A CORRELATION BETWEEN URINARY STEROID METABOLITES AND PATHWAYS OF STEROIDOGENESIS IN HUMAN BREAST-TUMOUR TISSUE

1163 A CORRELATION BETWEEN URINARY STEROID METABOLITES AND PATHWAYS OF STEROIDOGENESIS IN HUMAN BREAST-TUMOUR TISSUE

J. B. ADAMS M.Sc.

Sydney,

Ph.D. New South Wales

M. S. F. WONG B.Sc. New South Wales

University of New South Wales, Kensington 2033, Sydney, Australia

From the School of Biochemistry,

The ability of participate in

breast-carcinoma tissue to steroid-hormone synthesis in vitro, led to the determination of certain steroidhormone metabolites in urine. In particular 16-hydroxylated derivatives of dehydroepiandrosterone (D.H.E.A.) were selected since possession of a steroid 16&agr; hydroxylase was a characteristic feature of breast-carcinoma tissue. Androst-5-ene-3&bgr;, 16&agr;, 17&bgr;-triol (&agr;-triol) was found to be elevated in premenopausal women with breast cancer or benign breast disease and, when expressed as a ratio to dehydroepiandrosterone or to the sum of D.H.E.A., 16&agr;-hydroxy-D.H.E.A., and 16-oxoandrostenediol, the above group gave results highly significantly different from controls. A castrated, adrenalectomised woman with carcinoma of the breast was found to excrete D.H.E.A., 16&agr;-hydroxy-D.H.E.A., and 16-oxoandrostenediol in her urine. After perfusion of D.H.E.A. sulphate high levels of the above compounds, together with &agr;-triol, were found in the urine. Introduction IN-viTRO experiments have shown that human breastcarcinoma tissue contains enzymes which can participate in the biosynthesis of steroid hormones and which are normally confined to adrenals, ovaries, testes, and placenta. Cholesterol is converted to pregnenolone and progesterone, 17-hydroxyprogesterone to androstenedione (Adams and Wong 1968b), pregnenolone to progesterone, dehydroepiandrosterone (D.H.E.A.) to androstenedione, and testosterone to oestriol (Adams and Wong 1968a). One striking feature was the high 16fx-hydroxylating ability of the breast-carcinoma tissue: D.H.E.A. was converted in relatively high yield by breast-carcinoma tissue to androst-5-ene-3p,16
Sum ary

Premenopausal Patients Histological diagnosis was established for breast carcinoma (seven patients), fibroadenosis (five), and fibroadenoma (one). One other patient had inoperable breast cancer and was undergoing radiotherapy: as far as could be ascertained this was the only patient in this group who was using oral contraceptives. Ages ranged from 34 to 51 years (mean 42). Single 24-hour urine collections were made before operation, and stored at 5°C in the presence of toluene until analysed. The controls consisted of student nurses, aged 17-19, and

healthy was

30;

women

aged 26-43. The

none was

taking

oral

mean

age of all the controls

contraceptives.

Patients The ages of the cancer

Postmenopausal

patients in this group ranged from 52 to 84 years (mean 68). Four of the nine patients had had radical mastectomies some years before. The remainder were admitted for hormonal ablation or radiotherapy. Controls were inpatients aged 52-83 (mean 62). Chemical Methods Each 24-hour urine specimen was diluted to 1-71 if necessary, adjusted to pH 6-0 and boiled for 2 hours. 3&bgr;-hydroxy-&Dgr;5steroid sulphates were selectively hydrolysed under these conditions (Fotherby 1958). Solid ammonium sulphate (50 g. per 100 ml.) was dissolved in the cooled urine and the latter extracted three times with an equal volume of chloroform. The chloroform was washed twice with one-fifth of its own volume of 0-17V sodium hydroxide and twice with one-fifth volume of water, dried over anhydrous sodium sulphate and evaporated to dryness. The residue was dissolved in 40 ml. of 70% (v/v) aqueous methanol and extracted with an equal volume of n-hexane. The hexane was back-extracted with 90% (v/v) aqueous methanol and the pooled methanol was evaporated to dryness. Separation into the ketonic fraction and non-ketonic fraction was achieved by Girard’s reagent T (Fotherby et al. 1957). The 3:5-cyclo-6-hydroxysteroids which were formed during the initial boiling of the urine were converted back to 3-hydroxy-A-steroids, in the case of the ketonic fraction, during the acid treatment of the Girard complex. The nonketonic fraction was dissolved in 50 ml. of a water/ethanol (9/1, v/v) acidified to pH 1 with 5N sulphuric acid and after 1 hour the mixture was extracted three times with two volumes of chloroform (Fotherby 1958). The chloroform was washed twice with O1N sodium hydroxide and twice with water; and after drying over sodium sulphate, it was evaporated to dryness. The residue was then partitioned between benzene/petroleumether (1/1, v/v) and water, oc-triol was extracted from the water phase with ether (Adams and Wong 1968a). In later experiments one-tenth volumes of 24-hour urines were used for analysis. These were refluxed for 2 hours. Results obtained were in good agreement with those obtained using the complete urine specimen. Components of the ketonic fraction were separated and quantitated on 20 x 20 cm. silica-gel thin-layer plates by the method of Shackleton and Mitchell (1967). tx-triol was determined on separate plates. After spraying with antimony trichloride, the plates were heated until the background was a uniform white opacity. Scanning was then carried out immediately with a Joyce Loebl densitometer. Areas under the curves were determined by weighing. Four concentrations of each steroid to be analysed were run on each plate, and these were used to construct a standard curve for analysis of the samples spotted on the same plate. In keeping with the results of Shackleton and Mitchell (1967), Beer’s law was obeyed within the range of 0-5 g. Preliminary runs gave the necessary information as regards subsequent dilution of samples. Normally the equivalent of 1% of the 24-hour urine was applied. Reproducibility of the scanning technique was tested by spotting a sample of ketonic fraction 9 times.Values obtained for D.H.E.A. were 40::1::3(S.D.) g. per 24 hours

