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Effect of lipid peroxides and antioxidants on glycation of hemoglobin: An in vitro study on human erythrocytes
Clinica Chimica Acta 366 (2006) 190 – 195 www.elsevier.com/locate/clinchim
Effect of lipid peroxides and antioxidants on glycation of hemoglobin: An in vitro study on human erythrocytes N. Selvaraj, Zachariah Bobby *, V. Sathiyapriya Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, 605 006, India Received 9 September 2005; received in revised form 29 September 2005; accepted 4 October 2005 Available online 1 December 2005
Abstract Background: Glycation and lipid peroxidation are two important processes known to play a key role in complications of many pathophysiological process. We sought to assess the possibility of an interaction between these processes in vitro and to examine the effect of lipoic acid and taurine on the glycation of hemoglobin and lipid peroxidation. Methods: Human erythrocytes in phosphate buffered saline (pH 7.4) were incubated with 5 or 50 mmol/l glucose. To study the effect of antioxidants on glycation of hemoglobin, erythrocytes were incubated with either lipoic acid or taurine and then exposed to glucose concentration of either 5 or 50 mmol/l. To clarify if lipid peroxides per se enhances the glycated hemoglobin level, an in vitro study was performed by incubating erythrocyte suspension containing either 5 or 50 mmol/l glucose with or without MDA. Lipid peroxides and glycated hemoglobin levels were determined in the glucose treated cells. Results: Glycated hemoglobin levels were higher in erythrocytes incubated with 50 mmol/l glucose concentrations than in erythrocytes incubated with 5 mmol/l glucose. The increase in glycated hemoglobin levels was blocked significantly when erythrocytes were pretreated with either lipoic acid or taurine. Both the antioxidants used in the present study markedly reduced the MDA levels. The level of glycated hemoglobin in erythrocyte incubated in the presence of MDA was increased significantly when compared to erythrocyte incubated with glucose alone. Conclusions: Lipid peroxides per se may have a role to play in glycation of hemoglobin and antioxidants (lipoic acid and taurine) can partially inhibit the formation of glycated hemoglobin by lowering the levels of lipid peroxides. D 2005 Elsevier B.V. All rights reserved. Keywords: Glycation; Malondialdehyde; Lipoic acid; Taurine
1. Introduction Spontaneous nonenzymatic modifications of protein are commonly reported in tissues with slow turnover and they are considered by several authors as possible common mechanism involved in the progression of many pathological conditions [1,2]. Among the nonenzymatic processes, oxidative stress and nonenzymatic glycation have aroused a particular interest in the recent past [3,4]. Glycation is the nonenzymatic reaction of glucose with susceptible amino groups in the side chains of amino acid residues (usually lysine) in proteins. Although the initial * Corresponding author. Tel.: +91 413 2273078; fax: +91 413 2372067. E-mail address: [email protected] (Z. Bobby). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.10.002
reaction, the formation of fructose – lysine, is reversible, further rearrangement of the protein side chain can lead to more stable products [5]. It has been proposed that the tissue accumulation of advanced glycated end products participate in the alterations of structure and function of long-lived proteins responsible for cellular or tissue damage [6]. Similarly, free radicals that induce lipid peroxidation are another promoter event thought to be important in the development of several pathological conditions [7]. A combination of oxidative stress and glycation underlies most cases of diabetes, chronic renal failure (CRF) and atherosclerosis [1 – 4]. While the interplay of these two impairments is believed to be important in the development and progression of these pathophysiological states, the mechanisms involved are unclear.
N. Selvaraj et al. / Clinica Chimica Acta 366 (2006) 190 – 195
We previously reported a significant association between malondialdehyde and fructosamine in non-diabetic nephrotic syndrome and chronic renal failure patients [8,9]. In addition, we have also recently observed a parallel increase of glycated hemoglobin and malondialdehyde in hyperthyroid and chronic renal failure patients, with significant correlation between their respective increments [10,11]. These evidences support the hypothesis that glycation and oxidation processes may be mutually dependent in vivo. Therefore, further in vitro exploration of the role of lipid peroxidation and antioxidants on glycation of hemoglobin was deemed pertinent.
2. Materials and methods Blood was collected from normal healthy volunteers into tubes containing EDTA according to a protocol approved by the Institution Human Ethics Review Committee. The blood was centrifuged and the clear plasma and buffy coat were discarded. The cells were washed with cold physiological saline and the cells were suspended to 10% hematocrit in phosphate-buffered saline (PBS containing 0.016 mol/ l Na2HPO4, 0.001 mol/l NaH2PO4 and 0.14 mol/l NaCl, pH 7.4). Penicillin G and streptomycin were added to the incubating erythrocyte – PBS suspension to vitiate any microbial growth. All analyses were done in triplicate. 2.1. In vitro incubation with glucose The washed erythrocytes suspended to 10% hematocrit in PBS were treated with freshly prepared stock glucose solution. Glucose concentrations were expressed in terms of the total cell suspension. Erythrocyte treated with 5 mmol/ l glucose was considered as controls. The contents were incubated in a shaking water bath at 37 -C for 24 h. The percentage of hemolysis was < 2% in all incubations. Glucose-treated erythrocytes were washed with PBS (pH 7.4) before biochemical analyses. 2.2. In vitro treatment with lipoic acid In order to examine the protective role of lipoic acid against glycation of hemoglobin, erythrocytes were incubated with lipoic acid of different concentrations (50, 100, 150, 200 Amol/l) for 1 h at 37 -C. Nonenzymatic glycation of hemoglobin was initiated by incubating the pretreated (with lipoic acid) erythrocyte with glucose of either 5 or 50 mmol/l. The contents were incubated in a shaking water bath at 37 -C for 24 h. The percentage of hemolysis was < 2% in all incubations. 2.3. In vitro treatment with taurine Before treatment with glucose (concentration of either 5 or 50 mmol/l), erythrocytes were pretreated with taurine of
