The Effect of Glutathione Modulation on the Concentration of Homocysteine in Plasma of Rats

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1 C Pharmacology & Toxicology 2000, 87, Printed in Denmark. All rights reserved Copyright C ISSN The Effect of Glutathione Modulation on the Concentration of Homocysteine in Plasma of Rats Kjell K. Øvrebø 1 and Asbjørn Svardal 2 1 Surgical Research Laboratory, Department of Surgery, and 2 Department of Clinical Biochemistry, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway (Received February 21, 2000; Accepted April 28, 2000) Abstract: Elevated plasma homocysteine concentration in humans is associated with increased risk of arteriosclerosis and ischaemic heart disease. We studied whether the plasma homocysteine concentration could be changed by administration of drugs that modulate the concentration of glutathione in both plasma and tissue. Male wistar rats received reduced glutathione (0.5 mmol/kg), N-acetylcysteine (0.5 mmol/kg), L-buthionine-[S,R]-sulfoximine (2 mmol/kg) or Ringer acetate intravenously. Twenty min. later an arterial blood sample was drawn for the measurement of homocysteine and other thiols in the plasma. The thiols were quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The total homocysteine concentration in plasma of fasted rats was mm. Intravenous administration of reduced glutathione or N-acetylcysteine reduced the homocysteine concentration in plasma significantly by 51% to mm and 63% to mm, respectively (P 0.05). In contrast, L-buthionine-[S,R]-sulfoximine increased the concentration of homocysteine by 41% to mm (P 0.05). The glutathione concentration in plasma was mm in controls and was unchanged by N-acetylcysteine administration. Reduced glutathione increased plasma glutathione to mm (P 0.05), whereas L-buthionine-[S R]-sulfoximine lowered the plasma glutathione concentration to mm. Homocysteine was negatively correlated to the glutathione (rωª0.399, P 0.01) and the cysteine (rωª0.52, P 0.01) concentrations in plasma. Our conclusion is that modulation of the glutathione levels influences the concentration of homocysteine in plasma of rats. An elevated concentration of homocysteine in plasma constitutes an independent risk of vascular disease (Graham et al. 1997; Robinson et al. 1998). It seems clear that homocysteine damages venous and arterial endothelial cells, which is probably related to the production of hydrogen peroxide during oxidation of homocysteine to homocystine. The cellular damage can be prevented by catalase (Wilcken & Dudman 1989). Homocysteine also has mitogenic properties on vascular smooth muscle cells and reduces the cell proliferation of the endothelium (Tsai et al. 1994). However, the mechanisms by which homocysteine induces atherosclerotic disease, are not fully clarified. Factors that influence the plasma homocysteine level are therefore of interest for chemoprevention of atherosclerotic heart disease. Folic acid, vitamin B 6 and B 12, alone or in combination, lower the plasma level of homocysteine both among patients with a pathologically high plasma homocysteine level (Lobo et al. 1999) and in persons with plasma homocysteine levels within normal range (Dierkes et al. 1998). N-acetylcysteine lowers the plasma level of homocysteine also in patients with a high level of lipoprotein (a) (Wiklund et al. 1996) and in healthy volunteers (Hultberg et al. 1994). N-Acetylcysteine is often used clinically as a safe substrate Author for correspondence: Kjell K. Øvrebø, Surgical Research Laboratory, Department of Surgery, Haukeland University Hospital, N-5021 Bergen, Norway (fax π , Kjell.Ovrebo/kir.uib.no). for glutathione, which might imply a role for glutathione modulation in the intervention against an elevated plasma homocysteine level. However, it is not established whether the effect of N-acetylcysteine is the result of changes in the plasma level of glutathione and cysteine or a pharmacological effect of the drug. If the effect of N-acetylcysteine is the result of glutathione modulation, we would expect an equivalent effect on the concentration of homocysteine in plasma by administering glutathione instead of N-acetylcysteine. Moreover, depletion of glutathione should inversely increase the concentration of homocysteine in plasma. We investigated this hypotheses in an experimental rat model. Reduced glutathione and the specific g-glutamylcysteine synthetase inhibitor, L-buthionine-[S,R]-sulfoximine, were used as the principle intervention. Rats injected with N- acetylcysteine or Ringer acetate served as controls for earlier reported effects and as base controls, respectively. Materials and Methods Animals. Forty-four male Mol: WIST rats weighing g (mean S.D.) were supplied by Møllegård Breeding (Ejby, Denmark). The rats were housed in groups of five in MAK IV cages on aspen bedding. They had ad libitum access to water and RM1 expanded diet (Special Diets Services, Witham, UK). The rats were categorized on arrival according to the FELASA accreditation scheme (Federation of European Laboratory Animals Science Association, Utrecht, Netherlands). The animals arrived at the facilities ten days before the commencement of the experiments. The experimental animal board of the Norwegian Department of Agriculture approved the experiments.

