High-pressure liquid chromatographic and other assays for biphenyl hydroxylation compared [proceedings].

McPherson et al. (1974) have suggested that the stimulation in uitro of liver microsomal biphenyl 2-hydroxylation activity is a characteristic and potentially diagnostic property of chemical carcinogens. Many carcinogens, e.g. benzo[a]pyrene, are metabolized by microsomal preparations to compounds that show a very strong fluorescence in the same spectral region as 2-hydroxybiphenyl. A similarity in partition coefficients precludes the separation of 2-hydroxybiphenyl from many carcinogen metabolites by simple solvent extraction, and therefore the normal fluorimetric assay for 2-hydroxybiphenyl (Creaven ef al., 1965) can be complicated by interference from fluorescent carcinogen metabolites. Since the biphenyl test (sic) for carcinogens generally requires that the carcinogens be metabolized, very careful controls are necessary to avoid misinterpretation of the results if the standard fluorimetric assay is used in the test. For this reason, we have developed high-pressure liquid chromatographic and g.1.c. assays that measure 2-, 3and 4-hydroxybiphenyls without interference from carcinogen metabolites. Liver microsomal fractions (2mg of protein) from immature (1OOg) male Wistar albino rats were incubated, for lOmin at 37°C in an air atmosphere, with biphenyl (3 .25m) and an NADPH-generating system as previously described (Burke et al., 1975), except that biphenyl was added as a lop1 portion of a 0 . 6 5 ~ solution in dimethylformamide. The reaction was stopped with 0.5ml of ~ M H C I and the mixture was extracted with 7ml of 2,2,4-trimethylpentane (iso-octane) for 10 min. The organic layer (6.5ml) was evaporated to dryness in a lOml pear-shaped flask at 40°C on a rotary evaporator. The residue was redissolved in lml of trimethylpentane and extracted with 3ml of 0.1M-NaOH for 10min. After aspirating off the upper organic layer, the aqueous phase was acidified with 0.2ml of ~ M H C ~ and then extracted with 9ml of ethyl acetate for l0min. The ethyl acetate extract (8.5ml) was dried over anhydrous Na2S04 and filtered through a 0.45pm Teflon filter (Millipore). The filtered extract was evaporated to dryness as before and stored at 0°C until anal ysed. For high-pressure liquid chromatography the residue was redissolved in Sop1 of trimethylpentane/acetonitrile/3-methylbutan-l-o1 (25 : 1 : 1, by vol.). A 3Opl portion of this was injected on to a column (25cm) of bonded-phase silica-NH, (Phase Separations Ltd.) attached to a high-pressure liquid-chromatography instrument (Laboratory Data Controls). The sample was eluted with the above solvent (flow rate lSml/min) at room temperature and was analysed at 254nm in a flow-through spectrophotometer. 2,s-Xylenol was added, as internal standard, to the acidterminated biphenyl incubation mixture immediately before extraction. A representative chromatogram of biphenyl metabolites, formed with immature rat liver microsomal fraction, is shown in Fig. 1. In order to elute and measure dihydroxybiphenyls, 10 % (v/v) ethanol was incorporated into the high-pressure liquidchromatography solvent, by means of a 5 min linear gradient immediately after the elution of 4-hydroxybiphenyl. Elution times were 12 and 15min for 2,2'and 44'-