Studies on some free and conjugated steroids in urine

Abstract

Studies on the steroids excreted in the urine have played a fundamental part in building up the present knowledge of sex hormone metabolism. The isolation of some of the quantitatively more important urinary steroids preceded, and facilitated, the isolation of the primary hormones from gonadal sources. In the two decades which have passed since the first crystalline steroid was isolated from urine, a considerable amount of information has been acquired on the nature of the steroids excreted by normal individuals.

Following on the observations of Butler and Marrian (1937), Burrows, Cook, Roe and Warren (1937) and others, on the steroid excretion in cases where the adrenal cortex was hyperactive, and of Callow and Callow (1940) and Hirschmann (1940) on the 17- ketosteroid excretion of male and female castrates, it became obvious that the adrenal cortex, as well as the gonads, contributed to the urinary steroids.

Several groups of workers have studied the steroids excreted in the urine in cases of hyperplasia or tumour of the adrenal cortex, but although this work has led to the isolation of a number of new compounds, very little light has been thrown on the routes of steroid metabolism. Nor have such studies proved so far to be of differential diagnostic value: the steroid excretion picture varies so greatly from patient to patient, that no conclusions as to the nature of the hyperactivity can be drawn.

The apparent diversity of steroid excretion in these pathological conditions may be due, in part, to the small number of cases thoroughlyinvestigated, and to the different, and imperfect, techniques of hydrolysis and isolation used. While isolation techniques have now reached a very high level of perfection (Dobriner, Lieberman and Rhoads, 1948), the introduction of specific and accurate estimation techniques is overdue for many of the urinary steroids, since isolation procedures, however good, can. never be regarded as quantitative, and require a considerable expenditure of time and labour.

It must be admitted, however, that the study of urinary steroids can give but a limited knowledge of metabolic/ metabolic processes, since it is well known that the recovery of administered steroids as recognisable metabolic products in the urine is not usually more than 10-20%, and often less. It is unlikely that losses during hydrolysis and isolation procedures represent more than a small fraction of the unaccounted for material. For this reason the more direct approach to the problems of steroid metabolism which has recently been initiated in the introduction of the tissue slice technique, e.g. Samuels, McCaulay and Sellers, 1947; Clark, Kochakian and Lobotsky, 1947) is of particular interest, as are also the results reported on the excretion of steroids in bile (e.g. Pearlman and Cerceo, 1948; Pearlman and Rakoff, 1949). Nevertheless, practigally all the definite knowledge we at present possess of steroid, metabolism has been acquired by studying the excreted products following massive administration experiments. At one time it appeared as if the useful knowledge to be gained from the study of the urinary steroids had been practically exhausted by the intensive investigation which this subject has undergone. The discovery of the presence in urine of substances possessing cortin activity (Weil and Browne, 1639; Dorfman, Hoirwitt and Fish, 1942), followed by the isolation of some of the more highly oxygenated steroids, has opened up a whole new field of research. Since many of these compounds are acid-labile, the recent Introduction of the use of enzyrdc methods for the hydrolysis of steroid conjugates is of great importance. In addition, estimation results reported following enzymic hydrolysis (Cohen and Bates, 1949; Buehler, Katzman, Doisy and Doisy, 1949) suggest that even in the case of the oestrogens and neutral 17-ketosteroids interesting discoveries may yet remain to be made. It seems, certain that enzymic hydrolysis procedures will be much used in the future: possibly the day will come when the quantitative results obtained following acid hydrolysis will be treated with as little regard as is at present given to results obtained before such hydrolysis procedures were employed. In the past, the study of the conjugated steroids has been neglected, possibly because of the difficulties experienced in purifying these compounds, compared with the free steroids. The specific nature of enzyme hydrolysis obviously makes a more extensive knowledge of this subject desirable, and even necessary.

Using improved techniques of hydrolysis and estimation, studies on urinary steroids may still be expected to make important contributions to the knowledge of steroid metabolism.

The work presented in this thesis is divided into five sections, two of which (Sections II and V) are devoted to attempts to devise estimation techniqu for the free steroids, pregnane~3α:17α:20α-triol and. pregnan-3α-ol~20-one. Sections III and IV deal with the isolation of conjugated steroids, one of which, sodium pregnane-3α:20α-diol glucuronidate, has previously been isolated in an impure form; the other, sodium. pregnan-3α-ol~20-one glucuronidate, has been isolated for the first time. The final section (VI) is concerned with experiments on the hydrolysis of sodium pregnanediol glucuronidate with ox-spleen ß-glucuronidase.