191
50, 100, 150 and 200 Amol/l for 1 h at 37 -C. The in vitro glycation of hemoglobin was carried out for 24 h at 37 -C in a shaking water bath. The percentage of hemolysis was < 2% in all incubations. Erythrocytes were washed with PBS (pH 7.4) before biochemical analyses. 2.4. In vitro treatment with MDA To examine if MDA per se has any role in glycation of hemoglobin, erythrocytes were incubated with different concentrations of glucose (5 and 50 mmol/l), with or without MDA of 0.01, 0.1, 1 and 10 mmol/l concentration. The contents were incubated in a water bath at 37 -C for 24 h. At the end of the incubation the cells were washed with PBS before biochemical analyses. The percentage of hemolysis was <2% in all incubations. To exclude the possible influence of anionic products of MDA and hemoglobin in the estimation of glycated hemoglobin by ion exchange chromatography, erythrocytes were incubated with glucose (5 and 50 mmol/l) and 1 mmol/ l MDA. The incubation was carried out for a period of 24 h at 37 -C. At the end of the incubation the cells were washed with PBS and glycated hemoglobin was estimated by affinity chromatography method. 2.5. Malondialdehyde synthesis Malondialdehyde was prepared by acid hydrolysis of malondialdehyde bis (dimethyl acetate) [12]. Malondialdehyde bis (dimethyl acetate) was dissolved in 200 ml of double distilled water at 10 2 mol/l and incubated with 1 ml 1 mol/l HCl at 50 -C for 2 h. The reaction was stopped by cooling the solution at 4 -C. A portion of the acidic solution was diluted with 0.1 mol/l Tris – HCl (pH 8.0) and adjusted to pH 7.4 with 5 mol/l NaOH to prepare neutral 400 mmol/ l MDA solution for use. 2.6. Measurement of glycated hemoglobin (HbA1C) Glycated hemoglobin was measured by ion exchange chromatography using hemoglobin A1C microcolumn (BioRad, Hercules, CA; Catalog number 192 80000/192 8091) and expressed as the percentage of total hemoglobin. To rule out the possible influence of MDA –hemoglobin adducts in the estimation of glycated hemoglobin by ion exchange chromatography, glycated hemoglobin was also estimated by affinity chromatography using the aminophenylboronic acid gels purchased from Millipore [13]. One milliliter of gel was packed in a plastic column. The gel was pre-equilibrated with 5 ml of phosphate buffer (50 mmol/l, pH 9.2) and hemolysate in a volume of 200 Al was loaded onto the column and then washed with 10 ml of the same buffer. Non-glycated hemoglobin was eluted in this fraction (peak 1) and glycated hemoglobin that remained attached to the column was eluted with the same phosphate buffer containing 100 mmol/l sorbitol (peak 2). Hemoglobin
192
N. Selvaraj et al. / Clinica Chimica Acta 366 (2006) 190 – 195 0.6
*
7
**
**
**
6.5
MDA (nmol/mg Hb)
Glycated Hemoglobin (%)
8 7.5
**
6 5.5 5 4.5
*
0.5
**
**
**
0.4
**
0.3 0.2 0.1
4 0
3.5
Glucose (mmol/l)
5
5
5
50
50
50
50
50
Glucose (mmol/l)
5
5
5
50
50
50
50
50
Lipoic acid (µmol/l)
0
25
50
0
25
50
100
150
Lipoic acid (µmol/l)
0
25
50
0
25
50
100
150
Fig. 1. Effect of increasing concentrations of lipoic acid on the glycation of hemoglobin in erythrocytes treated with 5 or 50 mmol/l glucose. Values are means T S.D. of 4 different experiments. The pretreatment of erythrocytes with lipoic acid before incubating with 50 mmol/l glucose suppressed the glycation of hemoglobin (* vs. **p value < 0.05).
Fig. 2. Effect of lipoic acid on the levels of MDA in erythrocytes incubated with 5 and 50 mmol/l glucose. Values are means T S.D. of 4 different experiments. Significant decrease in levels of MDA was observed in erythrocytes preincubated with lipoic acid when compared to erythrocytes incubated with 50 mmol/l glucose alone (* vs. **p < 0.05).
concentration in peak 1 and peak 2 was estimated by reading the optical density at a wavelength of 430 nm.
of MDA – TBA complex 1.56 105 l mol 532 nm and expressed as nmol/mg Hb.
2.7. Determination of malondialdehyde (MDA)
2.8. Statistical analysis
Malondialdehyde was measured using the established thiobarbituric acid (TBARS) method [14]. This assay is based on the formation of red adduct in acidic medium between thiobarbituric acid and malondialdehyde, a colorless product of lipid peroxidation, measured at 532 nm. For this purpose, 0.2 ml of cells were suspended in 0.8 ml phosphate buffered saline (8.1 g NaCl, 2.302 g Na2HPO4 and 0.194 g NaH2PO4/l, pH 7.4) and 0.025 ml of butylated hydroxytoluene (88 mg/10 ml absolute alcohol). Thirty percent trichloroacetic acid (0.5 ml) was then added. Tubes were vortexed and allowed to stand in ice for at least 2 h. Tubes were centrifuged at 2000 rpm for 15 min. One milliliter of supernatant was transferred to another tube. To this, 75 Al of 0.1 mol/l ethylenediaminetetraacetic acid and 0.25 ml 1% TBA in 0.05 mol/l NaOH was added. Tubes were mixed and kept in a boiling water bath for 15 min. The MDA values were calculated using the extinction coefficient
Data are expressed as mean T standard deviation (S.D). The statistical significance of difference between groups was evaluated using Student’s t-test. The p value of 0.05 levels was selected as the point of minimal statistical significance.
Treatment 5 mmol/l glucose 5 mmol/l glucose + 25 Amol/l lipoic acid 5 mmol/l glucose + 50 Amol/l lipoic acid 50 mmol/l glucose 50 mmol/l glucose + 25 Amol/l lipoic acid 50 mmol/l glucose + 50 Amol/l lipoic acid 50 mmol/l glucose + 100 Amol/l lipoic acid 50 mmol/l glucose + 150 Amol/l lipoic acid
HbA1C values (in %) 5.48 T 0.35 5.41 T 0.27 5.46 T 0.26 6.81 T 0.25 6.62 T 0.26* 6.61 T 0.22* 6.62 T 0.39* 6.14 T 0.34*
Values are the mean T standard deviation of 4 experiments. *p < 0.05 when compared with HbA1C values of RBC treated with 50 mmol/l glucose alone.
cm
1
at
3. Results Fig. 1 and Table 1 show that glycated hemoglobin levels were significantly lower in erythrocytes pretreated with lipoic acid when compared to erythrocytes incubated with 50 mmol/l glucose alone. No difference in the level of
7.5
Glycated Hemoglobin (%)
Table 1 Effect of lipoic acid on glycated hemoglobin in erythrocytes treated with glucose (5 or 50 mmol/l)
1
*
7
**
6.5
**
**
**
6 5.5 5 4.5 4 3.5
Glucose (mmol/l)
5
5
5
50
50
50
50
50
Taurine (µmol/l)
0
25
50
0
25
50
100
150
Fig. 3. In vitro effect of taurine in erythrocytes treated with 5 and 50 mmol/ l glucose. Values are means T S.D. of 4 different experiments. The pretreatment of erythrocytes with taurine before incubating with 50 mmol/l glucose suppressed the glycation of hemoglobin (* vs. **p value < 0.05).
