Some new aspects of 17alpha-estradiol metabolism in man.

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.

Practical thin layer chromatography techniques for diaminopimelic acid and whole cell sugar analyses in the classification of environmental actinomycetes.

For the determination of diaminopimelic acid (DAP) stereoisomers in whole cell hydrolysates as chemotaxonomic markers of actinomycetes, two new pyridine-free solvent systems for TLC on cellulose sheets have been introduced: methanol/0.05 m potassium hydrogenphthalate buffer pH 4 2:1 (v/v) and methanol/0.12 M dimethylaminopyridine (DMAP) in H(2)O pH 6 2:1 (v/v). The commercial (Sigma) DAP standard can be separated by repeated TLC into its three stereoisomers. Addition of a single stereoisomer to the samples supported the detection (presence or absence) of the relevant stereoisomer by HPLC. In the study on whole cell sugars, TLC both on cellulose and on silica gel revealed to be successful in the separation of the components in modified pyridine-free solvent systems. The procedures of DAP and sugar analyses are summarized in two flow-charts.