2 104 KJELL K. ØVREBØ AND ASBJØRN SVARDAL Chemicals. Reduced glutathione (GSH) 98% pure, N-acetylcysteine 99% pure, and L-buthionine-[S,R]-sulfoximine (BSO) were purchased from Sigma Chemical Co., St. Louis, MO, USA. Deionized water was used as solvent to produce a solution isoosmolar with serum and to avoid side effects from buffering solutions. GSH (0.5 mmol/kg) and N-acetylcysteine (0.5 mmol/kg) were dissolved in 0.5 ml of deionized water resulting in a GSH solution with mean osmolality of 253 mosmol/kg and a ph of 2.89, and a N-acetylcysteine solution with mean osmolality of 265 mosmol/kg and ph of BSO (2 mmol/kg) was dissolved in 2 ml deionized water with a resulting mean osmolality of 297 mosmol/kg and ph of Ringer acetate was preferred for vehicle (deionized water) in controls to avoid complications from hypoosmolar injections. The drugs were injected immediately after preparation to avoid in vitro oxidation of the supplied reduced glutathione (GSH). Animal preparation. The animals received tap water only and were kept individually in wire-bottom cages the last 24 hr before the experiment. Anaesthesia was started with halothane followed by an intraperitoneal injection of 60 mg kg ª1 pentobarbital-na. Subsequent increments of pentobarbital were given intravenously upon return of the corneal reflex or pedal withdrawal response and under supervision of spontaneous ventilation. Body temperature was maintained at æ (mean S.D.) by a thermal blanket. A polyethylene catheter (o.d mm) was inserted through the right femoral artery into the abdominal aorta for monitoring blood pressure and for blood sampling. The catheter was connected to a SensoNor 840 pressure transducer (SensoNor, Horten, Norway) and a Gould WindoGraf 980 recorder (Cleveland, Ohio, USA) for continuous monitoring of mean aortic blood pressure and heart rate. Ringer acetate was infused at a rate of 1 ml 100 g ª1 hr ª1 in a second catheter inserted via the left femoral vein into the inferior caval vein. The animals were allowed 30 min. to stabilize and recover from surgery. The animals were distributed at random into four groups, each group containing eleven animals. After surgery, stabilization, and 20 min. before sacrifice, the animals in the GSH group received an intravenous injection of reduced glutathione (0.5 mmol/kg) in 0.5 ml of deionized water. The animals in the N-acetylcysteine group received an intravenous injection of N-acetylcysteine (0.5 mmol/kg) dissolved in 0.5 ml of deionized water, and the animals in the control group received 0.5 ml of Ringer acetate in the same time interval. Due to slow emptying of GSH from tissue stores (Martensson et al. 1990), the BSO group of animals received L-buthionine-[S,R]- sulfoximine, 2 mmol/kg in 2 ml deionized water, intraperitoneally 3 hr and 12 hr earlier, and Ringer acetate (0.5 ml) at the same time as in the control group. The drugs, dosages, and time intervals were tried out in a previous experiment (Øvrebø et al. 1997). Arterial blood samples for the measurement of blood gases and thiol concentrations were drawn at the end of the experiment. The animals were sacrificed by severing the inferior caval vein. The various thiol species were routinely measured in duplicates. Derivatised samples were stored at ª80 æ until chromatographic analysis and the samples were frozen and thawed only once. In the Results and the Discussion sections, the total free homocysteine, glutathione, cysteine and cysteinylglycine forms (reduced formπdisulfide formπsoluble mixed disulfide form) are for short referred to as homocysteine, glutathione, cysteine and cysteinylglycine. Statistics. The data analyses were