We characterized the NADPH-dependent metabolism of estrone (E 1 ) by liver microsomes of 21 male and 12 female human subjects. The structures of 11 hydroxylated or keto metabolites of E 1 formed by human liver microsomes were identified by chromatographic and mass spectrometric analyses. 2-Hydroxylation of E 1 was the dominant metabolic pathway with all human liver microsomes tested. E 1 is more prone to form catechol estrogens (particularly 4-OH-E 1 ) than 17␤-estradiol (E 2 ) and the average ratio of E 1 4-hydroxylation to 2-hydroxylation (0.24) was slightly higher than the ratio of E 2 4-to 2-hydroxylation (0.20, P Ͻ 0.001). An unidentified monohydroxylated E 1 metabolite (y-OH-E 1 ) was found to be one of the major metabolites formed by human liver microsomes of both genders. 6␤-OH-E 1 , 16␣-OH-E 1 , and 16␤-OH-E 1 were also formed in significant quantities. 16␣-Hydroxylation was not a major pathway for E 1 metabolism. The overall profiles for the E 1 metabolites formed by male and female human liver microsomes were similar, and their average rates were not significantly different. Hepatic CYP3A4/5 activity in both male and female liver microsomes correlated strongly with the rates of formation of several hydroxyestrogen metabolites. The dominant role of hepatic CYP3A4 and CYP3A5 in the formation of these hydroxyestrogen metabolites was further confirmed by incubations of human CYP3A4 or CYP3A5 with [ 3 H]E 1 and NADPH. Notably, human CYP3A5 has very high relative activity for E 1 4-hydroxylation, exceeding its activity for E 1 2-hydroxylation by ϳ100%. It will be of interest to determine the potential biological functions associated with any of the E 1 metabolites identified in our present study.The endogenous estrogens 17␤-estradiol (E 2 ) and estrone (E 1 ) undergo extensive metabolism in the body (their structures and possible sites for oxidative metabolism are illustrated in Fig.

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Acetylcholine-rich synaptic vesicles were isolated from myenteric plexus-longitudinal muscle strips derived from the guinea pig ileum by the method of Dowe, Kilbinger, and Whittaker [J. Neurochem. 35, 993-1003 (1980)] using either unstimulated preparations or preparations field-stimulated at 1 Hz for 10 min using pulses of 1 ms duration and 10 V . cm-1 intensity. The organ bath contained either tetradeuterated (d4) choline (50 microM) or [3H]acetate (2 muCi . ml-1); d4 acetylcholine was measured by gas chromatography-mass spectrometry. As with Torpedo electromotor cholinergic vesicle preparations made under similar conditions the distribution of newly synthesized (d4 or [3H]) acetylcholine in the zonal gradient from stimulated preparations was not identical with that of endogenous (d0, [1H]) acetylcholine, but corresponded to a subpopulation of denser vesicles (equivalent to the VP2 fraction from Torpedo) that had preferentially taken up newly synthesized transmitter. The density difference between the reserve (VP1) and recycling (VP2) vesicles was less than that observed in Torpedo but this smaller difference can be accounted for theoretically by the difference in size between the vesicles of the two tissues. At rest, a lesser incorporation of labelled acetylcholine into the vesicle fraction was observed, and the peaks of endogenous and newly synthesized acetylcholine coincided. Stimulation in the absence of label followed by addition of label did not lead to incorporation of labelled acetylcholine, suggesting that the synthesis and storage of acetylcholine in this preparation and its recovery from stimulation is much more rapid than in Torpedo.

A capillary gas chromatography/mass spectrometry (GC/MS) assay for the simultaneous quantitation of arecoline (ARE), acetylcholine (ACh), and choline (Ch) in biological tissue has been developed. The method utilizes hexadeuterated ARE and nonadeuterated ACh and Ch as internal standards. The compounds were ion-pair extracted from tissue using sodium tetraphenylboron in 3-heptanone. GC/MS analysis was achieved using capillary GC and electron impact mass spectrometry. Quantitation was accomplished using selected ion monitoring at m/z 140 and 146 for non-deuterated and deuterated arecoline respectively, and m/z 58 and 64 for non-deuterated and deuterated ACh and Ch respectively. The method easily detected 25 pmol of all three compounds taken through the assay, and was linear through 50 nmol.

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