The purification of acidic glutathione <i>S</i>-transferases from pea seeds and their activity with lipid peroxidation products

fractionated on a C18-Lichrosorb column a t low pressure into unmetabolized parent compound, intermediateand high-polarity metabolites eluted with 60%, 30-50% and 20% (v/v) MeCN/water, respectively. Metabolites were further separated on 5 and 3pm ODs-Hypersil h.p.1.c. columns eluted with 40% (v/v) MeCN/water. Metabolites collected from the column were freeze-dried, dissolved in C'HCI, and identified by H-n.m.r. (Bruker WM360) and ammonia-CI mass spectrometry (Finnigan 10-20 with desorption probe). The retention times of identified metabolites were measured relative to the respective parent compounds and compared with those measured in a similar way for radiolabelled fly metabolites. Both lipid amides penetrated rapidly into flies with very similar penetration curves (Fig. la). However, a 3-fold greater internal concentration was achieved for I than for I1 (Fig. lb). From h.p.1.c. of the extracts ofwashed flies, I1 was metabolized more extensively than I giving rise to a more complex pattern of metabolites. In rat liver microsomes, major metabolites were produced by epoxidation of either the 6,7or 8,9-double bonds of 11, hydration of these epoxides to diols and also aliphatic hydroxylation, principally a t C4. It seems likely that the extra unsaturation in I1 compared to I leads to more rapid metabolism via initial epoxidation since the relative retention times of fly metabolites strongly suggest the production of epoxides and diols. Although I1 was metabolized faster than I , there was no difference in the rate of excretion (Fig. la) implying a rate-limiting step in metabolism and excretion. This may be caused by slow conjugation of primary metabolites: highly polar metabolites which could not be identified were a major component in fly extracts.