Aflatoxin B1 hydroxylation: sensitive monooxygenase microassay suitable for liver biopsy specimens

A method for measuring the rate of monooxygenase catalyzed hydroxylation of aflatoxin B1 to aflatoxin Q1 has been developed. It is suitable for use with small amounts of tissue obtained by needle or surgical biopsy of liver. Liver samples are homogenized in buffered saline containing a glucose-6-phosphate:NADPH generating system. 14C-AFB, is added to initiate the 10 min oxidation period. Quick-freezing stops oxidations, and AFB1 and AFQ1 are extracted using CHCl3. After solvent removal, the residue is resuspended in benzene-acetonitrile (98:2) and applied to silica gel thin-layer chromatography plates which are developed twice in chloroform-acetone-n-hexane (85:15:20). AFB1 and AFQ1 were located by fluorescence and scraped into vials for scintillation counting. The reaction was linear for 10 min using 5-10 mg liver tissue per 0.2 ml incubation. Aflatoxin M1 and 3 other metabolites more polar than AFB1 were also produced. The apparant Km for AFQ1 formation was 8.6x10(-4)M and Vmax was 287 picomoles AFQ1 mg protein-1 min-1 using bonnet monkey Macaca radiatea liver. The procedures should be valuable in comparative biochemical studies, especially those using liver biopsy specimens of primates.