N. Selvaraj et al. / Clinica Chimica Acta 366 (2006) 190 – 195 10.5
HbA1C values (in %)
5 mmol/l glucose 5 mmol/l glucose + 25 Amol/l taurine 5 mmol/l glucose + 50 Amol/l taurine 50 mmol/l glucose 50 mmol/l glucose + 25 Amol/l taurine 50 mmol/l glucose + 50 Amol/l taurine 50 mmol/l glucose + 100 Amol/l taurine 50 mmol/l glucose + 150 Amol/l taurine
5.47 T 0.30 5.52 T 0.27 5.44 T 0.30 6.71 T 0.24 6.48 T 0.21* 6.27 T 0.25* 6.11 T 0.24* 5.96 T 0.30*
glycation of hemoglobin was observed between erythrocytes treated with 5 mmol/l glucose alone and erythrocytes incubated with lipoic acid along with 5 mmol/l glucose. As depicted in Fig. 2 lipoic acid was found to decrease the levels of lipid peroxides at all the concentrations used in the present experiments. Similar to the effect of lipoic acid, taurine was also found to suppress the HbA1C levels in erythrocytes incubated with 50 mmol/l glucose concentration (Fig. 3 and Table 2). The levels of MDA were also found to be low in erythrocytes pretreated with taurine when compared to erythrocytes incubated with 50 mmol/l glucose alone (Fig. 4). Fig. 5 and Table 3 illustrates the effect of MDA on glycation of hemoglobin. HbA1C levels were estimated by the ion exchange column. The addition of MDA to washed erythrocytes incubated with 50 mmol/l glucose caused an increase in glycation of hemoglobin when compared to erythrocytes incubated with glucose. In view of the possibility that MDA –hemoglobin adduct can interfere in the estimation of glycated hemoglobin by ion exchange, erythrocytes were treated with and without
0.6
MDA (nmol/mg Hb)
*
** ** **
0.4
**
9.5 8.5
**
**
** *
7.5 6.5 5.5 4.5 3.5
Values are the mean T standard deviation of 4 experiments. *p < 0.05 when compared with HbA1C values of RBC treated with 50 mmol/l glucose alone.
0.5
Glycated Hemoglobin (%)
Table 2 Effect of taurine on glycated hemoglobin in erythrocytes treated with glucose (5 or 50 mmol/l) Treatment
193
Glucose (mmol/l)
5
5
50
50
50
50
50
MDA (mmol/l)
0
1
0
0.01
0.1
1
10
Fig. 5. Effect of different concentrations of MDA on glycated hemoglobin values in erythrocytes treated with glucose (5 or 50 mmol/l). Values are means T S.D. of 4 different experiments. At all the tested concentration of MDA the level of glycated hemoglobin was found to be enhanced when compared with erythrocytes incubated with only 50 mmol/l glucose (* vs. **p < 0.05).
1 mmol/l MDA and glycated hemoglobin was estimated by affinity chromatography. HbA1C was 6.54 T 0.36 with 50 mmol/l glucose and MDA in contrast to 5.8 T 0.47 with 50 mmol/l glucose alone. This difference was statistically significant.
4. Discussion Many clinical and experimental observations have highlighted the important role played by the enhanced nonenzymatic glycation of circulating and structural proteins in contributing to the pathogenesis related to diabetes, CRF, atherosclerosis and aging [1– 4]. Much effort has thus been spent searching for substances capable of either preventing or arresting the progression of glycation-dependent complications. Two inhibitory agents to be first proposed were aspirin and ibuprofen [15 – 17]. However, with regard to a possible therapeutic use of these compounds, it must be stressed that it is not yet sufficiently clear what would be the effect of irreversibly bound foreign chemical groups in
**
0.3
Table 3 Effect of different concentrations of MDA on glycated hemoglobin in erythrocytes treated with glucose (5 or 50 mmol/l)
0.2 0.1 0
Glucose (mmol/l)
5
5
5
50
50
50
50
50
Taurine (µmol/l)
0
25
50
0
25
50
100
150
Fig. 4. Effect of taurine on the levels of MDA in erythrocytes incubated with 5 and 50 mmol/l glucose. Values are means T S.D. of 4 different experiments. Levels of MDA were suppressed in erythrocytes preincubated with taurine when compared with erythrocytes incubated with 50 mmol/ l glucose alone (* vs. **p < 0.05).
Treatment
HbA1C values (in %)
5 mmol/l glucose 5 mmol/l glucose + 10 mM MDA 50 mmol/l glucose 50 mmol/l glucose + 0.01 mM MDA 50 mmol/l glucose + 0.1 mM MDA 50 mmol/l glucose + 1 mM MDA 50 mmol/l glucose + 10 mM MDA
5.63 T 0.25 5.79 T 0.53 6.71 T 0.42 7.23 T 0.33* 7.68 T 0.38* 8.25 T 0.43* 9.17 T 0.34*
Values are the mean T standard deviation of 4 experiments. *p < 0.05 when compared with HbA1C values of RBC treated with 50 mmol/l glucose alone.
