group II and Group III were under medication.

The age and sex matched 43 normal subjects were selected randomly as the normal control who had no history of diabetes and no other co-morbid illness. Moreover, the controls had the fasting and p.p. blood glucose levels within the normal range of 80 – 120 mg/dL

Blood samples (5mL/subject) from the diabetic and con-trol subjects were collected aseptically and distributed into 2 sterile vials, one containing fluride (1 ml) for fast-ing blood glucose estimation and the other was free from any anticoagulant (4ml). The blood glucose level was estimated within 1h of collection by glucose-oxidase method using commercial kit, Transasia. On the same day the p.p. blood glucose levels of both the diabetic and con-trol subjects were estimated after collection of the blood samples at 2nd h of the main meal.

Serum samples were obtained by centrifuging the clotted blood samples at 5000 rpm for 5 min and processed for the estimation of the following biochemical parameters:

Serum Catalase activity was measured by following the spectrophotometric method of Goth L [25]. Serum SOD activity was estimated by a spectrophotometric assay based on NBT system [26,27]. Serum reduced glutathione level was estimated spectrophotometrically by DTNB method [28]. Serum total Antioxidant status (TAS) was measured spectrophotometrically using the method as described by Rice-Evans and Nicholas [29] based on ABTS+ Assay. Serum Methyl Glyoxal (MG) level was measured by using the method of Kooper [30], based on 2, 4, DNPH in alkaline condition and serum MDA level was determined by following the method of Ohkawa [31], a spectrophotometric method based on TBA (thioberbi-turic acid).

Statistical Analysis

The assay results of both the diabetic and control subjects were taken for statistical evaluation. The statistical analysis was done by using student’s t-test and the significance level was considered up to the p value less than 0.05. The significance levels of the different parameters were also compared.

Results

The results of different parameters of the diabetic and control subjects are summarised in Table 2.

The mean concentration of serum catalase activity, SOD activity and reduced glutathione levels were found to be reduced in all the 3 groups of diabetic patients when compared with the control (Table 3).

Table 2: Mean (± S.E.M) concentrations of different anti oxidants and oxidative stress parameters of 3 diabetic groups and control

Table 2

(For larger image of table, click here)

Table 3: Shows the levels of statistical significance of mean (± S.E) concentrations of different antioxidant and oxidant parameters between control vs. 3 groups of diabetic patients and diabetic Group II vs. Group I and Group III.

Table 3

(For larger image of table, click here)

n.s – not significant

Comparative analysis of Total Antioxidant Status

Fig 1: Comparative analysis of Total Antioxidant Status (TAS) between the control and diabetes patients TAS expresses the overall picture of antioxidant status of serum and it is actually the sum of all the various antioxidants in serum regardless of their origin. TAS levels were significantly decreased in all the 3 diabetic groups compared to control (Fig 1). NormalGroup IGroup IIGroup III0123456781Malon Di Aldehyde (MDA) Level (nmol/L)

Comparative analysis of MDA levels

Fig 2: Comparative analysis of MDA levels between control and diabetic groups.

Methyl glyoxal levels as well as serum MDA levels of all the 3 diabetic groups were found to be significantly increased than the control group.

The results showed that the mean concentrations of serum catalase activity, reduced glutathione levels and TAS levels of Group I and Group III were very much attenuated compared to Group II. Only the antioxidant parameter,

Corelation between serum MG levels and reduced glutathione levels

Fig 3: Corelation between serum MG levels and reduced glutathione levels (GSH) of the control and 3 diabetic groups serum SOD activity of group II was found to be less than the Group I and Group III, though the result was not statistically significant (Table 3). However, the methyl glyoxal and the cellular damage marker serum MDA levels were found to be significantly elevated in Group I and Group III in comparison to Group II (Fig. 2).

Discussion

There is a wide spread of belief that hyperglycaemia probably plays some major role in the development of chronic complications of diabetes and that the degree of risk factor which hyperglycaemia confers is proportional to the chronic level of blood glucose. Thus, we have subdivided diabetic patients into three major groups according to their fasting blood glucose level. But paradoxical findings are recorded when the different biochemical parameters of the subgroups of diabetic patients are ana-lysed.

Results from the Table 2 show a significant low levels of serum catalase, SOD, reduced glutathione and TAS asso-ciated with elevated levels of lipid peroxidation end prod-uct (indicated by MDA levels) and Methyl Glyoxal (MG) levels in all groups of the diabetic patients compared to the control. The oxidant and antioxidant imbalance in diabetes mellitus has previously been reported, however, the striking evidences have been found to compare these serum variables in between the diabetic subgroups.

