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Effects of some oral hypoglycaemic drugs on erythrocyte nicotinamide adenine dinucleotide hydrogen diaphorase (E.C. activity of Wistar albino rats (Rattus rattus).

The red cell nicotinamide adenine dinucleotide hydrogen (NADH) dependent methaemoglobin reductase or diaphorase I enzyme (Breaking et al., 1951; Gibson and Harrison, 1947), protects the erythrocyte from an accumulation of methaemoglobin. Methaemoglobin is haemoglobin in which the group (iron II) is oxidized to iron III (Sauve, 2008). Small amounts of methaemoglobin are produced continuously but the proportion of total haemoglobin that is present as methaemoglobin is maintained at about 1% by the action of an NADH dependent methaemoglobin reductase; also called NADH diaphorase II (Neuwrit and Ponka, 1977) and NADH ferricynide reductase (Board, 1981) and NADH cytochrome 65 reductase.

A methaemoglobin reductase concentration that is greater than 1% occurs if the rate of methaemoglobin formation exceeds its rate of reduction. Also methaemoglobin is not just incapable of binding oxygen, the oxidation of one or more of the heme iron atoms in the tetramer distort the tetramers structure (Ajamar et al., 2012; Haymond et al., 2005). As a result, the remaining non-oxidized heme sub-units bind oxygen avidly and also release it less efficiently.

The deficiency of NADH methaemoglobin reductase is genetically transmitted as an autosomal recessive characteristic chromosome (Jaffe, 1959). Also biochemical variants of this enzyme has been reported (West et al., 1967).

Oral hypoglycaemic drugs vary in their mode of excretion and duration of action. Chloropropamide (diabenase) has a fairly prolonged biological action and increased potency/ weight ratio (Kaln and Schechter, 1993). The biguanides are oral hypoglycaemic drugs and their effects are also directed on oxidative phosphorylation (Ellenhorn and Barceloux, 1994).

The oral hypoglycaemic drugs, diabenese, daonil and glucophage used in this study were obtained from Nigeria-German drugs, Plc (Lagos, Nigeria). Other chemicals used for the in vitro analysis were from BDH (Poole Dorset, U.K) and Sigma Chemical Company (St. Louis, Missouri, USA).

Wistar albino rats, (Rattus rattus) 12-14 weeks, weighing between 200-220 g and derived from a colony maintained at the animal house unit of the Department of Biochemistry, University of Port Harcourt, were used for the experiment. The animals which were kept in cages (within a temperature of 25 [+ or -] 2 [degrees]C) were fed with standard laboratory chow (Pfizer feeds Plc, Nigeria) and water ad libitum. The animals were allowed to acclimatize for two weeks.

For the in vivo test, a total of 144 rats (with average weight of 210.15 [+ or -] 10.2 g) were used. Rats were divided into three groups; diabenese group, daonil group and glucophage group. Each group had 48 test rats while 12 rats served as control. Each of the drugs was administered to the rats at four different concentrations of 0.00 g/mg (control), 0.01 mg, 0.02 mg and 0.03 mg per 200 g body weight. The administration of the drugs to the rats was orally by intubations. The drugs at each of the concentrations were administered to the rats at day one, two, six and fourteen. Since water was used for the solubization of the drugs, the control rats were administered the equivalent volume (0.2 mL) of water in each case.

On each of the day(s) interval and at 3 h after the administration of the drug, three rats from each of the drug concentration groups were sacrificed after blood collection. Rats from the 0.00 mg concentration group served as control. Blood from the rats were collected by cardiac puncture into heparinized anticoagulant bottle and used for analysis as required.

NADH determination. The technique used for NADH assay is based on the method described by Board (1981). The assay mixture of 2 mL contained 0.1m tri HCl with 0.5 mM EDTA, PH 8.0, 0.2 mM NADH, 0.2 mM k3Fc (CN)6 and an aliquot (0.02 mL) of haemolysate; A tris buffer NADH mixture was first incubated for 10 min at 30 [degrees]C. The reaction was then initiated by the addition of 0.22 mL of ferricyanide-haemolysate mixture (in the ratio 10:1) pre-mixed a minute before addition. The rate of decrease in optical density of the system at 340 nm was measured for 10 min at 30 sec intervals against the blank containing the reaction mixture without haemolysate. The reaction was carried out at 30[degrees]C rather than the usual 37[degrees]C because the enzyme is unstable at higher temperature (Beutler, 1984).

Statistical analysis. Results (Tables 1-3) of Biochemical estimations were reported as mean [+ or -] SD and statistical analysis was performed using the students t-test of statistical significance at 95% confidence level (P = 0.05) (Brookes et al., 1979). Data were also analyzed by one-way analysis of variance (ANOVA) using SPSS/PC package and difference between means were compared using Duncan's (1955) multiple range test.