194
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place of glucose in very long-lived protein structures. Thus, we could speculate that such groups might be chemically even more reactive than the stable glucose– protein adducts, the formation of which they have helped to prevent. Chronic therapy with aspirin and ibuprofen would be contraindicated, owing to its effects on the gastric mucosa. Of late, in vitro studies have demonstrated that vitamin E and vitamin C can inhibit the process of protein glycation [18,19]. But the results from the in vivo studies are discordant with this hypothesis. While Shoff et al. have shown that there is no significant relationship between vitamin E intake and glycated hemoglobin values [20], Ceriello et al. showed that vitamin E intake has an inverse relationship with the levels of glycated hemoglobin [21]. Similarly contradictory reports have been documented in the literature regarding the intake of vitamin C and the levels of glycated hemoglobin [20 –22]. Recently lipoic acid has attracted interest because of its diverse biological actions attributed to its chemical properties [23,24]. It is both lipid and water soluble [25] and has a potent antioxidative capacity in a wide variety of experimental system. Previous investigations have shown that lipoic acid exhibits metabolic effects on glucose transport and utilization. In cell culture (L6 myotubes and 3T3-L1 adipocytes), lipoic acid at concentrations exceeding 0.5 mmol/l exerted insulin-like effects, as indicated by the activation of the insulin signaling cascade leading to translocation of glucose transporters [26]. In animal models of diabetes, treatment with lipoic acid resulted in improved peripheral glucose utilization stimulated by insulin, and prevented diabetes related reduction in skeletal muscle content of GLUT 4 [27]. We found that lipoic acid caused decrease in the levels of glycated hemoglobin in erythrocytes incubated with 50 mmol/l glucose. Pretreatment with lipoic acid also significantly reduced the formation of lipid peroxides in erythrocytes. Our results are consistent with the observations of Kawabata and Packer that supplementation of lipoic acid in vitro can prevent glycation of bovine serum albumin [28]. Taurine is an intracellular amino acid that is present in millimolar concentrations in the plasma, tissues and interstitial media [29]. Taurine has been shown to exert beneficial effects in diabetes related conditions in which oxidative stress was suggested to have a pathological role. Experimental evidences have found a beneficial effect of taurine in preventing diabetic neuropathy, chronic puromycin aminonucleoside nephropathy and retinopathy [30 – 32]. In accordance to this, using animal models, taurine has been shown to prevent hyperglycemia-induced insulin resistance [33]. When the erythrocytes were pretreated with taurine, the glycation of hemoglobin was significantly attenuated. Similar to the effect of lipoic acid, preincubation of erythrocytes with taurine significantly inhibited the lipid peroxide formation.
The exact mechanism by which lipoic acid and taurine exert their protective effect against glycation of hemoglobin is difficult to entangle from the present study. But, the simultaneous inhibition of glycation and on the accumulation of lipid peroxides by lipoic acid and taurine suggest that lipid peroxide levels can have a role in the modulation of glycation reaction. This hypothesis was confirmed by our in vitro study where in MDA of different concentrations were co-incubated with glucose of 50 mmol/l concentration. In the present study, a concentration dependent increase in the extent of in vitro nonenzymatic glycation of hemoglobin was found in RBC incubated with glucose and MDA when compared with RBC incubated with glucose alone. Malondialdehyde is known to react with normal hemoglobin to form ionic components. As these compounds can interfere in the estimation of glycated hemoglobin by ion exchange chromatography, it seemed prudent to provide data comparing the results obtained with ion exchange chromatography with affinity chromatography method. When erythrocytes were incubated with 50 mmol/l glucose, the formation of glycated hemoglobin were significantly higher in cells treated with MDA and glucose than in cells treated with glucose alone. Thus, emphasizing the possible role of MDA per se on the glycation of hemoglobin. Consonant with our study, Jain and Palmer [34] using affinity chromatography method for the estimation of glycated hemoglobin have proposed that MDA per se can increase glycation of hemoglobin and vitamin E can suppress this process. No consensus has been reached as to the mechanism underlying the modulation of glycation of hemoglobin by MDA. Previously suggested mechanism includes the potential ability of the aldehyde groups of MDA to act as an anchor between sugar and hemoglobin moieties [34]. MDA at pathological concentration has been found to react with lysine to give N (-h-lysine amino acroline (h-LAA) [35]. h-LAA, the product of lysine with MDA, has been postulated to have the potential to react with an aldehyde group of sugar molecule, thereby bridging lysine and sugar molecules [36]. In conclusion, this study has shown that, in vitro, lipoic acid and taurine can inhibit the nonenzymatic glycation of hemoglobin. In vivo studies in normal animals and in animals with artificially induced diabetes are now required. This will help to define the physiological, pharmacological, chemical, and toxicity indices that might allow these antioxidant to be used safely and efficiently in humans in an attempt to minimize the nonenzymatic glycation of proteins, the hallmark of diabetic hyperglycemia and other chronic pathophysiological states. Acknowledgements This work was financially supported by the Indian Council of Medical Research (ICMR), New Delhi, India, in the form of Junior Research Fellowship to Mr. N. Selvaraj
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and by Council of Scientific and Industrial Research New Delhi, India, in the form of Junior Research Fellowship to Ms. V. Sathiyapriya. References [1] Kennedy AL, Lyons TJ. Glycation, oxidation, and lipoxidation in the development of diabetic complications. Metabolism 1997;46: 14 – 21. [2] Baynes JW, Thorpe SR. Glycoxidation and lipoxidation in atherogenesis. Free Radic Biol Med 2000;28:1708 – 16. [3] Miyata T, Wada Y, Cai Z, et al. Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure. Kidney Int 1997;51:1170 – 81. [4] Hunt JV, Dean RT, Wolff SP. Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and aging. Biochem J 1988;256:205 – 12. [5] Spicer KM, Allen RC, Hallett D, Buse MG. Synthesis of hemoglobin A1C and related minor hemoglobins by erythrocytes: in vitro study of regulation. J Clin Invest 1979;64:40 – 8. [6] Brownlee M. Negative consequences of glycation. Metabolism 2000;49(suppl. 1):9 – 13. [7] Yagi K. Lipid peroxides and related radicals in clinical medicine. Adv Exp Med Biol 1994;366:1 – 15. [8] Balamurugan R, Bobby Z, Selvaraj N, Nalini P, Koner BC, Sen SK. Increased protein glycation in non-diabetic pediatric nephrotic syndrome: possible role of lipid peroxidation. Clin Chim Acta 2003; 337:127 – 32. [9] Selvaraj N, Bobby Z, Das AK, Ramesh R, Koner BC. An evaluation of concentrations of oxidative stress and protein glycation in nondiabetic, undialyzed chronic renal failure patients. Clin Chim Acta 2002;324:45 – 50. [10] Mohan Kumar KM, Bobby Z, Selvaraj N, et al. Possible link between glycated hemoglobin and lipid peroxidation in hyperthyroidism. Clin Chim Acta 2004;342:187 – 92. [11] Selvaraj N, Bobby Z, Koner BC, Das AK. Reassessing the increased glycation of hemoglobin in nondiabetic chronic renal failure patients: a hypothesis on the role of lipid peroxides. Clin Chim Acta 2005;360: 108 – 13. [12] Michiels C, Remacle J. Cytotoxicity of linoleic acid peroxides, malondialdehyde and 4-hydroxynonenal towards human fibroblast. Toxicology 1991;66:225 – 34. [13] Jain SK. Hyperglycemia can cause membrane lipid peroxidation and osmotic fragility in human red blood cells. J Biol Chem 1989; 264:21340 – 5. [14] Yue DK, McLennan S, Church DB, Turtle JR. The measurement of glycosylated hemoglobin in man and animals by aminophenylboronic acid affinity chromatography. Diabetes 1982;31:701 – 5. [15] Huby R, Harding JJ. Non-enzymatic glycosylation (glycation) of lens proteins by galactose and protection by aspirin and reduced glutathione. Exp Eye Res 1988;47:53 – 9. [16] Ajiboye R, Harding JJ. Glycosylation of bovine lens proteins by glucosamine and its inhibition by aspirin, ibuprofen and glutathione. Exp Eye Res 1989;49:31 – 41. [17] Raza K, Harding JJ. Non-enzymatic modification of lens proteins by glucose and fructose: effects of ibuprofen. Exp Eye Res 1991; 52:205 – 12.
