THE EFFECTS OF DRUGS ON ERYTHROCYTES in vitro: HEINZ BODY FORMATION, GLUTATHIONE PEROXIDASE INHIBITION AND CHANGES IN MECHANICAL FRAGILITY

Abstract

1The relationship between mechanical fragility, glutathione peroxidase inhibition and Heinz body formation, in erythrocytes exposed to oxidant drugs in vitro, has been investigated. All drugs tested caused Heinz body formation, and with the exception of acetyl salicylic acid and salicylic acid, also caused increased erythrocyte mechanical fragility. 2 There was a direct relationship between mechanical fragility and drug concentration. Mechanical fragility increased in parallel with Heinz body formation, with primaquine, gentisic acid, ascorbic acid and potassium chlorate. In contrast Heinz body formation occurred at drug concentrations which did not cause a marked increase in mechanical fragility in the case of menadione, acetyl phenylhydrazine and phenylhydrazine. 3 The degree of inhibition of glutathione peroxidase was directly related to increased mechanical fragility with menadione, gentisic acid and potassium chlorate. However other substances causing increased mechanical fragility resulted in little or no loss of glutathione peroxidase activity. 4 The results show that there is no constant relationship between mechanical fragility caused by drugs, the formation of Heinz bodies and the inhibition of glutathione peroxidase. The factors contributing to oxidant drug-induced haemolysis appear to be variable and depend upon the drug concerned.

Proposed Insights in the Pharmaco-Dynamic Theory of Ascorbate Pro-Oxidant Activity

“…5C and D). The present study explains the mystery noticed by Hopkins and Tudhope (1974) who has shown that there is no consistent relationship between mechanical fragility, glutathione peroxidase inhibition and HB formation (Hopkins and Tudhope (1974). The clinical relevance of these findings is that the pharmacologic doses of AA may involve heavily the first pathway of oxidative stress and make cells more sensitives to a physical attack like radiotherapy; this is of high interest in a cells with active metabolism like cancer cells (Fig.…”

“…First of all, the total antioxidant capacity of different AA dilutions was assessed by the Oxygen Reactive Absorbance Capacity (ORAC) assay as described by Ou et al (2002). The effects of the AA on the RBCs were assessed by counting the blit cells and osmotic fragility for the membrane deformability as described by Hopkins and Tudhope (1974); Malanodialdehyde and 4-HNE for the lipid peroxydation as described by Quintanilha et al (1982) and Kinter (1996) respectively, Heinz bodies were measured by two assays; the colorometric assay of Winterbourn (1990) and the microscopic one of Stephen and Low (1985). Finally we have evaluated the activity of catalase, peroxydase and superoxide dismutase as described by Beutler (1982;Glatzle et al, 1974;Marklund and Marklund, 1974) respectively.…”

“…2A). The incubation was performed at 8°C during 18 h. (Hopkins and Tudhope, 1974;Stephen and Low, 1985;Beutler, 1982;Marklund and Marklund, 1974;Glatzle et al, 1974;Dodge et al, 1963;Winterbourn, 1990): Mechanical, biochemical and enzymatic assessment were performed for each cell suspension. First of all, the total antioxidant capacity of different AA dilutions was assessed by the Oxygen Reactive Absorbance Capacity (ORAC) assay as described by Ou et al (2002).…”

Spectrophotometric Method Applicable to <i>in vitro </i>Studies of Heinz Body Formation in Erythrocytes

“…Several dyes have been used, including crystal violet [1,4], methyl violet [3], brilliant green [11], rhodanile blue [13], brilliant cresyl blue [6], nile blue sulphate [10] and new methylene blue [8]. Cruz [2] haemolyzed blood with distilled water and used the turbidity of the resulting haemolysate as a measure of Heinz body formation.…”

“…Estimation of Heinz bodies has been used as a measure of the effect of oxidant drugs on intraerythrocytic haemoglobin [1,4,9,15], and in studies of erythrocytic changes leading to haemolysis [6,12].…”