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

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.