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Effect of lipid peroxides and antioxidants on glycation of hemoglobin: An in vitro study on human erythrocytes N. Selvaraj, Zachariah Bobby *, V. Sathiyapriya Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, 605 006, India Received 9 September 2005; received in revised form 29 September 2005; accepted 4 October 2005 Available online 1 December 2005
Abstract Background: Glycation and lipid peroxidation are two important processes known to play a key role in complications of many pathophysiological process. We sought to assess the possibility of an interaction between these processes in vitro and to examine the effect of lipoic acid and taurine on the glycation of hemoglobin and lipid peroxidation. Methods: Human erythrocytes in phosphate buffered saline (pH 7.4) were incubated with 5 or 50 mmol/l glucose. To study the effect of antioxidants on glycation of hemoglobin, erythrocytes were incubated with either lipoic acid or taurine and then exposed to glucose concentration of either 5 or 50 mmol/l. To clarify if lipid peroxides per se enhances the glycated hemoglobin level, an in vitro study was performed by incubating erythrocyte suspension containing either 5 or 50 mmol/l glucose with or without MDA. Lipid peroxides and glycated hemoglobin levels were determined in the glucose treated cells. Results: Glycated hemoglobin levels were higher in erythrocytes incubated with 50 mmol/l glucose concentrations than in erythrocytes incubated with 5 mmol/l glucose. The increase in glycated hemoglobin levels was blocked significantly when erythrocytes were pretreated with either lipoic acid or taurine. Both the antioxidants used in the present study markedly reduced the MDA levels. The level of glycated hemoglobin in erythrocyte incubated in the presence of MDA was increased significantly when compared to erythrocyte incubated with glucose alone. Conclusions: Lipid peroxides per se may have a role to play in glycation of hemoglobin and antioxidants (lipoic acid and taurine) can partially inhibit the formation of glycated hemoglobin by lowering the levels of lipid peroxides. D 2005 Elsevier B.V. All rights reserved. Keywords: Glycation; Malondialdehyde; Lipoic acid; Taurine
1. Introduction Spontaneous nonenzymatic modifications of protein are commonly reported in tissues with slow turnover and they are considered by several authors as possible common mechanism involved in the progression of many pathological conditions [1,2]. Among the nonenzymatic processes, oxidative stress and nonenzymatic glycation have aroused a particular interest in the recent past [3,4]. Glycation is the nonenzymatic reaction of glucose with susceptible amino groups in the side chains of amino acid residues (usually lysine) in proteins. Although the initial * Corresponding author. Tel.: +91 413 2273078; fax: +91 413 2372067. E-mail address: [email protected] (Z. Bobby). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.10.002
reaction, the formation of fructose – lysine, is reversible, further rearrangement of the protein side chain can lead to more stable products [5]. It has been proposed that the tissue accumulation of advanced glycated end products participate in the alterations of structure and function of long-lived proteins responsible for cellular or tissue damage [6]. Similarly, free radicals that induce lipid peroxidation are another promoter event thought to be important in the development of several pathological conditions [7]. A combination of oxidative stress and glycation underlies most cases of diabetes, chronic renal failure (CRF) and atherosclerosis [1 – 4]. While the interplay of these two impairments is believed to be important in the development and progression of these pathophysiological states, the mechanisms involved are unclear.
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We previously reported a significant association between malondialdehyde and fructosamine in non-diabetic nephrotic syndrome and chronic renal failure patients [8,9]. In addition, we have also recently observed a parallel increase of glycated hemoglobin and malondialdehyde in hyperthyroid and chronic renal failure patients, with significant correlation between their respective increments [10,11]. These evidences support the hypothesis that glycation and oxidation processes may be mutually dependent in vivo. Therefore, further in vitro exploration of the role of lipid peroxidation and antioxidants on glycation of hemoglobin was deemed pertinent.