performed with a personal computer and the SPSS ver. 8.0 statistical package. The plasma homocysteine, glutathione, cysteine or cysteinylglycine concentrations were the dependent variables. The variables were compared by oneway analysis of variance with experimental group as the grouping factor. Newman Keuls multiple range tests were applied whenever justified by the analysis of variance. Two-tailed bivariate correlation analyses were used to investigate associations between variables. Pearson correlation coefficient was presented for the significant associations. A P-value less than 0.05 was regarded as statistically significant. Unless otherwise indicated, data are presented as mean S.E.M. Results The central haemodynamics did not change significantly during the experiments. The mean arterial pressure was mmhg and the heart rate beats/min. Group differences were not observed. The arterial blood gases (po kpa, pco kpa) and blood ph ( ) were unaffected by the interventions. However, base excess and HCO 3 ª were somewhat lower in the GSH group (BE ª mmol/l, HCO mmol/l) than in the BSO group (BE ª mmol/l, HCO mmol/l) (P 0.05). Thiol measurement. Sample collection. Blood samples were routinely collected from the aortic catheter before sacrifice and centrifuged immediately at 5,000 rpm for 3 min. (Heraeus Labofuge 400, Heraeus, Osterode, Germany). The plasma was separated and stored in liquid nitrogen for subsequent analyses. Total thiols in plasma. Reduced thiols react with monobromobimane and form fluorescent adducts, which were quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. Total amount of homocysteine, glutathione, cysteine and cysteinylglycine (reduced formπdisulfide formπsoluble mixed disulfide formπprotein-bound form) were obtained from plasma. NaBH 4 reduced free and protein-bound disulfides in the samples before the thiols were derivatized with monobromobimane. The thiol-bimane adducts were quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection (Svardal et al. 1990). Fig. 1. Plasma homocysteine concentration in the controls and in animals treated with reduced glutathione (GSH), N-acetylcysteine (NAC), or L-buthionine-[S,R]-sulfoximine (BSO). ANOVA P * P 0.05, different from the control group by Newman- Keuls multiple range test. P 0.05, different from all the other groups by Newman-Keuls multiple range test.

3 PLASMA HOMOCYSTEINE AND GLUTATHIONE MODULATION 105 Table 1. The concentration of thiols (sum of reduced and oxidized forms) in plasma of rats. Control GSH NAC BSO Groups A B C D ANOVA Glutathione (mm) a,c,d P Cysteine (mm) a,c,d a,b,d P Cysteinyl-glycine (mm) a,c,d P Abbreviations: GSH, group of animals treated with reduced glutathione; NAC, animals treated with N-acetylcysteine; and BSO, animals treated with L-buthionine-[S,R]-sulfoximine. The total concentrations (sum of reduced and oxidized forms) of glutathione, cysteine, and cysteinylglycine in plasma are presented. Lower case letters denote a significant difference from columns with corresponding capital letter, P 0.05 by Newman-Keuls multiple range tests. Homocysteine. The effects of the glutathione modulation on the concentrations of homocysteine in the plasma are summarized in fig. 1. GSH and N-acetylcysteine administration lowered the concentration of homocysteine in the plasma significantly with 51% (P 0.05) and 63% (P 0.05), respectively, from the level in the control group. BSO on the other hand increased significantly the concentration of homocysteine in plasma by 41% (P 0.05). Glutathione. The concentration of glutathione in plasma was significantly elevated in the GSH group (table 1). When all groups were