Copyright 2002 by Institute of Pharmacology Polish Academy of Sciences

Polish Journal of Pharmacology Pol. J. Pharmacol., 2002, 54, 275280 ISSN 1230-6002

EFFECTS OF SOME DRUGS ON RAT ERYTHROCYTE 6-PHOSPHOGLUCONATE DEHYDROGENASE: AN IN VITRO AND IN VIVO STUDY
Mehmet ifti1,2,#, kr Beydemir1, Hayrullah Ylmaz3, Ebubekir Bakan4
1

Atatrk University, Arts and Science Faculty, Department of Chemistry, 25240 Erzurum, Turkey, 2Atatrk University, Biotechnology Application and Research Center, 25240 Erzurum, Turkey, 3Dicle University, Faculty of Education, Department of Chemistry, 21000 Diyarbakr, Turkey, 4Atatrk University, Medicinal Faculty, Department of Biochemistry, 25240 Erzurum, Turkey

Effects of some drugs on rat erythrocyte 6-phosphogluconate dehydrogenase: an in vitro and in vivo study. M. IFTI, . BEYDEMIR, H. YILMAZ, E. BAKAN. Pol. J. Pharmacol., 2002, 54, 275280. The in vitro and in vivo effects of some drugs on rat erythrocytes 6-phosphogluconate dehydrogenase were investigated in this study. Rat erythrocyte 6-phosphogluconate dehydrogenase was partially purified with ammonium sulfate precipitation. The enzyme activity was determined by Beutlers method. Some drugs such as ampicillin, amikacin sulfate, and netilmicin sulfate inhibited the enzyme activity in in vitro conditions, while metamizole activated it. The I50 values of the inhibiting drugs were 66.2, 5.836, and 0.963 mM, respectively. For the drugs having low I50 values (drug concentrations which produce 50% inhibition) (amikacin sulfate and netilmicin sulfate), in vivo studies were performed in rats (Sprague-Dawley). Amikacin sulfate at 64 mg/kg inhibited the enzyme activity significantly (p < 0.05) 2 h after dosing. Netilmicin sulfate at 6.4 mg/kg also inhibited the enzyme significantly (p < 0.05) 4 h after dosing. Amikacin sulfate and netilmicin sulfate inhibited rat erythrocyte 6-phospogluconate dehydrogenase both in vivo and in vitro. The enzyme was inhibited in vitro by ampicillin and activated in vitro by metamizole. Key words: 6-phospogluconate dehydrogenase, erythrocytes, rat, drugs

correspondence; e-mail: ciftcim@atauni.edu.tr

M. ifti, . Beydemir, H. Ylmaz, E. Bakan

INTRODUCTION
6-Phosphogluconate dehydrogenase (E.C.1.1.1.44; 6PGD) is the third enzyme of pentose phosphate metabolic pathway, catalyzing the conversion of 6PGA to D-riboluse-5-phosphate in the presence of NADP+. This reaction yields NADPH, which protects the cell against the oxidant agents by producing reduced glutathione [5, 21]. NADPH is also a coenzyme participating in the synthesis of a number of biomolecules such as fatty acids, steroids, and some amino acids [16, 29]. In the case of NADPH lack, the concentration of reduced glutathione in living system declines, resulting in cell death. For this reason, 6PGD can be defined as an antioxidant enzyme [19, 28]. Many drugs are known to activate or inhibit several body enzymes in vivo [1, 12, 14], affecting the metabolic pathways. If any drug inhibits 6PGD, the decreased NADPH and GSH will cause cell damage especially in older erythrocytes, resulting in severe health problems [1, 14]. No studies could be found on the in vitro and in vivo effects of some drugs on erythrocyte 6PGD. Vitamin C has been reported to stimulate 6PGD [24]. This study was aimed to determine any possible effect of some commonly used drugs on red blood cell 6PGD activity.

Ammonium sulfate precipitation The hemolysate was subjected to precipitation with ammonium sulfate (between 10% and 90%). The enzyme activity was determined both in supernatant and in precipitate for each respective precipitation. The enzyme was observed to precipitate at 6080% precipitation step. The precipitated enzyme was dissolved in phosphate buffer (50 mM; pH = 7.0). The resultant solution was clear, and contained partially purified enzyme. Activity determination The enzymatic activity was measured by Beutlers method [3]. In this spectrophotometric measurement, the reaction medium maintained at 25C contained 0.1 mM Tris-HCl (pH = 8.0) with 0.5 mM EDTA, 10 mM MgCl2, 0.2 mM NADP+, and 0.6 mM 6-PGA in a total volume of 1 ml. NADPH produced in the reaction mixture was measured at 340 nm. One unit of enzyme (EU) activity was defined as the enzyme amount reducing 1 mmol NADP+ per 1 min at 25C, pH 8.0. Protein determination Protein content in ammonium sulfate supernatant was quantified spectrophotometrically at 595 nm according to Bradfords method [6], with bovine serum albumin used as a standard. SDS polyacrylamide gel electrophoresis (SDS-PAGE) The control of enzyme purity was carried out using Laemmlis procedure [20] with 3% and 8% acrylamide concentrations for running and stacking gel, respectively. The gel solution was supplemented with 10% SDS. The gel was stabilized in the solution containing 50% propanol + 10% TCA + 40% distilled water for 30 min. The gels were stained for about 2 h in the solution of 0.1% Coommassie Brillant Blue R-250 in 50% methanol + 10% acetic acid. Finally, the washing was carried out in the solution of 50% methanol + 10% acetic acid + 40% distilled water until protein bands were clear. Rabbit myosin (205,000), E. Coli b-galactosidase (116,000), rabbit phosphorylase B (97,400), bovine albumin (66,000), chicken ovalbumin (45,000), and bovine carbonic anhydrase (29,000) were used as standards (Sigma: MW-SDS-200).

