An in vitro demonstration of Pro-oxidant effect of Ascorbic acid in sheep erythrocyte hemolysate and purified hemoglobin preparations

In vitro studies have demonstrated pro-oxidant effect of ascorbic acid on sheep erythrocyte hemolysate and on purified ruminant hemoglobin preparations. Methemoglobin formation was concentration dependent over ca. 3.4 through 34 μmole ascorbic acid with sheep hemolysate (r=0.97, p<0.01, n= 3 each) and over ca. 5.68 through 22.7 μmole ascorbic acid with purified ruminant Hb preparations (r >0.99, p<0.01, n=6 each). In contrast, copper acetate was very potent showing linear increase in methemoglobin formation in Hb preparations over 1 through 5 μmole (r±S.E. = 0.998±0.002). Methemoglobin formation due to ascorbic acid or copper acetate has occurred only at alkaline pH and failed to occur at acidic pH. Comparative evaluation of regression estimates of copper acetate and ascorbic acid revealed that on molar basis ascorbic acid was nearly 1/11 as potent as copper in inducing methemoglobin formation. The effect of ascorbic acid occurred even in presence of copper when the copper ions got reduced and precipitated out by ascorbic acid at alkaline test conditions. Increase in methemoglobin formation with combined copper and ascorbic acid was almost equal to that of copper alone and nearly three folds more than that due to ascorbic acid alone. Key-words: Ascorbic acid, Sheep hemolysate, Ruminant hemoglobin, Pro-oxidant, Methemoglobin.