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N. DESHPANDE and R. D. BULBROOK

SUMMARY

(1) Total 17-hydroxycorticosteroids (17-OHCS) and 17-oxosteroids have been determined simultaneously in plasma from 76 normal men.

(3) The 17-OHCS are positively correlated with age and negatively with weight. The 17-oxosteroids are negatively correlated with age.

(3) The age-adjusted mean 17-OHCS levels are identical in men and women but the age relationship differs significantly. Conversely, the slopes of the regressions of 17-oxosteroids on age are the same but the adjusted mean level is higher in men than in women.

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N. DESHPANDE and R. D. BULBROOK

SUMMARY

The method of Deshpande & Bulbrook (1964) for the determination of total 17-oxosteroids and 17-hydroxycorticosteroids (17-OHCS) in blood has been employed in a study of fifty-two normal women of different ages. The results show that there is a significant correlation between age and the plasma levels of 17-oxosteroids and 17-OHCS. Contrary to similar studies on urines, no other parameter gave a significant correlation. Factors which might control the relative amounts of plasma 17-oxosteroids and 17-OHCS are discussed.

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N. DESHPANDE and R. D. BULBROOK

SUMMARY

A simple method for the simultaneous estimation of 17-hydroxycorticosteroids and 17-oxosteroids in human plasma is described. It involves extraction of free corticosteroids, hydrolysis of glucuronosides with β-glucuronidase and solvolysis of sulphates, chromatography on silica gel and colorimetry using microcells. Recoveries of added steroids, using appropriate conjugated compounds were over 80%.

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D. Y. WANG and R. D. BULBROOK

SUMMARY

The binding by plasma from man, rabbit and rat of the sulphate esters of dehydroepiandrosterone, testosterone, 17-acetoxypregnenolone and pregnenolone has been studied using equilibrium dialysis.

The plasma of these species had a large capacity for binding dehydroepiandrosterone sulphate and testosterone sulphate. A large capacity for binding pregnenolone sulphate and 17-acetoxypregnenolone sulphate was observed with plasma from man and rabbit.

The percentage binding of the steroid sulphates studied was higher for human plasma than for rabbit plasma and, in the case of the sulphates of dehydroepiandrosterone and testosterone, for rat plasma. The higher percentage binding by human plasma was confirmed using multiple equilibrium dialysis.

Evidence is presented that suggests that the sulphate esters of dehydroepiandrosterone, testosterone and pregnenolone are bound to the same sites. Dehydroepiandrosterone sulphate was displaced by non-steroidal organic sulphates, suggesting that the binding sites involved are not specific for steroid sulphates.

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R. A. A. SALOKANGAS and R. D. BULBROOK

SUMMARY

1. The purification step described by Ittrich (1958) has been incorporated in the method of Brown, Bulbrook & Greenwood (1957b) for estimating urinary oestrogens.

2. When the Kober colours given by urinary oestrone, oestradiol-17β and oestriol are extracted with tetrachloroethane containing p-nitrophenol, the decrease in the absorption due to impurities is such that the Kober reaction can be scaled down and micro-cells used in the measurement of the final colours.

3. Amounts of urinary oestrogens as low as 0·5 μg./24 hr. specimen can be measured with a reasonable degree of accuracy and precision.

4. The results with the new method show that the method of Brown et al. (1957b) gives over-estimates of approx. 1 μg. oestrone and 1 μg. oestriol/24 hr. urine specimen when used for the estimation of urinary oestrogens of low titre.

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J. H. OTTAWAY and R. D. BULBROOK

SUMMARY

Growth hormone has been reported to cause either a depression or a stimulation of the glucose uptake of isolated rat diaphragm. The present paper describes further work on the two effects.

1. Anaerobic conditions during the preparation of the diaphragm for incubation affect the glucose uptake and alter the response of the muscle to growth hormone. By controlling the oxygen tension in the diaphragm immediately after excision, variation of the glucose uptake and the effect of the hormone is reduced.

2. Solutions of growth hormone were found to be extremely labile, but, by rigidly standardizing the method of preparing solutions, consistent results were obtained.

3. The relationship between the concentration of growth hormone and its effect on the glucose uptake of isolated diaphragm was investigated separately for muscle saturated with oxygen and with nitrogen. With oxygenated muscle at high concentrations the hormone stimulates, and at low concentrations depresses, the rate of glucose uptake.

4. The mode of action of growth hormone in vitro and in vivo is discussed.

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Y. ANDRÉE LEON and R. D. BULBROOK

SUMMARY

The specificity of the method of Stern [1957] for the determination of urinary pregnanetriol has been examined using chromatography on alumina columns and paper. The pregnanetriol fractions were found to contain chromogenic impurities, most of which could be removed by the addition of a pre-wash of 3% ethanol/benzene to the system described by Stern for the elution of pregnanetriol from alumina. The reliability of the modified method has been assessed with special reference to specificity.

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D. Y. WANG, STRETTON YOUNG and R. D. BULBROOK

SUMMARY

(1) The incorporation of [1,2-3H]testosterone in vivo into various tissues of virgin, pregnant, post-partum and tumour-bearing female rats was studied.

(2) In virgin female rats the clearance of radioactivity from mesenteric fat, mammary gland, uterus, spleen, lung and blood was similar. This similarity in the rates of clearance of radioactivity for all the tissues examined was also found for the tissues of pregnant, post-partum, and tumour-bearing rats.

(3) After the administration of [1,2-3H]testosterone different amounts of radioactivity were found in each of the tissues examined. In virgin rats the levels of incorporation were fat > uterus ≥ mammary gland > lung > blood ≥ spleen. This pattern was also obtained in post-partum and tumour-bearing animals; the tumours in the latter behaved in a similar way to normal mammary glands. In the pregnant rat, the foetus incorporated the least amount of radioactivity.

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D. Y. WANG, VICKY AMOR and R. D. BULBROOK

In experiments on the effect of insulin on the stimulation of incorporation of radioactive thymidine into the DNA of pregnant mouse mammary gland explants in culture, considerable variation between the results of successive experiments was noticed. Dependence of the extent of incorporation on the stage of pregnancy was investigated as a possible cause.

Cultures of explants of mammary gland were made (Trowell, 1959) from Schneider mice at various stages of their first pregnancy. The length of pregnancy was taken from the time of appearance of a vaginal plug. Insulin (Sigma Chemical Co., 24·1 i.u/mg.) was used at a concentration of 5 μg./ml. medium. After 24 hr. of incubation the cultures were pulsed for 4 hr. with [methyl-3H]thymidine (18·3 c/m-mole; Radiochemical Centre, Amersham) at a concentration of 0·5 μc/ml. medium (Stockdale & Topper, 1966). The tissue was removed and weighed, the acid-insoluble material hydrolysed (Mahin & Lofberg, 1966), and

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D. Y. WANG, R. D. BULBROOK, A. SNEDDON and T. HAMILTON

SUMMARY

The disappearance of isotopically labelled dehydroepiandrosterone, testosterone and their sulphates from the peripheral circulation of man, rabbit and rat has been investigated.

Metabolic clearance rates, distribution volumes and half-lives have been determined for these compounds in the above species.

In man, the steroid sulphates have a much lower metabolic clearance rate than the corresponding free steroids. This large difference stems from longer half-lives and lower distribution volumes of the former.

In the rabbit or rat the steroid sulphates and the appropriate free steroids do not show such marked differences in their metabolic clearance rates: the half-lives and distribution volumes are comparable.