Methylglyoxal increase in uremia with special reference to snakebite-mediated acute renal failure

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Clinica Chimica Acta 391 (2008) 13 – 17 www.elsevier.com/locate/clinchim

Methylglyoxal increase in uremia with special reference to snakebite-mediated acute renal failure Soma Mukhopadhyay ⁎, Arpita Ghosh, Manoj Kar Department of Biochemistry, NRS Medical College and Hospital, Kolkata, India Received 23 March 2007; received in revised form 1 January 2008; accepted 14 January 2008 Available online 24 January 2008

Abstract Background: Advanced glycation and lipoxidation endproducts (AGEs and ALEs) due to oxidative and carbonyl stress are involved in pathogenesis of several diseases including uremia. Methylglyoxal, a dicarbonyl compound is a metabolic hazard and potent glycating agent in the body, which is an important precursor of AGE and ALE. Methylglyoxal has been reported to be increased in uremia, but there is no report of MG status in snake venom mediated acute renal failure cases (SARF). We investigated the carbonyl and oxidative stress as well as the methylglyoxal concentration in SARF where renal clearance is rapidly shut down. Methods: We studied serum carbonyl compounds, methylglyoxal, total antioxidant status, GSH and cellular damage marker thiobarbituric acid reacting substances (TBARS) and intracellular erythrocytic GSH concentration following standard methods of 45 SARF and 56 normoglycemic chronic renal failure cases (CRF) and compared with 81 normal controls. Result: Methylglyoxal concentration has been found to be significantly increased in SARF associated with decreased concentration of serum as well as erythrocytic GSH and other antioxidant markers , in comparison with CRF and normal control. The cellular damage (TBARS concentration), is also found increased in SARF. Conclusion: MG increase as well as accumulation due to GSH depletion may play a pivotal role in their rapid pathophysiological complicacies in SARF. © 2008 Elsevier B.V. All rights reserved. Keywords: Methylglyoxal; Snakebite mediated acute renal failure; Normoglycemic chronic renal failure; Reduced glutathione; Total antioxidant status

1. Introduction In uremia, the retention of a variety of toxic and nontoxic compounds results into a deficient renal clearance. These retention solutes are taken to induce biochemical disorders characteristic of uremic complications [1]. Advanced glycation endproduct (AGE) and advanced lipoxidation endproduct (ALE) have been reported recently to play the major role in the complication and pathogenesis of uremia [2]. AGE and ALE are

⁎ Corresponding author. C/O Dr. Manoj Kar, Department of Biochemistry, Academy Building, NRS Medical College and Hospital, 138, A J C Bose Road, Kolkata, West Bengal, Pin 700014, India. Tel.: +913324486362, +919433070825, +919433900400, +9122276161; fax: +913322295628. E-mail addresses: [email protected], [email protected] (S. Mukhopadhyay). 0009-8981/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.01.013

formed during nonenzymatic glycation and oxidation of proteins with reducing sugar or lipid peroxidation endproduct [3,4]. Thus uremia may be described as a state of “carbonyl stress” [2]. Methylglyoxal (MG), a body metabolite is a major precursor of AGE and ALE. It is an endogenous metabolic hazard and derived nonenzymically from amine catalyzed sugar fragmentation reaction [5], triose phosphate decomposition [6], and oxidative decomposition of fatty acids [7] threonine [8] and acetone [9] or enzymatically by methylglyoxal synthetase [10]. Being a reactive dicarbonyl compound, MG reacts with lysine and arginine residues of proteins and also with MDA in Malliard reaction, yielding AGE and ALE. Among these, glyoxal-lysine dimer (GOLD), methylglyoxal-lysine dimer (MOLD), pentosidine etc. have been reported to be increased in diabetic nephropathy and also in normoglycemic uremic patients, suggest in a relationship between advanced glycation and lipoxidation reactions and the pathophysiology of renal diseases [11].