MATERIALS and METHODS

Materials 6PGA, NADP+, Tris, and the other chemicals were from Sigma Chem. Co., and the drugs were purchased from Hoechst Marian Roussel (Turkey). Preparation of the hemolysate and hemoglobin determination Fresh blood samples from the rats, collected to EDTA-containing tubes, were centrifuged (15 min, 2,500 g) and plasma and buffy coat (leucocytes) were removed. The pack of red cells was washed three times with KCl (0.16 M) and hemolyzed with 5 volume of ice-cold water and then centrifuged (+4C, 10,000 g, for 30 min) to remove the ghosts and intact cells. Hemoglobin (Hb) concentration in hemolysate was determined by cyanmethemoglobin method [2, 11, 23, 26].

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In vitro drug effect In order to determine the effects of some drugs on 6-PGD, some concentrations of ampicillin (8.17, 40.85, 57.19, 81.7, and 122.55 mM), amikacin sulfate (2.13, 4.26, 6.4, 8.52, 12.78, and 21.3 mM), netilmicin sulfate (0.069, 0.208, 0.52, 1.04, 1.56, and 2.6 mM), and metamizole (1.42, 2.84, 4.26, 8.53, and 12.80 mM) were added to separate tubes containing purified enzyme. The enzyme activity was measured in these tubes taking the tubes containing no drug as control (100% activity). The I50 values were obtained after activity (in %) was plotted vs. drug concentration. In vivo drug effect In vivo drug effect studies were conducted only for those having low I50 values, i.e. amikacin sulfate and netilmicin sulfate. Each drug-treated group comprised 10 animals which were kept under special conditions (in a windowless room, at temperature of 22C, with light on for 14 h) for 2 months. Once the rats reached a weight of about 180 20 g, the study begun. Before intramuscular drug injection, the control blood samples (0.5 ml of whole blood with EDTA) were obtained from the animals; 64 mg/kg of amikacin sulfate was injected to one group, and 6.4 mg/kg of netilmicin sulfate to the other. The blood samples were taken from each group 2, 4, and 6 h after injection. Hemolysates were prepared from all blood samples as mentioned above. Hb levels and 6PGD activity were determined in these hemolysates. Statistical analysis The mean values and standard deviations of the results were given. Student t-test was used for statistical evaluation of the difference, with the p value less than 0.05 considered significant.
Fig. 1. SDS-PAGE bands of 6PGD (Lane 1: supernatant sample after precipitation with 060% ammonium sulfate. Line 2: standard proteins; rabbit myosin (205,000), E. Coli b-galactosidase (116,000), rabbit phosphorylase B (97,400), bovine albumin (66,000), chicken ovalbumin (45,000), and bovine carbonic anhydrase (29,000). Lane 3: precipitate sample after precipitation with 6080% ammonium sulfate)

250 200

Activity %

150 100 50 0 0 2 4 6 8 10 12 14

[Metamizole] mM
Fig. 2. Activity (%) vs. metamizole concentration regression analysis graphs for 6PGD in the presence of 5 different metamizole concentrations

120 100

RESULTS
The enzyme 6PGD of the rat erythrocytes was partially purified 78.5 fold by 6080% ammonium sulfate precipitation. The degree of purity of the enzyme was controlled with SDS-PAGE (Fig. 1). Figures 2, 3, 4, and 5 show the in vitro effects of the selected drugs on the enzyme activity. I50 values were estimated at 66.2, 5.82, and 0.96 mM for ampicillin, amikacin sulfate, and netilmicin sulfate, respectively. The results of in vivo effects of amika-

Activity %

80 60 40 20 0 0 5 10 15 20 25

[Amikacin sulfate] mM
Fig. 3. Activity (%) vs. amikacin sulfate concentration regression analysis graphs for 6PGD in the presence of 6 different amikacin sulfate concentrations

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120 100 80 60 40 20 0 0 20 40 60 80 100 120 140

