An improved technique for the assay of red blood cell superoxide dismutase (SOD) activity

Transcription

1 ELSEVIER Clinica Chimica Acta 247 (1996) 1-6 An improved technique for the assay of red blood cell superoxide dismutase (SOD) activity L.E. Ilouno, E.N. Shu*, G.E. Igbokwe Department of Applied Biochemistry, Nnamdi Azikwe University, P.M.B. 5025, Awka, Nigeria Received 1 May 1995; revision received 25 September 1995; accepted 16 October 1995 Abstract Superoxide dismutase (SOD) activity of erythrocytes from normal pregnant women (controls) and from pre-eclamptic toxaemia (PET) cases was determined by the method of Winterbourn et al. (J Lab Clin Med 1975;85: ) with slight modifications. Instead of the Michaelis-Menten type of plot that is usually obtained and which these authors have found to be imprecise, a linear plot was obtained when the reciprocal values for the percentage inhibition of nitroblue tetrazolium (NBT) reduction were plotted against the corresponding volumes of red blood cell (RBC) extract. This plot was found to give precise values for the volumes of RBC extracts required for 50% inhibition of NBT reduction. Consequently, a clearer understanding is beginning to emerge regarding the SOD activity of PET cases compared with that of normal pregnant women. Keywords: Superoxide dismutase; Pre-eclamptic toxaemia; Pregnancy; Nitroblue tetrazolium; Red blood cells 1. Introduction Superoxide dismutase (SOD; EC ), a free radical-metabolising enzyme, protects the cell membrane from damage by the highly reactive superoxide free radicals [1-3]. Such damage results in the formation of * Corresponding author, Department of Pharmacology and Therapeutics, College of Medicine, University of Nigeria Teaching Hospital, P.M.B , Enugu, Nigeria /96/$ Elsevier Science B.V. All rights reserved SSDI ! (95)06193-H

2 2 L.E. llouno et al./clinica Chimica Acta 247 (1996) 1-6 malonyldialdehydes (MDAs) which are thought to play a role in the aetiology of pre-eclamptic toxaemia (PET) of pregnancy [4]. SOD, which is a copper enzyme, occurs very widely in cells and tissues such as the erythrocytes, liver and brain. Hence it can be variously referred to as erythrocuprein (erythrocyte SOD), hepatocuprein (liver SOD), and cerebrocuprein (brain SOD). These are all commonly referred to as cytocuprein [5-7]. Various methods have been used to determine SOD activity in biological fluids [8-11], some of which measure the volume of cell extract required for the half-maximal inhibition of reduction of nitroblue tetrazolium (NBT). However, the hyperbolic curve that is usually obtained by this procedure has been found by these authors [12] to generate imprecise results. There was thus a need for a more precise procedure and it necessitated the mofidication of the original procedure as undertaken in the present study. 2. Materials and methods 2.1. Subjects Non-pregnant females aged between 18 and 30 years, all students of the Midwifery School, University of Calabar Teaching Hospital (UCTH) were included in this study. These subjects were not taking oral contraceptives. Normal pregnant women were from the out-patient department of the Maternity Service, UCTH. Their blood pressures were within normal limits and the subjects had no proteinuria or visible oedema. In addition, they were in their third trimester of pregnancy and were aged between 18 and 40 years, with no more than 3 previous deliveries. The PET cases were those diagnosed as having high blood pressure (30 mmhg and 15 mmhg above the normal values of systolic and diastolic blood pressure respectively) and having oedema of the face and lower limbs and/or proteinuria. These women were in their third trimester of pregnancy and aged between 18 and 42 years. Individual subjects gave their informed consent for the study Red cell SOD assay Venous blood (10 ml) was collected from each subject into an anticoagulant, sodium citrate. The samples were centrifuged at 3000 rev./min for 10 min. The plasma and the buffy coat were then removed and the red cells were washed 3 times with saline and packed by centrifugation. Chloroform-ethanol extracts of red blood cell (RBC) haemolysates were

3 L.E. llouno et al./ Clinica Chimica Acta 247 (1996) then used to determine SOD activity according to the method of Winterbourn et al. [9]. The reaction mixture for each sample contained varying volumes (/zl) of red cell extract, plus 0.2 ml 0.1 mold EDTA containing 1.5 mg sodium cyanide/100 ml; 0.1 ml 1.5 mmol/1 NBT; 0.05 ml 0.12 mmol/l riboflavin and mol/1 phosphate buffer, ph 7.8, to a final volume of 3 ml. Absorbances were measured at 560 nm Calculation of results and linearisation of curve Values of percent inhibition of NBT reduction were obtained by the method of Winterbourn et al. [9]. In the case of these previous workers, they plotted the values obtained above directly against the respective volumes of RBC extract. Since it is difficult to obtain objective values for maximal inhibition of NBT reduction (and consequently the half-maximal values) from the resulting hyperbolic curve, the SOD activity obtained by this procedure is not usually precise. In the modified procedure described in the present study, the reciprocal values for the percentage inhibition of NBT reduction were plotted against their corresponding volumes of RBC extract to give a linear curve. In this modified technique, the first point on the graph (that belonging to the tube containing l0 ~l RBC extract) has been found to overshoot in most of the cases studied and so should be discarded. The rest of the points (those corresponding to 20 #l up to 100 ~l RBC extract) give a straight line. The resulting line is extrapolated onto the Y-axis and the volume corresponding to the mid-point value of the intercept is taken as the amount of RBC extract required for half-maximal inhibition of NBT reduction. When a factor of is divided by this volume, SOD activity in units per g haemoglobin is obtained. Table 1 RBC SOD levels in PET of pregnancy Group Mean SOD S.D. P value CV (%) level (units/g Hb) 1. Non-pregnant females (n = 25) 2. Normal pregnant (2) vs. (1) 4.9 females (n = 20) P > PET of pregnancy (2) vs. (3) 3.8 (n = 17) P < 0.05