analyzed together, neither N-acetylcysteine nor BSO altered the plasma concentration of glutathione. However, since the concentration of glutathione in plasma of the GSH group represents an intervention and not a metabolic effect, the ANOVA was repeated without this group. The concentration of glutathione was then significantly lower in the BSO group than in the control and in the N-acetylcysteine group (P 0.05). When all were analyzed together, the concentration of glutathione was negatively correlated to the concentration of homocysteine in plasma (rωª0.399, P 0.01). The scatter diagram revealed a clustering around many low and some high glutathione concentrations represented by the GSH group. No association was observed between glutathione and homocysteine in the control, GSH, or N-acetylcysteine groups. In the BSO group, however, the concentration of glutathione and the concentration of homocysteine in plasma were positively correlated (rω 0.613, PΩ0.045). Cysteine. Both the GSH and the N-acetylcysteine administration increased the concentration of cysteine in the plasma (table 1). GSH increased the concentration of cysteine in plasma significantly more than N-acetylcysteine (P 0.05). BSO, however, did not influence the concentration of cysteine in plasma. The concentration of cysteine in plasma was negatively correlated to that of homocysteine (rωª0.52, P 0.01). When each group was analyzed separately, no association was seen between the cysteine and the homocysteine concentrations in the control, GSH, or N-acetylcysteine groups. In the BSO group, however, the concentration of cysteine was positively correlated to that of homocysteine (rω0.637, PΩ0.035). Cysteinylglycine. GSH significantly increased the concentration of cysteinylglycine in plasma whereas the BSO and the N-acetylcysteine administration lowered significantly the plasma concentrations of cysteinylglycine (table 1). Discussion Antioxidants are believed to protect against vascular disease. We found that modulation of the glutathione level significantly changed the concentration of homocysteine in plasma, which is directly related to the risk of cardiovascular disease (fig. 1). The concentrations of homocysteine and glutathione in plasma of fasted controls (fig. 1 and table 1) were consistent with former observations (Griffith & Meister 1979; Hagen et al. 1990; Hum et al. 1991; Boston et al. 1995). The central haemodynamics and the blood gasses were stable and unaffected by the GSH, N-acetylcysteine, or BSO administration. GSH and N-acetylcysteine dissolved in deionized water are weak acids, and GSH lowered the HCO 3 level and the base excess content in the blood. However, the blood ph did not change. The acidity of GSH and N-acetylcysteine was therefore of minor importance to the animals and it is unlikely that the acidity of the drugs was a confounding factor. It was difficult to preserve and quantify thiols in the reduced form in this model. The thiols are instantly oxidized outside the body (Øvrebø et al. 1997). Therefore, only the total concentration of the thiols (reduced and oxidized forms) in the plasma was evaluated in this study. GSH, N-acetylcysteine and BSO are modulators of the glutathione level in both plasma and tissues. Glutathione takes part in conjugation of endogenous and exogenous compounds, the reduction of H 2 O 2 and O 2 ª, provides the cells with a reduced local environment and protects the cell membrane lipids against oxidation, and causes a reductive cleavage of disulfides linkages (Meister & Anderson 1983). GSH and BSO changed the concentration of glutathione and homocysteine in the plasma but in opposite directions.