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Thornalley et al. [12] reported the increase of MG in diabetes mellitus. So AGE or ALE formation in diabetic nephropathy may be clearly understood but the mechanism of AGE increase in normoglycemic uremia remains poorly understood [2]. Several lines of evidences indeed suggested that chronic uremia appears to be a state of increased oxidative stress [13–15]. We previously reported MG increase in free radical mediated diseases like diabetes mellitus [16] and rheumatoid arthritis [17] and the correlation between increased concentration of MG with increased oxidative stress in these diseases. MG has been reported recently to enhance ROS production in neutrophils through a process involving p38 MAPK-dependent exocytosis of intracellular storage granules [18]. Although the MG concentration has been reported to be increased in end-stage renal disease patients subjected to peritoneal dialysis [19] but there is no report of status of MG in snake bite mediated acute renal failure (SARF) cases. In this study we concentrated on the status of carbonyl compounds and MG concentration in SARF and normoglycemic chronic renal failure (CRF). The snakes of Vipera sp. are commonly found in the rural areas of West Bengal, India and a large number of people are admitted for their treatment especially in the period of June to August (monsoon period) whose venom cause hemolysis, resulting into acute renal failure. We tried to understand the sudden and rapid metabolic renal functional changes due to snake venom in SARF. So we included snake venom mediated uremic patients in our study. We also studied the total antioxidant status (TAS) and cellular damage of both CRF and SARF cases. GSH helps to detoxify MG by converting it to D-lactate through glyoxalase system [20]. Thus it may play a great regulatory role in accumulation of MG in biological system [20]. So we also estimated the concentration of GSH, to better understand its role in uremia. 2. Materials and methods 2.1. Sample collection Blood samples were collected both in clotted and heparinated vials from 81 normal subjects, randomly selected, with no proteinuria (51 males, 30 females, 38.3 ± 8.3 y), 56 normoglycemic chronic renal failure (35 males, 21 females, 51.2 ± 7.9 y) and 45 snake venom (Vipera sp.)-mediated acute renal failure patients (27 males, 18 females, 39.9 ± 11.3 y). Serum was separated by centrifuging the clotted blood at 5000 rpm for 5 min. Stroma-free hemolysate was prepared from

heparinated blood by removing serum and buffy coat, washing thrice by 0.15 mol/l NaCl solution and treating with hypotonic phosphate buffer followed by centrifugation at 10,000 rpm for 30 min. None of the patients were on lipid lowering or antioxidant drugs. Clinical histories of all patients were taken which showed the normoglycemic status of SARF patients also before the snakebite. All patients were with renal failure (serum creatinine concentration N7 mg/dl). Blood samples of the patients were collected prior to hemodialysis.

2.2. Estimation of biochemical parameters Serum concentrations of the following parameters of both normal and uremic patients were estimated. TAS was estimated according to the method of Re et al. [21], based on the inhibition of radical cation ABTS+, which has characteristic long wavelength absorbance maxima at 734 nm. Serum concentration of reduced glutathione is measured according to the method of Miao-Lin-Hu [22] by spectrofluorimetric method based on a fluorescence dye o-phthalaldehyde and fluorescence intensity was measured at 420 nm, exciting at 350 nm. Intracellular erythrocytic GSH concentration has been estimated by DTNB method spectrophotometrically. The stroma-free hemolysate was prepared from heparinated blood sample of both SARF and CRF patients and normal control and intracellular GSH was estimated [22] and expressed against per gram of hemoglobin. Carbonyl compounds were measured spectrophotometrically using 2,4 DNPH (Dinitro phenyl hydrazine) reagent in alkaline condition [23]. MG was estimated spectrophotometrically , based on 1,2 Di-amino benzene following Ghosh et al. method [24] which is a modified method of Cordeiro et al. [25]. The cellular damage was measured by estimating thiobarbituric acid reacting substances (TBARS). This is a spectrophotometric assay based on thiobarbituric acid (TBA) reaction, read at a wavelength of 532 nm following Okhawa method [26]. Creatinine was measured using commercial kit of Transasia.

2.3. Statistical analysis The results of different assays of uremic patients and normal control were undergone to statistical evaluation. Two sample student t-test were performed and significance was considered at p b 0.05.

