Departments of Pharmacology and
1 Biochemistry, Medical Faculty and
2 Biotechnology Application and Research Center, Atatürk University, Erzurum, Turkey
Received 12 July 2001; in revised form 25 January 2002; accepted 13 February 2002
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ABSTRACT |
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INTRODUCTION |
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G6PD (d-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49) is the key enzyme which catalyses the first step of the pentose phosphate metabolic pathway (Shreve and Levy, 1977). A major role of NADPH in erythrocytes is regeneration of reduced glutathione, which prevents haemoglobin denaturation, preserves the integrity of red blood cell membrane sulphydryl groups, and detoxifies hydrogen peroxide and oxygen radicals in and on the red blood cells (Deutsch, 1983
; Weksler et al., 1990
). A decrease of G6PD results in NADPH and reduced glutathione deficiency in erythrocytes; scarcity of reduced glutathione causes early haemolysis in spleen (Andrews and Mooney, 1994
).
Ethanol is a widely consumed sedativehypnotic drug throughout the world (Lee and Becker, 1989). It has been shown that ethanol intake may lead to oxidative damage in several tissues such as brain, stomach, liver or erythrocyte (Bondy and Guo, 1994
; Sozmen et al., 1994
; Lindi et al., 1998
; Hernandez-Munoz et al., 2000
). Ethanol increases the generation of reactive oxygen species in these tissues and its acute intake decreases reduced glutathione levels in plasma and erythrocytes (Loguercio et al., 1997
).
Since ethanol has oxidant effects in erythrocytes, it was considered important to reveal the effect of ethanol consumption on erythrocyte G6PD activity, which has not been studied before. Therefore the objective of this study was to investigate the effect of ethanol on G6PD enzyme activity in vitro in human, and in vivo in rat, erythrocytes.
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MATERIALS AND METHODS |
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In vitro studies
Preparation of the haemolysates. Fresh human blood collected in tubes with EDTA (5 mM) was centrifuged at 2500 g for 15 min and the plasma and leucocyte coat were removed by aspiration. The packed red cells were washed with 0.16 M KCl solution thrice each time, the samples were centrifuged at 2500 g and the supernatants were removed. One vol of erythrocytes was haemolysed with 5 vol of ice-cold water and centrifuged at 4°C and 10 000 g for 30 min to remove the ghosts and intact cells (Shreve and Levy, 1977; Ninfali et al., 1990
; Weksler et al., 1990
).
Ammonium sulphate fractionation and dialysis. Ammonium sulphate (3565%) precipitation was performed on haemolysates. Ammonium sulphate was slowly added to complete dissolution. The mixture was centrifuged at 5000 g for 15 min and the precipitate was dissolved in 50 mM phosphate buffer (pH 7.0), then dialysed at 4°C in 50 mM K-acetate/50 mM K-phosphate buffer (pH 7.0) for 2 h with two changes of buffer (Ninfali et al., 1990).
Preparation of affinity gels. Two grams of dried 2',5'-ADPSepharose 4B gel were used for a 10 ml column volume. The gel was washed with distilled water to remove foreign bodies and air in the swollen gel was eliminated. The gel was suspended in 0.1 M K-acetate/0.1 M K-phosphate buffer (pH 6.0), then packed in a small column (1 x 10 cm) and equilibrated with the same buffer. The gel was washed with equilibration buffer. The flow rates for washing and equilibration were adjusted by a peristaltic pump to 50 ml/h (Ninfali et al., 1990).
Purification of G6PD by affinity chromatography. The dialysed sample was loaded on the 2',5'-ADPSepharose 4B affinity column and the gel was washed with 25 ml of 0.1 M K-acetate/0.1 M K-phosphate (pH 6.0), with 25 ml of 0.1 M K-acetate/0.1 M K-phosphate (pH 7.85), and finally with 0.1 M KCl/0.1 M K-phosphate (pH 7.85) buffer. Washing continued up to an absorbance of 0.05 at 280 nm. Elution was carried out with 80 mM K-phosphate + 80 mM KCl + 0.5 mM NADP+ + 10 mM EDTA (pH 7.85) solution at 20 ml/h flow rate. Eluates were collected in 2 ml tubes and the activity of each was separately calculated. Active fractions were collected. All procedures were performed at 4°C (Morelli et al., 1978; Delgado et al., 1990
; Ninfali et al., 1990
).
Protein determination. Quantitative protein determination was performed spectrophotometrically at 595 nm by the method of Bradford (1976), with bovine serum albumin as standard.
Sodium dodecyl sulphate (SDS)polyacryamide gel electrophoresis. This was performed after the purification of enzyme by the method of Laemmli (1970). It was carried out in 3% and 10% acrylamide concentrations for gel stacking and running respectively, containing 0.1% SDS.
In vitro inhibitor studies
Ethanol was used as the inhibitor. Activities were measured at 0.242, 0.484, 0.968, 1.936 and 2.904 mM cuvette concentrations of ethanol. Drug concentrations which produce 50% inhibition (I50) were calculated from graphs drawn from data with five different ethanol concentrations.
Measurements of G6PD activity
G6PD activity was measured at 37°C by the method of Beutler (1971), which depends on the reduction of 2 mM NADP+ by G6PD, in the presence of glucose 6-phosphate. The activity measurement was made by monitoring the increase in absorption at 340 nm at 37°C. One enzyme unit was defined as the reduction of 1 µmol of NADP+/min at 37°C, pH 8.0.
In vivo inhibitor studies
Ten adult male SpragueDawley rats with a weight of 200250 g were used for the experiment. The animals were housed individually and were fed with standard laboratory chow and water before the experiment. The animal laboratory was windowless with controlled temperature (22 ± 1°C) and lighting controls (14 h light/10 h dark cycles). Twenty-four hours before the experiments, the rats were starved, but were allowed access to water ad libitum. For control measurements, a 0.5 ml blood sample was taken from a tail-vein before drug administration. Then, 2 ml/kg of ethanol (96%) was administered by gavage. At 1, 3 and 6 h after ethanol administration, 0.5 ml blood samples were taken again. All blood samples were added to EDTA tubes. Haemolysates were prepared as described for the in vitro studies. G6PD activity was measured at 37°C according to Beutler's method (Beutler, 1971).
Statistical analysis
Results are given as means ± SD. Data were analysed by Student's t-test and analysis of variance (ANOVA). P < 0.05 was considered significant.
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RESULTS |
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DISCUSSION |
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After ethanol intake, its blood level rises to a peak within 3090 min (Laurence et al., 1997). The half-life of ethanol in plasma is highly variable, but a single dose of ethanol will not all be disposed of for 68 h or even more (Laurence et al., 1997
). In our study, maximal inhibition of G6PD activity was within 1 h after ethanol administration, and inhibition continued significantly after 3 h (Table 2
). These results suggest a correlation between plasma peak level of ethanol and maximal inhibition of G6PD enzyme activity, and between the plasma half-life of ethanol and continuity of inhibition. Moreover, the results of our study show that the ethanol itself is a highly potent inhibitor of erythrocyte G6PD enzyme activity in vitro. Based on our results, we believe that the inhibitory effect of ethanol on G6PD enzyme activity at least may be one of the causes of ethanol-induced haemolysis.
In conclusion, the present study suggests that patients with G6PD deficiency should avoid ethanol intake. If they consume any ethanol, they should not be administered oxidant drugs such as analgesics and antipyretic, which frequently inhibit G6PD activity. If it is required to give these drugs to the patient with G6PD deficiency, their dosage should be strictly determined to minimize the haemolytic side-effects.
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FOOTNOTES |
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