2. Materials and methods Blood was collected from normal healthy volunteers into tubes containing EDTA according to a protocol approved by the Institution Human Ethics Review Committee. The blood was centrifuged and the clear plasma and buffy coat were discarded. The cells were washed with cold physiological saline and the cells were suspended to 10% hematocrit in phosphate-buffered saline (PBS containing 0.016 mol/ l Na2HPO4, 0.001 mol/l NaH2PO4 and 0.14 mol/l NaCl, pH 7.4). Penicillin G and streptomycin were added to the incubating erythrocyte – PBS suspension to vitiate any microbial growth. All analyses were done in triplicate. 2.1. In vitro incubation with glucose The washed erythrocytes suspended to 10% hematocrit in PBS were treated with freshly prepared stock glucose solution. Glucose concentrations were expressed in terms of the total cell suspension. Erythrocyte treated with 5 mmol/ l glucose was considered as controls. The contents were incubated in a shaking water bath at 37 -C for 24 h. The percentage of hemolysis was < 2% in all incubations. Glucose-treated erythrocytes were washed with PBS (pH 7.4) before biochemical analyses. 2.2. In vitro treatment with lipoic acid In order to examine the protective role of lipoic acid against glycation of hemoglobin, erythrocytes were incubated with lipoic acid of different concentrations (50, 100, 150, 200 Amol/l) for 1 h at 37 -C. Nonenzymatic glycation of hemoglobin was initiated by incubating the pretreated (with lipoic acid) erythrocyte with glucose of either 5 or 50 mmol/l. The contents were incubated in a shaking water bath at 37 -C for 24 h. The percentage of hemolysis was < 2% in all incubations. 2.3. In vitro treatment with taurine Before treatment with glucose (concentration of either 5 or 50 mmol/l), erythrocytes were pretreated with taurine of
191
50, 100, 150 and 200 Amol/l for 1 h at 37 -C. The in vitro glycation of hemoglobin was carried out for 24 h at 37 -C in a shaking water bath. The percentage of hemolysis was < 2% in all incubations. Erythrocytes were washed with PBS (pH 7.4) before biochemical analyses. 2.4. In vitro treatment with MDA To examine if MDA per se has any role in glycation of hemoglobin, erythrocytes were incubated with different concentrations of glucose (5 and 50 mmol/l), with or without MDA of 0.01, 0.1, 1 and 10 mmol/l concentration. The contents were incubated in a water bath at 37 -C for 24 h. At the end of the incubation the cells were washed with PBS before biochemical analyses. The percentage of hemolysis was <2% in all incubations. To exclude the possible influence of anionic products of MDA and hemoglobin in the estimation of glycated hemoglobin by ion exchange chromatography, erythrocytes were incubated with glucose (5 and 50 mmol/l) and 1 mmol/ l MDA. The incubation was carried out for a period of 24 h at 37 -C. At the end of the incubation the cells were washed with PBS and glycated hemoglobin was estimated by affinity chromatography method. 2.5. Malondialdehyde synthesis Malondialdehyde was prepared by acid hydrolysis of malondialdehyde bis (dimethyl acetate) [12]. Malondialdehyde bis (dimethyl acetate) was dissolved in 200 ml of double distilled water at 10 2 mol/l and incubated with 1 ml 1 mol/l HCl at 50 -C for 2 h. The reaction was stopped by cooling the solution at 4 -C. A portion of the acidic solution was diluted with 0.1 mol/l Tris – HCl (pH 8.0) and adjusted to pH 7.4 with 5 mol/l NaOH to prepare neutral 400 mmol/ l MDA solution for use. 2.6. Measurement of glycated hemoglobin (HbA1C) Glycated hemoglobin was measured by ion exchange chromatography using hemoglobin A1C microcolumn (BioRad, Hercules, CA; Catalog number 192 80000/192 8091) and expressed as the percentage of total hemoglobin. To rule out the possible influence of MDA –hemoglobin adducts in the estimation of glycated hemoglobin by ion exchange chromatography, glycated hemoglobin was also estimated by affinity chromatography using the aminophenylboronic acid gels purchased from Millipore [13]. One milliliter of gel was packed in a plastic column. The gel was pre-equilibrated with 5 ml of phosphate buffer (50 mmol/l, pH 9.2) and hemolysate in a volume of 200 Al was loaded onto the column and then washed with 10 ml of the same buffer. Non-glycated hemoglobin was eluted in this fraction (peak 1) and glycated hemoglobin that remained attached to the column was eluted with the same phosphate buffer containing 100 mmol/l sorbitol (peak 2). Hemoglobin
192
N. Selvaraj et al. / Clinica Chimica Acta 366 (2006) 190 – 195 0.6
*
7
**
**
**
6.5
MDA (nmol/mg Hb)
Glycated Hemoglobin (%)
8 7.5
**
6 5.5 5 4.5
*
0.5
**
**
**
0.4
**
0.3 0.2 0.1
4 0
3.5
Glucose (mmol/l)
5
5
5
50
50
50
50
50
Glucose (mmol/l)
5
5
5
50
50
50
50
50
Lipoic acid (µmol/l)
0
25
50
0
25
50
100
150
Lipoic acid (µmol/l)
0
25
50
0
25
50
100
150
Fig. 1. Effect of increasing concentrations of lipoic acid on the glycation of hemoglobin in erythrocytes treated with 5 or 50 mmol/l glucose. Values are means T S.D. of 4 different experiments. The pretreatment of erythrocytes with lipoic acid before incubating with 50 mmol/l glucose suppressed the glycation of hemoglobin (* vs. **p value < 0.05).
Fig. 2. Effect of lipoic acid on the levels of MDA in erythrocytes incubated with 5 and 50 mmol/l glucose. Values are means T S.D. of 4 different experiments. Significant decrease in levels of MDA was observed in erythrocytes preincubated with lipoic acid when compared to erythrocytes incubated with 50 mmol/l glucose alone (* vs. **p < 0.05).
concentration in peak 1 and peak 2 was estimated by reading the optical density at a wavelength of 430 nm.
of MDA – TBA complex 1.56 105 l mol 532 nm and expressed as nmol/mg Hb.
2.7. Determination of malondialdehyde (MDA)
2.8. Statistical analysis
Malondialdehyde was measured using the established thiobarbituric acid (TBARS) method [14]. This assay is based on the formation of red adduct in acidic medium between thiobarbituric acid and malondialdehyde, a colorless product of lipid peroxidation, measured at 532 nm. For this purpose, 0.2 ml of cells were suspended in 0.8 ml phosphate buffered saline (8.1 g NaCl, 2.302 g Na2HPO4 and 0.194 g NaH2PO4/l, pH 7.4) and 0.025 ml of butylated hydroxytoluene (88 mg/10 ml absolute alcohol). Thirty percent trichloroacetic acid (0.5 ml) was then added. Tubes were vortexed and allowed to stand in ice for at least 2 h. Tubes were centrifuged at 2000 rpm for 15 min. One milliliter of supernatant was transferred to another tube. To this, 75 Al of 0.1 mol/l ethylenediaminetetraacetic acid and 0.25 ml 1% TBA in 0.05 mol/l NaOH was added. Tubes were mixed and kept in a boiling water bath for 15 min. The MDA values were calculated using the extinction coefficient
Data are expressed as mean T standard deviation (S.D). The statistical significance of difference between groups was evaluated using Student’s t-test. The p value of 0.05 levels was selected as the point of minimal statistical significance.