Table 1. In vivo effects of amikacin sulfate and netilmicin sulfate on rat red blood cell 6-phosphogluconate dehydrogenase activity (n = 10) Drug Amikacin sulfate Time (h) Control 2 4 6 Netilmicin sulfate Control 2 4 6 X SD (EU/gHb) 2.356 0.154 1.082 0.257 1.848 0.342 2.150 0.116 2.647 0.040 1.408 0.245 1.441 0.173 2.370 0.119 p < 0.05 > 0.05 > 0.05 < 0.05 < 0.05 > 0.05

Activity %

[Ampicillin] mM
Fig. 4. Activity (%) vs. ampicillin concentration regression analysis graphs for 6PGD in the presence of 5 different ampicillin concentrations
120 100 80 60 40 20 0 0 0.5 1 1.5 2 2.5 3

[Netilmicin sulfate] mM
Fig. 5. Activity (%) vs. netilmicin sulfate concentration regression analysis graphs for 6PGD in the presence of 6 different netilmicin sulfate concentrations

cin sulfate and netilmicin sulfate are presented in Table 1. In amikacin sulfate-treated group of animals, the control enzyme activity was 2.356 0.154 EU/gHb, while the respective values determined 2, 4 and 6 h after drug administration were: 1.082 0.257 EU/gHb, 1.848 0.342 EU/gHb, and of 2.150 0.257 EU/gHb. On the other hand, in netilmicin sulfate-treated group of animals, the control enzyme activity was 2.647 0.04 EU/gHb, and 2, 4, and 6 h after drug treatment it amounted to 1.408 0.245 EU/gHb, 1.441 0.173 EU/gHb, and 2.370 0.119 EU/gHb, respectively.

DISCUSSION
As it is seen in Figure 1, when the supernatant sample after precipitation with 060% ammonium sulfate (Lane: 1) and precipitate sample obtained after precipitation with 6080% ammonium sulfate (Lane: 3) were compared, impurities were significantly eliminated.

It is well known that many drugs have adverse effects on the organism when used for therapeutic or other purposes [13]. These effects may be dramatic and systematic [10]. A good example for this situation is that in 1926 pamaquine used for malaria treatment caused severe adverse effects in the patients within several days, resulting in black urination, hyperbilirubinemia, dramatic decrease in blood Hb levels, and finally death, which occurred in cases of severe G6PD deficiency [18]. Similarly, acetazolamide inhibits carbonic anhydrase (CA), giving rise to severe diuresis [31]. Amikacin sulfate and netilmicin sulfate are commonly used aminoglycoside antibiotics. However, they have some side effects such as nephrotoxicity, ototoxicity, neurotoxicity, fever, bone marrow depression, and hemolytic anemia. Ampicillin is a b-lactam antibiotic, having many side effects (fever, allergy, epidermal eruption). Metamizole is a potent analgesic and is commonly used. Its side effects include bone marrow depression and water and salt retention [17]. Ampicillin inhibits both human red cells G6PD and CA, netilmicin sulfate inhibits human red cells G6PD, and metamizole inhibits human red cells G6PD and activates human red cells CA. In addition, it has been reported that ampicillin and metamizole inhibit the rat CA in vivo within 3 h [4, 810]. We could find no previous study related to amikacin sulfate and to the effects of these drug on 6PGD activity. GSH is used by antioxidant defence mechanisms and its production requires NADPH synthesized in pentose phosphate metabolic pathway, in which G6PD and 6PGD participate. For this rea-

Activity %

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son, one can consider 6PGD as antioxidant enzyme [25]. In the present study, the effect of some commonly used drugs on 6PGD has been investigated in an animal model and in vitro. Figures 2, 3, 4, and 5 show that metamizole activates 6PGD in vitro and amikacin sulfate, ampicillin, and netilmicin sulfate inhibit it. The I50 values of amikacin sulfate, ampicillin, and netilmicin sulfate are 66.2, 5.82, and 0.96 mM, respectively, with netilmicin sulfate being a potent inhibitor for 6PGD. The in vivo studies showed that netilmicin sulfate inhibited the enzyme by 46.8% at 2nd h (p < 0.05) and 46.5% at 4th h (p < 0.05), while the result obtained at 6th h after drug treatment was not statistically significant. Amikacin sulfate inhibited the enzyme by 54% at 2nd h (p < 0.05) while the effects observed at 4th and 6th h were not statistically significant (Tab. 1). The in vitro and in vivo results support each other. The half-life of amikacin sulfate is 2 to 3 h [17], which is supported by the fact that the enzyme activity has been begun to rise after 2nd h being related to the elimination of the drug. The same is also true for netilmicin sulfate. The doses of amikacin sulfate (64 mg/kg) and netilmicin (6.4 mg/kg) are consistent with the previous studies, since the doses of 37.5200 mg/kg and 10100 mg/kg have been reported for amikacin sulfate and netilmicin, respectively [7, 15, 22, 27, 30]. If it is required to give amikacin sulfate and netilmicin sulfate to the patient, their dosage should be very well ordered to decrease the hemolytic side effects, because, these drugs may worsen the health of patient and may lead to fatal outcome. REFERENCES
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Received: January 30, 2002; in revised form: March 5, 2002.

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