3. Result Table 1 shows the comparative study of biochemical parameters between the uremic patients and the normal control. TAS depicts the total antioxidant profile of serum, which is about 39% less in CRF, and 32% less in SARF when compared to normal control. Serum reduced glutathione concentration, an important antioxidant marker, has been found very significantly declined by 40% in CRF and 46% in SARF, in comparison to normal control. The intracellular GSH concentration is more important than the serum GSH concentration and found depleted by N 3 times in SARF and CRF both in comparison to normal

Table 1 Comparative study of mean concentration (±S.E.M.) of biochemical parameters (serum and intracellular level) and their significant levels of normal control, CRF and SARF patients Parameter

Group Normal

SARF

CRF

Normal vs SARF

Normal vs CRF

Serum creatinine (mg/dl) Serum TAS (mM) Serum GSH (µM) Intracellular erythrocytic GSH (µM/g of Hb) Carbonyl compounds (nmol/ml) MG (nmol/ml) TBARS (nmol/ml)

1.13 ± 0.13 1.31 ± 0.05 5.83 ± 0.29 64.8 ± 5.0 71.9 ± 2.27 4.7 ± 0.38 2.65 ± 0.1

8.91 ± 0.77 0.89 ± 0.03 3.13 ± 0.27 18.8 ± 2.42 93.6 ± 4.04 18.5 ± 1.6 5.41 ± 0.76

7.71 ± 0.58 0.80 ± 0.04 3.5 ± 0.43 19.3 ± 1.89 82.6 ± 4.33 17.9 ± 0.81 5.19 ± 0.63

p b 0.001 p b 0.001 p b 0.001 p b 0.0001 p b 0.001 p b 0.001 p b 0.001

p b 0.001 p b 0.001 p b 0.001 p b 0.0001 p b 0.05 p b 0.001 p b 0.001

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control. Carbonyl compounds were found very significantly increased not only in CRF but in SARF also. Strikingly, in SARF the serum concentration of carbonyl compounds are found to be higher than CRF (p b 0.05). The most important finding in our present study is the increase of MG in uremia. We found that serum MG concentration in SARF and in CRF is almost 4 and 3.8 fold higher respectively than the normal control concentration. The increased ROS attack the lipoproteins, deoxyribose etc. and produce MDA like compounds, which are measured by estimating TBARS. The TBARS concentration is about 2 fold higher in normoglycemic CRF and also similarly increased in SARF cases (slightly higher than CRF but statistically not significant). We estimated serum creatinine concentration of all uremic patients and normal control as surrogate biomarker of renal clearance. Here we tried to correlate serum MG and creatinine concentration of both CRF and SARF patients and found the correlation coefficients of 0.61 (p b 0.0001) and 0.72 (p b 0.0001) respectively. However we noticed from the clinical follow up of the patients that those with low antioxidant status like GSH and TAS and high carbonyl stress (MG concentration), had very poor prognosis and delayed recovery or in some cases no recovery at all. We analyzed serum MG and GSH of SARF patients and normal control and calculated the serum MG and GSH ratio. We found that serum MG, GSH ratio was ≤1.0 in case of normal control whereas it was within the range of 3 to 27 in SARF cases. Patient follow up data (n = 28) indicate that serum MG and GSH ratio is having good correlation with the disease prognosis. It has been observed when the ratio was within 3 to 4.5, patients had better prognosis and early recovery. The ratio was N 6 and within 8.5, the prognosis was not better and there was a delayed recovery. When the ratio was N 10, the prognosis was worse and although the crisis was over after continuous dialysis, the retention of complications persisted for a very prolonged period. Two among these group (MG:GSH N 10) and one patient having MG:GSH = 27, did not survive. The symptoms of the delayed recovery or no recovery were generally persistence of aneuria or oligouria, local edema, no control of serum urea and creatinine concentration, pericardial effusion, uremic encephalopathy and disorientation.