Treatment 5 mmol/l glucose 5 mmol/l glucose + 25 Amol/l lipoic acid 5 mmol/l glucose + 50 Amol/l lipoic acid 50 mmol/l glucose 50 mmol/l glucose + 25 Amol/l lipoic acid 50 mmol/l glucose + 50 Amol/l lipoic acid 50 mmol/l glucose + 100 Amol/l lipoic acid 50 mmol/l glucose + 150 Amol/l lipoic acid
HbA1C values (in %) 5.48 T 0.35 5.41 T 0.27 5.46 T 0.26 6.81 T 0.25 6.62 T 0.26* 6.61 T 0.22* 6.62 T 0.39* 6.14 T 0.34*
Values are the mean T standard deviation of 4 experiments. *p < 0.05 when compared with HbA1C values of RBC treated with 50 mmol/l glucose alone.
cm
1
at
3. Results Fig. 1 and Table 1 show that glycated hemoglobin levels were significantly lower in erythrocytes pretreated with lipoic acid when compared to erythrocytes incubated with 50 mmol/l glucose alone. No difference in the level of
7.5
Glycated Hemoglobin (%)
Table 1 Effect of lipoic acid on glycated hemoglobin in erythrocytes treated with glucose (5 or 50 mmol/l)
1
*
7
**
6.5
**
**
**
6 5.5 5 4.5 4 3.5
Glucose (mmol/l)
5
5
5
50
50
50
50
50
Taurine (µmol/l)
0
25
50
0
25
50
100
150
Fig. 3. In vitro effect of taurine in erythrocytes treated with 5 and 50 mmol/ l glucose. Values are means T S.D. of 4 different experiments. The pretreatment of erythrocytes with taurine before incubating with 50 mmol/l glucose suppressed the glycation of hemoglobin (* vs. **p value < 0.05).
N. Selvaraj et al. / Clinica Chimica Acta 366 (2006) 190 – 195 10.5
HbA1C values (in %)
5 mmol/l glucose 5 mmol/l glucose + 25 Amol/l taurine 5 mmol/l glucose + 50 Amol/l taurine 50 mmol/l glucose 50 mmol/l glucose + 25 Amol/l taurine 50 mmol/l glucose + 50 Amol/l taurine 50 mmol/l glucose + 100 Amol/l taurine 50 mmol/l glucose + 150 Amol/l taurine
5.47 T 0.30 5.52 T 0.27 5.44 T 0.30 6.71 T 0.24 6.48 T 0.21* 6.27 T 0.25* 6.11 T 0.24* 5.96 T 0.30*
glycation of hemoglobin was observed between erythrocytes treated with 5 mmol/l glucose alone and erythrocytes incubated with lipoic acid along with 5 mmol/l glucose. As depicted in Fig. 2 lipoic acid was found to decrease the levels of lipid peroxides at all the concentrations used in the present experiments. Similar to the effect of lipoic acid, taurine was also found to suppress the HbA1C levels in erythrocytes incubated with 50 mmol/l glucose concentration (Fig. 3 and Table 2). The levels of MDA were also found to be low in erythrocytes pretreated with taurine when compared to erythrocytes incubated with 50 mmol/l glucose alone (Fig. 4). Fig. 5 and Table 3 illustrates the effect of MDA on glycation of hemoglobin. HbA1C levels were estimated by the ion exchange column. The addition of MDA to washed erythrocytes incubated with 50 mmol/l glucose caused an increase in glycation of hemoglobin when compared to erythrocytes incubated with glucose. In view of the possibility that MDA –hemoglobin adduct can interfere in the estimation of glycated hemoglobin by ion exchange, erythrocytes were treated with and without
0.6
MDA (nmol/mg Hb)
*
** ** **
0.4
**
9.5 8.5
**
**
** *
7.5 6.5 5.5 4.5 3.5
Values are the mean T standard deviation of 4 experiments. *p < 0.05 when compared with HbA1C values of RBC treated with 50 mmol/l glucose alone.
0.5
Glycated Hemoglobin (%)
Table 2 Effect of taurine on glycated hemoglobin in erythrocytes treated with glucose (5 or 50 mmol/l) Treatment
193
Glucose (mmol/l)
5
5
50
50
50
50
50
MDA (mmol/l)
0
1
0
0.01
0.1
1
10
Fig. 5. Effect of different concentrations of MDA on glycated hemoglobin values in erythrocytes treated with glucose (5 or 50 mmol/l). Values are means T S.D. of 4 different experiments. At all the tested concentration of MDA the level of glycated hemoglobin was found to be enhanced when compared with erythrocytes incubated with only 50 mmol/l glucose (* vs. **p < 0.05).
1 mmol/l MDA and glycated hemoglobin was estimated by affinity chromatography. HbA1C was 6.54 T 0.36 with 50 mmol/l glucose and MDA in contrast to 5.8 T 0.47 with 50 mmol/l glucose alone. This difference was statistically significant.
4. Discussion Many clinical and experimental observations have highlighted the important role played by the enhanced nonenzymatic glycation of circulating and structural proteins in contributing to the pathogenesis related to diabetes, CRF, atherosclerosis and aging [1– 4]. Much effort has thus been spent searching for substances capable of either preventing or arresting the progression of glycation-dependent complications. Two inhibitory agents to be first proposed were aspirin and ibuprofen [15 – 17]. However, with regard to a possible therapeutic use of these compounds, it must be stressed that it is not yet sufficiently clear what would be the effect of irreversibly bound foreign chemical groups in
**
0.3
Table 3 Effect of different concentrations of MDA on glycated hemoglobin in erythrocytes treated with glucose (5 or 50 mmol/l)
0.2 0.1 0
Glucose (mmol/l)
5
5
5
50
50
50
50
50
Taurine (µmol/l)
0
25
50
0
25
50
100
150
Fig. 4. Effect of taurine on the levels of MDA in erythrocytes incubated with 5 and 50 mmol/l glucose. Values are means T S.D. of 4 different experiments. Levels of MDA were suppressed in erythrocytes preincubated with taurine when compared with erythrocytes incubated with 50 mmol/ l glucose alone (* vs. **p < 0.05).
