Pregnanetriols

One inset shows the quantification of THAldo against its 3/>5/Ttetrahydroaldosterone (THAldo) internal standard, and the second inset monitoring derivatized stigmasterol (SS) and its hydrolyzed counterpart, an indicator of derivative stability. Abbreviation An androsterone, CB cholesteryl butyrate, DHEA dehydroepiandrosterone, Et etiocholanolone, PT pregnanetriol... 

Pregnanetriol is formed by the reduction of 17-a-hydroxyprogest-erone, which is formed by hydroxylation at the 17- a-position of progesterone. Progesterone is produced by the corpus luteum of the ovary, the placenta, and the adrenal gland. Under normal circumstances, very little pregnanetriol appears to accumulate rather it is secreted as the glucosiduronate in the urine. 

The following procedure was developed for urinary pregnanediol, pregnanetriol, and 17-ketosteroids in urine. It is similar to the one presented earlier for pregnanediol (12). Hydrolysis is complete because sulfatase for the cleavage of sulfates is included with the B-glucuronidase. 

Other workers [358] carried out the acylation of estrogens in acetone and reported the following conditions as optimal for the preparation of tris-HFB-estriol 0.1—0.3 jul of acetone per 1 jug of the substrate and 0.05 ml of HFB anhydride at room temperature for 10 min. The use of a larger amount of another solvent (benzene, methylene chloride, dimethyl sulphoxide, diethyl ether, dioxane) was said to result in the formation of a number of by-products. Poole and Morgan [359], however, stated that the HFB derivatives of some steroids are not thermally stable and that only the decomposition product is detected, e.g., cholesta-3,5-diene is produced from cholesterol. This leads to a considerably lower ECD response, so that the detection limit, which under favourable circumstances can be as low as 0.005 ng, is usually not achieved. As steroids that form unstable HFB derivatives they reported cholesterol, lumisterol, ergosterol, estradiol, pregnanetriol and others. 

Technically, acid hydrolysis is often preferred (except for acid-labile hormones), because the process offers simplicity and speed and usually results in complete hydrolysis regardless of the nature of conjugates. Enzymatic hydrolysis requires special attention to factors such as optimal concentration and type of enzyme, pH, temperature, and duration of incubation. In addition, the possible presence of enzyme inhibitors, which vary in amount and nature with different specimens, may affect the completeness of hydrolysis. In spite of potential problems, enzymatic hydrolysis is used for plasma analysis of steroids that are labile in strong acid solution (e.g., pregnanetriol and corticosteroids), and because it prevents interference from substances that are produced by acid hydrolysis. Enzymatic hydrolysis, for example, has been used successfully to determme the urine concentration of estrone and the plasma concentration of estrone sulfate in postmenopausal women. 

This statement too may cause raised eyebrows but an objective assessment of errors and costs can support it. Thus, De Courcy s trichloroacetic acid reagent (D2) is capable of convenient application to the detection of pregnanetriol in the nonketonic fraction from one-sixteenth of a 24-hour collection from men (100-150 ml) on simple LLC paper chromatograms (a TLC method could no doubt also be devised for this). As she pointed out, the same quantity can be obtained from one-thousandth of a 24-hour collection (1.5-2 ml) of urine from some patients with adrenal hyperplasia. Quantitative estimation by the same reaction after elution from the paper or TLC chromatogram could also be used either by one of the many variations of the micro-17-ketogenic steroid methods or by the same fluorescence reaction (e.g., W7). Further confirmation of identity could be obtained by using a small fraction of the latter after the periodate oxidation for a paper LLC, or TLC, chromatogram and application of the Zimmerman reaction to demonstrate the formation of etiocholanolone (B32). 

Even quantitative assessment of pregnanetriol by visual comparison with standards would be adequate using carefully coded samples, and preferably duplicates. It is agreed from objective evaluations in a wide variety of fields that this method has a C.V. of 15 to 20% (B27). In the presence of 5- to 100-fold elevations of this steroid in the adrenogenital syndrome, the visual method would meet the usual requirements for a clinically valid discriminant (W3). 

It is probable that the increased amounts of pregnanetriol (H5), corticosterone, and 17-deoxycorticosteroids (E8, N6) and of Pettenkoffer chromogens (N2), also found in maternal urine, emanate from the fetoplacental unit if not from the fetus itself (M16). 

Congenital adrenal hyperplasia 21-Hydroxylase with and without salt loss High 17-08 excretion High 3/S-hydroxy-A steroid excretion (infants) High pregnanetriol excretion High testosterone excretion -1- Normal or low Normal or low... 

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