The serum catalase, SOD, reduced glutathione and TAS (Fig 1) are reduced where as the MDA levels (Fig 2) and MG levels are found to be increased in Group I compared to Group II (Table 2). Surprisingly the serum catalase, TAS and reduced glutathione levels have been found to be further elevated followed by low MDA levels, an index of cellular damage and the MG levels, the toxic gly-cating metabolite in Group II compared to Group I.

At the early stage of hyperglycaemic condition (Group I), the plasma glucose level at 200 mg/dL, may enhance oxidative stress due to sudden attack of free radicals, which is reflected in the elevation of the levels of MG and MDA. But in Group II where glucose levels are increased above the renal threshold, gene expression and repairing mechanism may be in favour of synthesizing more anti-oxidants to compensate oxidative and carbonyl stress-mediated cellular damage and possibly the cells try to cope up with the adverse situation. Results of Group III indicate that when blood glucose levels are above 300 mg/dL, the antioxidant barrier is drastically broken down and the cells cannot cope up with the situation as depicted in the Table 2 where catalase, TAS, reduced glutathione are shown to be significantly poor. On the other hand, highly significant levels of MG and MDA are recorded compared to Group I and Group II. Moreover, the level of SOD has been declined in Group II compared to Group I and Group III. SOD converts superoxide to H2O2 and catalase reduces H2O2 into neutral water. However, H2O2 itself acts as a precursor of dreadly damaging free radical *OH by performing the Fenton reaction with free reactive iron which has been reported to be increased [12, 13 ]. In Group I and Group III, though the mean concentrations of SOD are higher than in Group II, yet the catalase levels are found to be comparatively lower in both the groups.

High concentration of mean SOD may indicate that much more superoxides are converted into H2O2 but low levels of catalase cannot be able to convert the total H2O2 level into neutral water. Therefore, H2O2 concentrations may be increased in Group III and Group I diabetics which pro-duce *OH ( by Fenton chemistry with increased level of free reactive iron) and thus the cellular damage has been found to be significantly increased in Group III and Group I as indicated by the elevated levels of MDA (Fig 2, Table 3). Therefore, the low level of mean concentra-tion of SOD in Group II may not have the significant ef-fect on the cellular damage compared to the other 2 groups.

Reduced glutathione acts as a cofactor in MG catabolism. MG binds with reduced glutathione and produces hemimercaptal, an intermediate which turns into S-D Lac-toyl glutathione by glyoxalase I enzyme and finally converted to D-lactate by glyoxalase II. Fig 3 shows the increased level of MG is associated with the decreased level of reduced glutathione in diabetes patients. Reduced glu-tathione acts as an potent antioxidant molecule as well as it plays the role in xenobiotics in phase II reactions. When the oxidative stress increases in diabetes patients, reduced glutathione level is depleted automatically and as a result the MG is accumulated due to lack of reduced glutathione level. It is thus evident from the data that al-though the patients of Group I showed lower levels of glucose, compared to Group II, their MDA levels are significantly higher than Group II. However, the MG levels among all hyperglycaemic groups are proportionately and significantly elevated with the decreased levels of reduced glutathione and an increase in MDA levels.

Total antioxidant status is also proportionately declined according to inclined levels of MG in diabetic groups. Based on the above observations it may be concluded that free radical-mediated cellular damage in type II diabetic patients is correlated with MG level irrespective of hyperglycaemic condition. The accumulation of metabolic hazard, MG which increases oxidative stress is directly linked to the serum levels of reduced glutathione.

Estimation of the level of MG as a biomarker may give valuable guidelines to understand the prognosis of the diabetic secondary complications and pathophysiological conditions.

Acknowledgements

The authors acknowledge with gratitude the cooperation of Dr. Ashok Ghosh, Dr. Santanu Sen, Arpita Ghosh and the technical assistance of Mr. Sukanta Koner and Sou-mita Shome. The work has been supported by DST. Govt. of India (SR/ WOS-A/ LS-414/ 2004).

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Correspondence:
Soma Mukhopadhyay
C/o Dr. Manoj Kar.
Department of Biochemistry, NRS Medical College and Hospital
138, AJC Bose Road.
Kolkata 14, West Bengal, India
e-mail: somashis ( at ) vsnl.net
somashis1 ( at ) rediffmail.com

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