Pregnanediol urinary

Pregnanediol, 5-B-pregnane-3-a,20-ci-diol, is a major metabolite of progesterone excreted in the urine. The cyclical fluctuations in the urinary excretion of pregnanediol observed in normal ovulating women reflect changes in the secretion by the ovaries. 

The traditional method for the determination of urinary pregnanediol has generally been that of Klopper et al. (9). 

Cooper et al. (10) and Cox (11) were the first to report the gas chromatographic analysis of urinary pregnanediol. These methods normally involved hydrolysis, extraction, and the chromatographic analysis of the free pregnanediol, the acetate derivative, the trimethylsilyl ether derivative, or the bis-heptafluor-obutyrate derivative. 

Acid may be used to hydrolyze urinary pregnanediol conjugates in a reproducible manner with approximately 90% recovery. The overall advantage to this method is the amount of time saved. Acid hydrolysis normally takes about 15 min enzyme hydrolysis takes 24 hr. Since many steroid molecules can be broken by acid hydrolysis, enzyme hydrolysis is the preferred method. 

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. 

The presence, in the sample, of more than one glucuronic conjugate in any appreciable amount calls for a preliminary separation. The numerous methods suggested for determination of urinary pregnanediol glucosiduronic acid will not be reviewed. 

Fig. 2.2. Flowdiagram of the method for the simultaneous determination of urinary androstanediols and pregnanediols.

O Connor KA, Brindle E, Hohnan DJ> Klein NA, Soules MR, Campbell KL, et al. Urinary estrone conjugate and pregnanediol 3-glucuronide enzyme immunoassays for population research. Clin Chem 2003 49 1139-48. 

A direct chemiluminescence assay for urinary pregnanediol 3-glucuronide employs the isoluminol conjugate (31) as tracer, with monoclonal antibodies. Specificity is excellent, the assay being sensitive to 30 pg.143 A solid-phase chemiluminescence assay for plasma progesterone uses as tracer the isoluminol conjugate (32) derived from lla-hydroxyprogesterone.144... 

For some ten years the most useful method for measuring urinary pregnanediol was that of Klopper et al. (K8). The principal isomer excreted in human urine is the 5j8(H)-3a,20a -diol. Much smaller quantities of the 5a (H) epimer and of the 5a(H)-3j8-OH bis-epimer have been... 

J5. Jungmann, R. A., Calvary, E., and Schweppe, J. S., Quantitative analysis of urinary ll-deoxy-17-ketosteroids and pregnanediol by gas-liquid chromatography. 

Lll. Lipsett, G. B., and Kirschner, M. A., Analysis of urinary pregnanediol and pregnanediol by gas-liquid chromatography. In Gas Chromatography of Steroids in Biological Fluids (M. B. Lipsett, ed.), pp. 135-142 Plenum Press, New York, 1965. 

Sulimovici, S., Lunenfeld, B., and Shelesnyak, M. C., A practical method for estimation of urinary pregnanediol and allopegnanediol using thin layer chromatography. Acta Endocrinol. 49, 97-106 (1965). 

As Brooks (B25) has pointed out, urinary steroid estimations may be classified according to the amount of purification necessary prior to GLC. For example, in the estimation of pregnanediol in pregnancy a fairly simple extraction and clean-up of the hydrolyzed urine is all that is needed before application of GLC, whereas the analysis of estrogens in nonpregnancy urine requires extensive purification procedures prior to GLC. Estimation of pregnanetriol in nonpregnancy urine provides a case which is intermediate between these two examples. 

Darcey and Evenson (D4) have compared GLC and the Zimmerman reaction in the analysis of total 17-ketosteroids in urine. Urinary 17-keto-steroid GLC patterns have been studied by Cawley et al. (C5), who noted the presence of several peaks with retention times shorter than those of 17-ketosteroids. A comparison of different methods of hydrolysis has been made by Curtius and Muller (C18) in their study of the GLC of 17-ketosteroids and progesterone metabolites of urine. The simultaneous determination by GLC of pregnanediol and pregnanolone in urinary extracts has been reported by Guarnieri and Barry (G8). GLC was found to be the method of choice in a study by Barry et al. (B2) of four methods for the quantitative analysis of pregnanediol in urine. 

In a personal communication to the writer, van Kampen and Hoek have emphasized the two great problems involved in the determination of steroid excretion in man. First, there is uncertainty about the fate of steroids in (pathological) body fluids, e.g., losses via the skin, or transformation of degradation products. Secondly, difficulties are encountered in the isolation of steroids from urinary conjugates. Even in enzyme hydrolysis, losses of up to 60% can be found, e.g., in the determination of pregnanediol when the time of hydrolysis is prolonged from 30 to 180 minutes. 

G8. Guamieri, M., and Barry, R. D., Simultaneous determination of pregnanediol and pregnanolone in urinary extracts by gas chromatography. Clin, Chem. 14, 35-37 (1968). 

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