The in vivo study showed that rat erythrocyte NADH diaphorase activity was significantly elevated in the presence of the oral hypoglycaemic drugs, daonil, glucophage and diabenese. The maximal in vivo effect of the drugs on rat erythrocyte NADH diaphorase activity was obtained on the sixth day of drug administration with a significant (P < 0.05) decline on the fourteenth day (Tables 1-3). Comparatively, the effect of the three drugs on the enzyme was in the order: Glucophages > Daonil > Diabenese. The result of this work has shown that NADH diaphorase activity was enhanced following the administration of glucophage and daonil at concentration dependent manner. The effect of glucophage on NADH diaphorase activity was greater than that of daonil for the various concentrations. Also the effect was significantly different than that of the control.

On the other hand, diabenese had no significant effect on the erythrocytes enzyme activity. The activation of the enzyme by daonil and glucophage confirms the work of Chasseaud (1979) in which he proposed that erythrocyte enzyme functions to detoxify red cell xenobiotics. The increase in the rate of oxidation of haemoglobin leads to the accumulation of methaemoglobion which forms granules. This leads to an increase in the rate of its destruction by the spleen. As a result, the patient becomes anaemic (Mc Gilvery, 1979; Neutwrit and Ponka, 1977). Thus NADH diaphorase helps in curbing this effect as has been demonstrated by this work. This observation perfectly agrees with the suggested role of erythrocyte NADH diaphorase in xenobiotics detoxication (Mc Gilvery, 1979).


The authors are grateful to the Animal House Unit of the Department of Biochemistry, University of Port Harcourt, Nigeria for the provision of the experimental animals used for this work.


Ajamar, F., Gaetani, G., Garre, C., Goivanna, B., Salvido, E. 2012. Effect of primaquine on erythrocytes with NADH-methaemoglobin reductase deficiency and low glutathione reductose activity. British Journal of Haematology, 23: 333-341.

Beutler, E. 1984. Red Cell Metabolism. A Manual of Biochemical Method. pp. 3, 78-83, Grune and Stratton, New York, USA.

Board, P.G. 1981. NADH-ferricyanide reductase, a convenient approach to the evaluation of NADH-methaemoglobin reductase in human erythrocytes. Clinica Chemica Acta, 109: 233-237.

Breaking, V.K., Gibson, H., Harrison, D.C. 1951. Familial idiopathic methaemoglobinnaemia. Lancet I: 935-941.

Brookes, C.J., Bettley, I.G., Loxton, S.M. 1979. Fundamentals of Mathematics and Statistics for Students of Chemistry and Allied Subjects. pp. 382-384, John Wiley and Sons Ltd., NY, USA.

Chasseaud, L.F. 1979. The role of glutathione and glutathione-s-transferase in the metabolism of chemical carcinogens and other electrophilic agents. Advnce in CancerResearch, 29: 175-177.

Duncan, D.B. 1955. Multiple range and multiple F-test. Biometrics, 11: 1-10.

Ellenhorn, M.J., Barceloux, D.G. 1994. Medical Toxicology Diagnosis and Treatment of Human Poisoning. pp. 440-461, Charles Louisiana, USA.

Gibson, Q.H., Harrison, D.C. 1947. Familial idiopathic methaemoglobin. Five cases in one family. Lancet 2: 941-948.

Haymond, S., cariappa, R., Eby, C.S., Scott, M.G. 2005. Laboratory assessment of oxygenation in methemoglobinemia. Clinical Chemistry, 51: 434-444.

Jaffe, E.R 1959. Reduction of methaemoglobin in human erythrocytes incubated with purine neucleostides. Journal of Clinical Investigation, 38: 1555-1563.

Kaln, R.C., Schechter, Y. 1993. Insulin, oral hypoglycemic agents and the pharmacology of the endocrine pancreas. In: The Pharmacological Basis of Therapeutic, A. G. Gilman, T. W. Rall, A.S. Nies and P. Taylor (eds), pp. 1463-1484, 8th edition, New York, USA.

Mc Gilvery, R.W. 1979. Biochemistry, a Functional Approach. pp. 579-580, Holt Saunders International edition, Philadephia, USA.

Neuwrit, J., Ponka, P.J. 1977. The Regulation of Haemoglobin Synthesis. pp. 47, 67, 136, Martinus Nijhoff. The Hague, the Netherlands.

Sauve, A.A. 2008. NAD and Vitamin B3: from metabolism to therapies. Journal of Pharmacy and Experimental Therapy, 324: 883-930.