Treatment
HbA1C values (in %)
5 mmol/l glucose 5 mmol/l glucose + 10 mM MDA 50 mmol/l glucose 50 mmol/l glucose + 0.01 mM MDA 50 mmol/l glucose + 0.1 mM MDA 50 mmol/l glucose + 1 mM MDA 50 mmol/l glucose + 10 mM MDA
5.63 T 0.25 5.79 T 0.53 6.71 T 0.42 7.23 T 0.33* 7.68 T 0.38* 8.25 T 0.43* 9.17 T 0.34*
Values are the mean T standard deviation of 4 experiments. *p < 0.05 when compared with HbA1C values of RBC treated with 50 mmol/l glucose alone.
194
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place of glucose in very long-lived protein structures. Thus, we could speculate that such groups might be chemically even more reactive than the stable glucose– protein adducts, the formation of which they have helped to prevent. Chronic therapy with aspirin and ibuprofen would be contraindicated, owing to its effects on the gastric mucosa. Of late, in vitro studies have demonstrated that vitamin E and vitamin C can inhibit the process of protein glycation [18,19]. But the results from the in vivo studies are discordant with this hypothesis. While Shoff et al. have shown that there is no significant relationship between vitamin E intake and glycated hemoglobin values [20], Ceriello et al. showed that vitamin E intake has an inverse relationship with the levels of glycated hemoglobin [21]. Similarly contradictory reports have been documented in the literature regarding the intake of vitamin C and the levels of glycated hemoglobin [20 –22]. Recently lipoic acid has attracted interest because of its diverse biological actions attributed to its chemical properties [23,24]. It is both lipid and water soluble [25] and has a potent antioxidative capacity in a wide variety of experimental system. Previous investigations have shown that lipoic acid exhibits metabolic effects on glucose transport and utilization. In cell culture (L6 myotubes and 3T3-L1 adipocytes), lipoic acid at concentrations exceeding 0.5 mmol/l exerted insulin-like effects, as indicated by the activation of the insulin signaling cascade leading to translocation of glucose transporters [26]. In animal models of diabetes, treatment with lipoic acid resulted in improved peripheral glucose utilization stimulated by insulin, and prevented diabetes related reduction in skeletal muscle content of GLUT 4 [27]. We found that lipoic acid caused decrease in the levels of glycated hemoglobin in erythrocytes incubated with 50 mmol/l glucose. Pretreatment with lipoic acid also significantly reduced the formation of lipid peroxides in erythrocytes. Our results are consistent with the observations of Kawabata and Packer that supplementation of lipoic acid in vitro can prevent glycation of bovine serum albumin [28]. Taurine is an intracellular amino acid that is present in millimolar concentrations in the plasma, tissues and interstitial media [29]. Taurine has been shown to exert beneficial effects in diabetes related conditions in which oxidative stress was suggested to have a pathological role. Experimental evidences have found a beneficial effect of taurine in preventing diabetic neuropathy, chronic puromycin aminonucleoside nephropathy and retinopathy [30 – 32]. In accordance to this, using animal models, taurine has been shown to prevent hyperglycemia-induced insulin resistance [33]. When the erythrocytes were pretreated with taurine, the glycation of hemoglobin was significantly attenuated. Similar to the effect of lipoic acid, preincubation of erythrocytes with taurine significantly inhibited the lipid peroxide formation.
The exact mechanism by which lipoic acid and taurine exert their protective effect against glycation of hemoglobin is difficult to entangle from the present study. But, the simultaneous inhibition of glycation and on the accumulation of lipid peroxides by lipoic acid and taurine suggest that lipid peroxide levels can have a role in the modulation of glycation reaction. This hypothesis was confirmed by our in vitro study where in MDA of different concentrations were co-incubated with glucose of 50 mmol/l concentration. In the present study, a concentration dependent increase in the extent of in vitro nonenzymatic glycation of hemoglobin was found in RBC incubated with glucose and MDA when compared with RBC incubated with glucose alone. Malondialdehyde is known to react with normal hemoglobin to form ionic components. As these compounds can interfere in the estimation of glycated hemoglobin by ion exchange chromatography, it seemed prudent to provide data comparing the results obtained with ion exchange chromatography with affinity chromatography method. When erythrocytes were incubated with 50 mmol/l glucose, the formation of glycated hemoglobin were significantly higher in cells treated with MDA and glucose than in cells treated with glucose alone. Thus, emphasizing the possible role of MDA per se on the glycation of hemoglobin. Consonant with our study, Jain and Palmer [34] using affinity chromatography method for the estimation of glycated hemoglobin have proposed that MDA per se can increase glycation of hemoglobin and vitamin E can suppress this process. No consensus has been reached as to the mechanism underlying the modulation of glycation of hemoglobin by MDA. Previously suggested mechanism includes the potential ability of the aldehyde groups of MDA to act as an anchor between sugar and hemoglobin moieties [34]. MDA at pathological concentration has been found to react with lysine to give N (-h-lysine amino acroline (h-LAA) [35]. h-LAA, the product of lysine with MDA, has been postulated to have the potential to react with an aldehyde group of sugar molecule, thereby bridging lysine and sugar molecules [36]. In conclusion, this study has shown that, in vitro, lipoic acid and taurine can inhibit the nonenzymatic glycation of hemoglobin. In vivo studies in normal animals and in animals with artificially induced diabetes are now required. This will help to define the physiological, pharmacological, chemical, and toxicity indices that might allow these antioxidant to be used safely and efficiently in humans in an attempt to minimize the nonenzymatic glycation of proteins, the hallmark of diabetic hyperglycemia and other chronic pathophysiological states. Acknowledgements This work was financially supported by the Indian Council of Medical Research (ICMR), New Delhi, India, in the form of Junior Research Fellowship to Mr. N. Selvaraj
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