(range 36-45). Total (acid-hydrolysable) 17-ketosteroids in urine were determined by the method of Kraushaar et al. (1966). This method was also used for the collective determination of the ketonic fraction obtained from boiled urine. Statistical Methods The t test was used for comparison of means of two groups. When significant differences in variance were encountered, the analysis outlined by Snedecor (1956) was used. Results Steroids Identification of Urinary Characterisation of the urinary

metabolites

possessed identical chromatographic behaviour

as

which authen-

1164

Fig. 1-Mean values in premenopausal women of urinary acidhydrolysable 17-ketosteroids (left), and 3D-hydroxy-A6-steroids formed from ester sulphates by boiling the urine samples (right).

16a-hydroxy-D.H.E.A., and D.H.E.A. in the multidevelopmental systems of Shackleton and Mitchell (1967), was achieved by isolation from thin-layer plates and comparison of the chromatographic behaviour of the free steroids and their acetate derivatives, the sulphurictic a-triol,

acid spectra and spectra in the Eik-Nes reagent, and the retention-times of trimethylsilyl derivatives on gas chromatography. In each case the isolated compounds behaved identically to the authentic reference steroids. Shackleton and Mitchell (1967) had previously established the identity of these urinary metabolites and also 16-oxoandrostenediol by a combination of gas chromatography and mass spectroscopy. The identity of 16-oxoandrostenediol was confined, in our experiments, to comparison with authentic material on thin-layer plates.

Premenopausal

Fig.

Women

1 summarises the results obtained with

eight

Fig. 2-Mean values of urinary 3&bgr;-hydroxy- &Dgr;5-steroids in postmenopausal women.

patients, and six patients with benign breast disease, as compared with fourteen healthy controls. Total (acid-hydrolysable) 17-ketosteroid levels were significantly lower in the group with breast cancer or benign breast disease. The " ketonic fraction ", obtained from urine boiled for 2 hours to cleave selectively sulphate esters of 33shydroxy-05-steroids, was not significantly different (P=0’l) in the two groups, but the values in the patients tended to be low. Mean levels OfD.H.E.A. were significantly lower in the patients, but this no longer held when one of the controls, who had an abnormally high value of 525 µg. breast-cancer

hours, was excluded. Mean values for 16&hydroxy-D.H.E.A. and 16-oxoandrostenediol were not significantly different, but in the latter case the values again tended to be low in the cancer-benign group. By contrast the cx-triol levels were significantly higher in the patients (P=0.001); per 24

of the controls in the same age-range as the cancer group had levels lower than that detectable by the method (2 µg. per 24 hours), and for the purposes of calculation, this value has been adopted in these cases (see fig. 3). most

Fig. 3-a-triol levels and

ratios in

premenopausal

women.

(a) Individual values for urinary at-triol. (b) a-triol expressed as ratios to the sum

of D.H.E.A., 16at-hydroxyD.H.E.A., and 16-oxoandrostenediol. (c) at-triol as ratio to D.H.E.A. (d) a-triol as ratio to the ketonic fraction (see text) determined

colorimetrically. Individual values have been plotted in the abscissa for comparison.

same

sequence

along the

Fig. 4-Individual values in postmenopausal controls cancer patients (see fig. 3).