West. C.A., Gomperts, B.D., Huehns, E. R., Kessel, I., Ashby, J.R. 1967. Demonstration of an enzyme variant in a case of congenital methaemoglobin. British Medical Journal, 4: 212-214.

Samuel Chimezie Onuoha and Austin Amadike Uwakwe

Department of Biochemistry, University of Port Harcourt, PMB 5323 Choba, Nigeria

(received April 9, 2011; revised April 25, 2012; accepted May 2, 2012)

* Author for correspondence; E-mail:
Table 1. In vivo effect of Daonil on erythrocyte NADH
diaphorase activity of rat at pH 8.0 and 30[degrees]C NADH

Daonil (mg/200g   Day 1            Day 2
body weight)

                  X [+ or -] SD    X [+ or -] SD

0.00              6.13 (a)         6.19
                  [+ or -]  1.24   [+ or -] 0.81
0.01              6.66 (a)         7.83 (b)
                  [+ or -] 0.83    [+ or -] 0.59
0.02              7.00 (b)         8.50 (c)
                  [+ or -] 0.02    [+ or -] 0.01
0.03              7.43 (b)         9.41 (d)
                  [+ or -] 0.01    [+ or -] 0.14

Daonil (mg/200g   Day 6            Day 14
body weight)

                  X [+ or -] SD   X  [+ or -]  SD

0.00              6.70 (a)        6.91 (b)
                  [+ or -] 0.65   [+ or -] 0.42
0.01              10.17 (e)       10.35 (e)
                  [+ or -] 0.69   [+ or -] 0.96
0.02              10.35 (e)       11.01 (f)
                  [+ or -] 0.97   [+ or -]  1.41
0.03              11.44 (f)       7.04 (b)
                  [+ or -] 0.82   [+ or -]  1.03

Table 2. In vivo effect of Diabenese on erythrocyte NADH
diaphorase activity ofrat at pH 8.0 and 30[degrees]C.

Diabenes (mg/200g   Day 1            Day 2
body weight)
                    X [+ or -] SD    X [+ or -] SD

0.00                6.13 (a)         5.70 (a)
                    [+ or -]  1.24   [+ or -] 0.81
0.01                7.00 (b)         6.91 (b)
                    [+ or -]  1.40   [+ or -] 0.09
0.02                6.95 (b)         8.33 (d)
                    [+ or -] 1.04    [+ or -] 0.54
0.03                7.09 (b)         9.41 (d)
                    [+ or -] 0.05    [+ or -] 0.14

Diabenes (mg/200g   Day 6            Day 14
body weight)
                    X [+ or -] SD   X [+ or -]  SD

0.00                6.70 (b)        6.91 (b)
                    [+ or -] 1.16   [+ or -] 0.42
0.01                7.40 (c)        7.39 (e)
                    [+ or -] 0.01   [+ or -] 0.22
0.02                7.34 (e)        8.91 (d)
                    [+ or -] 0.45   [+ or -]  0.62
0.03                7.71 (c)        10.66 (f)
                    [+ or -] 0.98   [+ or -]  0.52

Table 3. In vivo effect of Glucophage on erythrocyte
NADH diaphorase activity ofrat at pH 8.0 and 30 [degrees]C.

Glucophage (mg/200 g   Day 1            Day 2
body weight)

                       X [+ or -] SD    X [+ or -] SD

0.00                   6.13 (a)         5.70 (a)
                       [+ or -]  1.24   [+ or -] 0.81
0.01                   8.18 (b)         12.06 (e)
                       [+ or -] 0.23    [+ or -] 0.69
0.02                   9.07 (b)         14.71 (f)
                       [+ or -] 0.06    [+ or -] 0.60
0.03                   10.56 (b)        18.20g
                       [+ or -] 0.67    [+ or -] 0.43

Glucophage (mg/200 g   Day 6            Day 14
body weight)

                       X [+ or -] SD    X [+ or -] SD

0.00                   6.70 (a)         6.91 (b)
                       [+ or -]  1.16   [+ or -]  0.42
0.01                   14.74 (f)        17.57g
                       [+ or -] 0.04    [+ or -] 0.08
0.02                   19.50h           17.87g
                       [+ or -] 0.15    [+ or -] 0.09
0.03                   20.55h           17.92g
                       [+ or -] 0.57    [+ or -] 0.15

Values with the same superscript letters (in Table 1-3) are not
statistically significant at 95% confidence level (P<0.05).
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Title Annotation:Short Communication
Author:Onuoha, Samuel Chimezie; Uwakwe, Austin Amadike
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Article Type:Report
Geographic Code:9PAKI
Date:Nov 1, 2012
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