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Concentration of ‘carbonyl compounds’ as a whole indicates the total ‘carbonyl stress’ in uremic serum including glyoxal, methylglyoxal and other carbonyl compounds. We found serum MG concentration is significantly increased in both SARF and CRF cases. The very high concentration of MG in SARF and CRF cases indicates the accumulation as well as the retention of MG in uremia. We also documented the decrease in GSH concentration, both intra- and extra-cellularly, very significantly in SARF and CRF than the normal control group. GSH, a thiol containing component functions in diverse role such as regulating antioxidant defences, xenobiotics and in the redox regulation of signal transduction [30]. As the altered redox regulation is related to increased glycoxidation reaction in uremia [31], the decrease in intra and extracellular GSH concentration in both SARF and CRF again gives the evidence of redox imbalance in uremia. GSH plays a pivotal role in MG detoxification [20,32]. MG reacts with GSH spontaneously to form hemimercaptal, the intermediate derivative. This derivative is the substrate of glyoxalase I

4. Discussion Recent evidence [27,28] showed the striking rise of AGE and ALE in normoglycemic uremic patients which indicate strongly that the factors other than hyperglycemia also determine the rate of formation of advanced lipoxidation and glycation endproducts and oxidative stress may play an important role in uremic complications. In our study we found the low profile of TAS and increased concentration of TBARS in SARF and CRF in comparison to the normal control, which further indicate the possibilities of increased oxidative stress. Plasma CML, an AGE species is reported to be highly correlated with plasma MDAlysine, an ALE species, in patients on chronic hemodialysis [29]. The increased concentration of TBARS, obtained from our study gives the direct evidence to cause the development of ALE in uremia.

Fig. 1. A. Correlation between serum MG and creatinine in CRF patients (n = 34, r = 0.61, y = 0.864x + 11.29. B. Correlation between serum MG and creatinine in SARF patients (n = 23, r = 0.72, y = 1.5x + 5.12).

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enzyme which converts it into S-D-Lactoyl glutathione and ultimately excreted as D-Lactate by glyoxalase II from the body. So GSH prevents the accumulation of MG, and minimizes its toxicity in the body. In our study we observed that in due course of oxidative stress, GSH concentration is dropped down in uremia, which facilitates oxidative damage and MG accumulation in body. Accumulated MG further uses GSH for its detoxification and may suppress the GSH pool in the body. The data of Table 1 indicate that serum MG concentration is inversely proportional to the serum GSH concentration. So decreased GSH concentration helps to understand the accumulation of MG in both SARF and CRF. Increased creatinine (Table 1) is the common index of insufficient renal clearance in uremia. Fig. 1 A and B demonstrates the positive correlation between creatinine and MG in both SARF and CRF. But the most surprising finding in our study is that carbonyl compounds and MG increased rapidly in SARF patients and the r value of MG-creatinine correlation analysis is greater than that of CRF. Antioxidant markers like TAS, GSH are found little bit less as well as cellular damage (TBARS concentration) is slightly higher in SARF than CRF. The long-term disease duration in CRF cases may be the prevalent reason behind the prolonged oxidative and carbonyl stress, which leads to glycoxidation product formation. But the people (age range 20–65 y) who might be considered as normal before the snake bite undergo the severe oxidative and carbonyl stress with a low TAS and GSH concentration along with very high TBARS and MG concentration in their serum just within 2–3 days after snakebite in comparison to normal people. It may be suggested that snake venom induces the sudden shock in the body which may trigger the immune response and reaction of neutrophils and macrophages may produce ROS that increase the oxidative stress. It is known that snake venom of Vipera sp. contains many proteases and lipases [33], which help in break down of RBC membranes and cause hemolysis. As a result of protein and lipid breakdown, the threonine, fatty acids, MDA like compounds (by the breakdown of arachidonic acid of RBC membrane) and carbonyl derivatives are produced rapidly which may be the precursor of MG synthesis [7,8] and the another source of MG accumulation in SARF. Though the AGE study has not been done in snakebite cases, still the increase of MG may indicate the insufficient renal clearance and increased pathogenesis. Further studies are required to better understand whether the decrease of thiol concentration in SARF derives only from oxidative stress or from other nonoxidative pathway and the rapid accumulation of MG occurs due to depleted GSH mediated detoxification only or snake venom induced altered metabolic pathway is linked to it. Our study suggests that MG may be a functional biomarker of renal clearance and pathogenesis of any kind of uremia. The therapeutic use of glutathione precursor, carbonyl compound quencher and antioxidant drugs may be taken under consideration in all uremic conditions. Acknowledgement The authors thank Drs. Ashis Mukhopadhyay of Netaji Subhas Chandra Bose Cancer Research Institute, Kolkata and

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