(a) Urinary a-triol. (b) Ratio of a-triol androstenediol.

to

and breast-

D.H.E.A. + 16a-hydroxy-D.H.E.A. + 16-oxo-

1165

Postmenopausal Women In fig. 2 the corresponding mean values are shown for a group of nine postmenopausal cancer patients and a group of nine controls. No significant differences were observed between these groups. Individual a-triol Values

The premenopausal carcinoma/benign group had 1 (fig. 3b). The difference between the mean values was highly significant (P>0-001). The segregation of the two groups was not quite as good when the ratios of a-triol to D:H.E.A. were calculated (fig. 3c), but the values for the means was still highly significant This also applied when ratios of 1 (fig. 4b). Since most patients undergoing breast surgery were admitted only 1 or 2 days before operation, urine had to be collected at this time so collection could not be synchronised with any phase of the menstrual cycle. Accordingly, urine from controls was collected randomly. Levels of x-triol in premenopausal controls did not seem to be highly dependent on the stage of the cycle, since concentrations below 2 jg. per 24 hours were found in collections made on days 3, 6, 10, and 17 of individual cycles. In one control, urine collections were made at four intervals ranging over two successive cycles, and complete analyses were done. The concentration of
3&bgr;-HYDROXY-Ll5_STEROIDS

IN AN ADRENALEC-

TOMISED AND CASTRATED WOMAN BEFORE AND AFTER INFUSION OF D.H.E.A. SULPHATE

*

Androst-5-ene-3P-16P-17p-triol tentatively identified by chromatographic comparison with authentic material.

determined in a breast-carcinoma patient (aged 41) who had undergone the following treatments at the previous time intervals indicated: left radical mastectomy (7 years), radiation therapy (5 years), oophorectomy (5 years), and bilateral adrenalectomy (2 years). This patient who had been maintained on cortisone, was given an infusion of D.H.E.A. sulphate (110 mg. per 24 hours) over a period of 13 days. Analytical results, obtained before infusion and on the first two days afterwards, are shown in the table. Discussion

The relative increase in the mean urinary concentration of a-triol in premenopausal women with breast cancer or benign breast disease compared with controls, provides a link between the pathways of steroidbiosynthesis established in vitro (Adams and Wong 1968 a, b) and the situation in vivo. When individual levels of 3&bgr;-hydroxyA5-steroids are analysed-e.g., by expressing results as ratios of D.H.E.A., ketonic fraction, and so on (fig. 3)then, despite the fact that a relatively small sample was studied, the results were highly significant. Although not statistically significant in the postmenopausal group, the trend was in the same direction. Of the two high «-triol values obtained with younger controls (fig. 3a), one (305 g. per 24 hours) could perhaps be explained by the very high total acid-hydrolysable 17-ketosteroid level (14-4 mg.), ketonic fraction (1-75 mg.), D.H.E.A. (525 .g.), and 16a-hydroxy-D.H.E.A. (318 tg.). When related to D.H.E.A., and so on as described above, values fell into the normal control ranges. However, the other girl (a-triol 470 µg.), whilst also showing high levels of 16’x-hydroxyD.H.E.A. and 16-oxoandrostenediol (125 and 320 µg., respectively), had normal values for total acid-hydrolysable 17-ketosteroids, ketonic fraction, and D.H.E.A. Furtherto D.H.E.A. and so on remained high more, ratios of
hydroxy-D.H.E.A.-3-sulphate and x-triol sulphate) in the fcetal-placental unit (Diczfalusy 1967). It has also been shown that premenopausal women with breast cancer and benign breast disease excreted a relatively greater proportion of oestrogen as cestriol, than corresponding "

sick " and " well " control groups (Marmorston et al. 1965b): the total cestrogens secreted by the cancer-benign group were not different from the controls, but significant decreases in oestrone and oestradiol masked a specific increase in cestriol. Elevation of urinary x-triol levels in breast cancer and benign breast disease would thus seem to parallel cestriol elevation, and this could reflect a pathway independent of the prior formation of oestrone and cestradiol. Persistency of oestrogens in the urine of castrated, adrenalectomised patients with breast carcinoma is well documented (Adams and Wong 1968a) and the breast (carcinoma) must now be considered as a likely source.