4 4 L.E. llouno et al / Clinica Chimica Acta 247 (1996) Data analysis The mean (± S.D.) of enzyme activity for the various groups was calculated and analysed for statistically significant differences by Student's t-test [13-15]. This test is very appropriate for data analysis especially when the number of samples studied is not very large. Coefficient of variation (CV) was calculated for each group [13]. 3. Results and discussion The SOD activities of non-pregnant females as well as those of normal and pre-eclamptic cases are shown in Table 1. Also, Fig. 1 shows a typical plot obtained when the reciprocal values for the percentage inhibition of NBT reduction were plotted against the volumes of RBC extracts. Fig. 2 on the other hand shows a typical hyperbolic curve that results from the method of Winterbourn et al. [9], while Table 2 summarises the results obtained from this less precise technique. As far as is known, no previous report has been made regarding the new procedure reported here for the determination of the volume of RBC extracts required for 50% inhibition of NBT reduction. Previous studies by one of the authors [12] showed how imprecise and insensitive the hyperbolic curve of Winterbourn et al. [9] could be. 0 X 2" _<.,~ io A ~o ~o ~ 7o so Volume of red blood cell extract (~t) Fig. 1. Reciprocal of % inhibition of NBT reduction as a function of volume of RBC extract.

5 L.E. llouno et al. / Clinica Chimica Acta 247 (1996) " C.o "6 60. r~ m Z 0 c.o c- c- o,~ ~ Volume of red blood cell exlracl (~L) Fig. 2. Inhibition of reduction of NBT as a function of volume of RBC extract. Variations in estimates of the volume of extracts required for 50% inhibition of NBT reduction could lead to a distortion of the true RBC SOD activity, especially in the case of the older procedure (Table 2). This explains why a difference between the mean SOD activities of PET cases and those of normal pregnancy could not be detected in earlier studies [12]. However, as seen Table 2 RBC SOD levels in PET of pregnancy Group Mean SOD S.D. P value CV (%) Level (units/g Hb) 1. Non-pregnant females (n = 25) 2. Normal pregnant (2) vs. (1) 13.8 females (n = 20) P > PET of pregnancy (2) vs. (3) 5.8 (n = 17) P < 0.05 Result obtained by the technique of Winterbourn et al. [9].

6 6 L.E. llouno et al./clinica Chimica Acta 247 (1996) 1-6 from the present study, a significant difference (P < 0.05) exists between the mean RBC SOD activity of normal pregnant women and that of the PET cases when the revised procedure is used (Table 1). This may explain the observed increased level of peroxidation products arising from membrane lipids such as MDA, levels of which have been reported to increase in PET cases compared to the normal pregnant women [4], The reason for the observed decrease in SOD activity in PET cases compared to normals is not yet clear. It is hoped that further studies in which PET cases are monitored for their SOD activity immediately after parturition and up to 3 months later will help to exclude constitutively low enzyme activity cases, if any. References [1] Halliwell B, Gutteridge JMC, Cross CC. Free radicals, anti-oxidants, and human disease: where are we now? J Lab Clin Med 1992;119(6): [2] Aruoma OI, Kaur H, Halliwell B. Oxygen free radicals and human diseases. J R Soc Health 1991;October: [3] Wever R, Oudega B, Van Golder BF. Generation of superoxide radicals during the autoxidation of mammalian oxyhaemoglobin. Biochem Biophys Act 1973;303: [4] Wickens D, Wilkins MH, Lunec J, Ball G, Dormandy TL. Free radical oxidation (peroxidation) products in plasma in normal and abnormal pregnancy. Ann Biochem 1981;18: [5] Carrell RW, Winterbourn CC, Rachmilewit EA. Activated oxygen and haemolysis. Br J Haematol 1975;30: [6] Carrico R J, Deutsch HF. Isolation of human hepatocuprein and cerebrocuprein: their identity with erythrocuprein. J Biol Chem 1969;244: [7] Stamp TCB. Mineral metabolism. In: Dickerson JWI, Lee HA, eds. Nutrition in chemical management of disease. London: Edward Arnold, 1970; [81 Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44: [9] Winterbourn CC, Hawkins RE, Brian M, Carrell RW. The estimation of red blood cell superoxide activity. J Lab Clin Med 1975;85: [10] Mirami M, Yoshikoowa MA. Simplified assay method of superoxide dismutase activity for clinical use. Clin Chem Acta 1979;92: [11] Sum Y, Oberley LW, Ying LA. A simplified method for clinical assay of superoxide dismutase. Clin Chem 1988;34(3): [12] Ilouno LE. Copper metabolism in pregnancy. PhD thesis. Calabar, Nigeria: University of Calabar, [13] Hill AB. A short text book of medical statistics. 10th edn. London: English Language Book Series and Hodder and Stoughton, 1977; [14] Armitage P. Statistical methods in medical research. New York: John Wiley & Sons, [15] Moss DW, Henderson AR, Kuchmar JF. Enzymes. In: Tietz N, ed. Fundamentals of clinical chemistry. London: W.B. Saunders Company, 1976.