4 106 KJELL K. ØVREBØ AND ASBJØRN SVARDAL N-Acetylcysteine effectively lowered the concentration of homocysteine in the plasma but without a concomitant change in the plasma glutathione concentration. The negative association between the glutathione concentration and the homocysteine concentration was weak, and probably not important due to scatter of the data. This lack of uniformity suggests that the concentration of glutathione in plasma is not the common denominator for the observed changes in the homocysteine level in plasma. The concentration of cysteine in plasma was negatively associated with the concentration of homocysteine. However, the correlation was again weak, and while GSH and N-acetylcysteine changed the cysteine and homocysteine concentration in plasma (table 1), neither of these groups revealed any correlation between cysteine and homocysteine. Within the BSO group, a significant correlation was observed between the cysteine and the homocysteine concentration in plasma but the BSO administration did not convey a group effect on the cysteine concentration. Thus it is also unlikely that the concentration of cysteine in plasma is the common denominator in the modulation of the homocysteine level in this study. Only GSH administration significantly changed the concentration of cysteinylglycine in the plasma (table 1). N- Acetylcysteine and BSO administration, which changed the concentration of homocysteine in opposite directions, did not alter the cysteinylglycine concentration. Cysteinylglycine therefore seems to be of little importance for the homocysteine level in plasma. A common plasma mediator for the effect of GSH, N- acetylcysteine, or BSO on the concentration of homocysteine in plasma of rats was not found in this study. It is possible, however, that N-acetylcysteine, GSH or BSO could mediate the changed plasma homocysteine level in a pharmacological manner, but we did not evaluate this possibility. N-Acetylcysteine and BSO both change the intracellular glutathione levels by acting as substrate for cysteine and thereby glutathione syntheses or by inhibiting g-glutamyl synthetase, respectively (Meister 1985). Earlier, we found that GSH also elevated the level of glutathione in the gastric mucosa, possibly by increasing plasma metabolites, which are translocated into the cells as substrate (Øvrebø et al. 1997). It is therefore possible that the observed changes in homocysteine concentration are a result of an altered intracellular level of glutathione and cysteine. Two mechanisms for the change in plasma homocysteine are generally accepted: 1) Altered release of homocysteine from tissue cells; and 2) altered clearance of homocysteine from plasma. These mechanisms regulate the half-life and the concentration of homocysteine in the plasma (Refsum et al. 1998). The export of homocysteine from cells is more pronounced in proliferating than in stationary cells, and increases with high levels of methionine and low levels of folate (Refsum et al. 1998). Hultberg et al. (1997) showed that to some extent N-acetylcysteine lowered a high intracellular homocysteine level in cell cultures when homocysteine was added in excess to the medium. A low intracellular level of homocysteine would explain a low plasma homocysteine concentration by reduced translocation of homocysteine out of cells. This could account for the GSH and N-acetylcysteine mediated reduction of plasma homocysteine in our study. However, the half-life of homocysteine in plasma is long (approximately 223 min. in man) (Guttormsen et al. 1993). It is therefore uncertain whether the time span of 20 min. in our study would be sufficient to lower intracellular homocysteine, its translocation out of cells, and lower the concentration of homocysteine in plasma by 51% or 63% by regular metabolism. In addition, a low intracellular level of glutathione does not influence the intracellular level of homocysteine in vitro (Djurhuus et al. 1990; Hultberg et al. 1995). Increased intracellular level of homocysteine and increased translocation of homocysteine out of cells could therefore not account for the observed effects of BSO on the plasma homocysteine concentration in our study. The principles of changed intracellular level of homocysteine and translocation out of cells were therefore probably unimportant for the effects observed in our study. A change of homocysteine clearance from the plasma is more likely. Hultberg et al. (1994) found that N-acetylcysteine lowered the total homocysteine level in plasma of healthy volunteers, whereas the amount of reduced homocysteine and free (non-protein bound) fraction of homocysteine increased. They suggested that N-acetylcysteine displaced homocysteine from their protein binding sites by disulfide interchange reactions. This leads to the formation of mixed low molecular weight cysteine and N-acetylcysteine disulfides with high renal clearance. Later Bostom et al. (1995) confirmed a substantial clearance of homocysteine by normal rat kidney. GSH, used in our study, should be equally effective in displacing homocysteine from plasma proteins. It is possible that displacement of homocysteine can explain the effects of GSH and N-acetylcysteine observed in our study. A reverse mechanism with increased protein binding of homocysteine in plasma of rats treated with BSO could result in a reduced clearance from plasma. An extrarenal metabolism or clearance of plasma homocysteine is also possible. N-Acetylcysteine alleviate homocysteinemia in uraemic patients, but without affecting the removal of homocysteine during haemodialysis (Bostom et al. 1996). Glutathione modulation might also effect these pathways of homocysteine clearance. This study supports a role for glutathione modulation in the treatment of homocysteinaemia and in the prevention of vascular disease. However, further studies involving the mechanisms involved, the safety, and the efficacy of glutathione modulation on the plasma concentration of homocysteine are needed. Acknowledgements We would like to thank Audun Høylandskjær for skilled technical assistance.

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