Effect of diltiazem isomers and thiamine on piglet liver microsomal peroxidation using dichlorofluorescein.

ABSTRACT

PURPOSE: We investigated a potential hepatoprotective role of d-cis diltiazem, l-cis diltiazem, thiamine and the combination d-cis diltiazem and thiamine against lipid peroxidation in a piglet liver microsomal model. A modified in vitro dichlorofluorescein assay was developed to assess the extent of peroxidative damage induced by reactive oxygen species in the piglet liver microsomal fraction. METHODS: Microsomal membrane fraction, obtained from 3 week old female piglets, was treated with either the biologically vasoactive d-cis diltiazem or the non-vasoactive stereoisomer l-cis diltiazem (5-1000 microM) for 1 hour at 37 degrees C followed by one hour incubation with the free radical generator AAPH (2,2'-azobis-(2-amidinopropane) dihydrochloride; 1 mM) to initiate lipid peroxidation. In a separate study, piglet liver microsomes were pre-treated with d-cis diltiazem (50 or 500 microM) and thiamine (10-100 microM) to assess the antioxidant activity of the combination. RESULTS: A dose dependant inhibition of membrane lipid peroxidation was observed with d-cis diltiazem (p<0.05) but not with l-cis diltiazem, suggesting that diltiazem is stereospecific in protecting against microsomal lipid peroxidation. Combining diltiazem with thiamine further protected microsomes against lipid peroxidation compared to use of individual drugs. CONCLUSION: We conclude that diltiazem and the combination of diltiazem and thiamine offers a hepatoprotective effect against free radicals.