1166 Formation of «-triol and 16(x-hydroxy-D.H.E.A. on administration of D.H.E.A. sulphate to a castrated and adrenalectomised patient (table), would at least eliminate the ovaries and adrenals as unique sites for the formation of these compounds. The liver and perhaps secondary breast carcinoma deposits and normal breast tissue would then be implicated. It is also of interest that this patient excreted D.H.E.A., 16
and Eetiocholanolone are metabolites of D.H.E.A. and an increase in urinary a-triol, itself derived from D.H.E.A. (table), would be expected to lower the levels of the former metabolites. Stoichiometry between increased (x-triol and decreased androsterone plus xtiocholanolone should not necessarily be expected since very little 3&bgr;hydroxy-&Dgr;5- steroids are present in urine compared to their 5x and 5 reduction products. The greater part of the xtriol formed in vivo may then be excreted as reduction products. This aspect is being investigated. It is perhaps also significant that a decrease in urinary- D.H.E.A. in postmenopausal women with breast cancer, accompanied a specific increase in oestriol as reported by Marmorston (1966). Marmorston et al. (1965b) also found that premenopausal women with breast cancer and benign breast disease excreted a significantly higher proportion of oestrogen as oestriol. This common behaviour of breast cancer and benign breast disease also applies to the relative proportion of a-triol amongst the 3&bgr;-hydroxy-il5steroids in the urine (fig. 3). We thank Dr. J. Halley and Dr. P. Fitzpatrick for helpful discussion, the New South Wales Cancer Council for financial support, Dr. A. Ryan and Dr. G. Holder for the use of the densitometer, Mr. K. Wynne for the use of gas-chromatography apparatus and for

provision of urine specimens from the patient receiving sulphate, and Dr. D. Wurth, Dr. C. Hambly, and the Prince Alfred Hospital and the Prince of Wales Hospital for help with provision of urine specimens.

D.H.E.A.

REFERENCES

Adams, J. B., Wong, M. S. F. (1968a) J. Endocr. 41, 41. (1968b) Proceedings of a meeting of the Australian Biochemical Society held in Canberra in May, 1968; p. 58. Allen, B. J., Hayward, J. L., Merivale, W. H. H. (1957) Lancet, i, 496. Brown, J. B. (1958) in Endocrine Aspects of Breast Cancer (edited by A. R. Currie); p. 197. London. Bulbrook, R. D. (1965) Vitamins Horm. 23, 329. Diczfalusy (1967) Mém. Acad. Méd. Belg. 6, 111. —

—

Fotherby, K. (1958) Biochem. J. 69, 596. Colas, A., Atherden, S. M., Marrian, G. F. (1957) ibid. 66, 664. Gutierrez, R. M., Williams, R. J. (1968) Proc. natn Acad. Sci., U.S.A. 59, 938. Kraushaar, L. A., Epstein, E., Zak, B. (1966) Clin. Chem. 12, 282. Marmorston, J. (1966) Ann. N.Y. Acad. Sci. 125, 959. — Crowley, L. G., Myers, S. M., Stern, E., Hopkins, C. E. (1965a) Am. J. Obstet. Gynec. 92, 447. — — — — — (1965b) ibid. p. 460. Nissen-Meyer, R., Sanner, T. (1963) Acta endocr., Copenh. 44, 334. Persson, B. H., Risholm, L. (1964) ibid. 47, 15. Shackleton, C. H. L., Mitchell, F. L. (1967) Steroids, 10, 359. Snedecor, G. W. (1956) Statistical Methods; p. 97. Iowa. Stern, E., Hopkins, C. E., Weiner, J. M., Marmorston, J. (1964) Science, N.Y. 145, 716. —

EFFECT OF POTASSIUM ON BLOOD-SUGAR AND PLASMA-INSULIN LEVELS IN PATIENTS UNDERGOING PERITONEAL DIALYSIS AND HÆMODIALYSIS Y. K. SEEDAT* N.U.I., F.C.P. (S.A.)

M.D.

From the

University Department of Medicine, Royal Infirmary, Manchester 13

of total urinary ll-deoxy-17-oxosteroids (or more specifically the main components of this fraction, androsterone and aetiocholanolone) were first reported by Allen et al. (1957). These findings were confirmed and extended by the stimulating and painstaking work of Bulbrook, Hayward and their associates (Bulbrook 1965). Further confirmation has been reported by Marmorston et al. (1965a) and by Gutierrez and Williams (1968). Both androsterone Lowered levels in breast-cancer

cases

The role of hypokalæmia in influencing the blood-sugar and plasma-insulin levels in patients undergoing chronic peritoneal dialysis and hæmodialysis has been studied. Ten patients who had peritoneal dialysis and hæmodialysis were subjected to a large concentration of glucose in the dialysate fluid. Four patients who developed hyperglycæmia during dialysis all had a low plasma-potassium level. After potassium

Summary

*

Senior physician and senior lecturer, Department of Medicine, University of Natal and King Edward VIII Hospital, Durban, South Africa.