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An improved method for the determination of individual urinary 17-oxosteroids is described. Both the advantages and the limitations of the published procedures have been taken into account at the elaboration of the method. Simultaneous mild acid hydrolysis and benzene extraction were employed for liberation of 17-oxosteroid esters. The individual urinary metabolites were separated from each other, from hydrolysis artefacts, from other 3-, 17- and 20-oxosteroids as also from interfering substances by means of a new paper Chromatographic system. The control of the method and other comparative examinations are also described.

A method for the simultaneous determination of free cortisol and of dehydroepiandrosterone and androsterone conjugates in human plasma is described. Chloroform extraction, subsequent paper chromatography and alkaline tetrazolium-blue reaction were used for measuring free cortisol values. For splitting of 17-oxosteroid conjugates, simultaneous acid hydrolysis and benzene extraction are employed. An extensive purification of the extract is achieved by thin-layer chromatography. Paper chromatography is used for separation, and in situ Zimmermann-reaction applied for quantitation of individual 17-oxosteroids. The control of the method, the normal values and the plasma steroid level in different endocrine disorders are presented.

The behaviour of model steroids, 17-ketosteroid formates, acetates, bromides, and dinitrophenylhydrazones, and of extracts obtained from biological material has been studied by means of thin-layer chromatography.

Dehydroepiandrosterone, androsterone and etiocholanolone sulphate esters have been determined in peripheral blood plasma of patients with prostatic carcinoma. A significant decrease in the plasma level of steroid was observed. A diminution in the excretion rate of certain 17-oxosteroid conjugates was also found in the same patients. There was no correlation between the steroid levels and orchidectomy or heterosexual hormone treatment. From the findings it is assumed that an insufficient production of dehydroepiandrosterone and/or its sulphate ester in adrenal may play a role in the low steroid content of peripheral blood and urine. However, the abnormality of steroid metabolism appears to be in no close relationship with the disease, rather, it may be the consequence of an aspecific stress reaction.

The dynamic parameters of dehydroepiandrosterone and dehydroepiandrosterone sulphate metabolism have been determined following a single injection of [4-14C] dehydroepiandrosterone and [7α-3H] dehydroepiandrosterone sulphate into normal female subjects. The urinary secretion and production rates, the ϱDDS and ϱDSD conversion factors, the metabolic clearance rates, blood production rates and distribution volumes were calculated. Although the comparison between urinary and blood parameters of steroid dynamics suggested certain limitations of the methods used, they were found suitable for gaining information on dehydroepiandrosterone and dehydroepiandrosterone sulphate metabolism. A poor extra-adrenal conversion of dehydroepiandrosterone to the sulphate, small dehydroepiandrosterone sulphate and large dehydroepiandrosterone distribution volumes or clearance patterns, and a possible correlation between the blood parameters of dehydroepiandrosterone sulphate and anthropomorphology of normal female subjects were found.

Follicle-stimulating hormone and thyrotropin administered perinatally to cockerels have an overlapping effect on the testis and on the thyroid gland. Both hormones when given in one single dose considerably increased the serum level of both thyroxin and testosterone. In the case of chronic treatment performed in the perinatal period, the thyroxin level will similarly increase, though to a smaller extent, while the testosterone level decreases. The experiments studying the direct effect on hormone secretion proved the overlapping effect of the hormones in the perinatal period.

Radioimmunological methods have been employed for the simultaneous determination of dehydroepiandrosterone, androstenedione, testosterone, oestrogens (oestradiol + oestrone), progesterone, 17 alpha-hydroxyprogesterone and cortisol in human adipose tissue and peripheral blood to compare the hormone pool of adipose tissue with that in the general circulation. Extremely high steroid concentrations in the adipose tissue and hormone pool in the fat of obese subjects were observed. For adipose tissue/serum steroid ratios, the highest values were obtained for dehydroepiandrosterone and the lowest ones for cortisol. A preliminary study showed a great accumulation of steroids in adjacent adipose tissue of breast tumors. Striking differences were observed in the adipose tissue steroid concentrations between benign and malignant mammary tumors. The present findings revealed that blood hormone determinations may be insufficient to consider the steroid hormone availability in various endocrinopathies or steroid responsive tumors, especially when the endocrine state of extremely obese subjects is observed.

A simple routine method is reported for the determination of progesterone in blood by gas-liquid chromatography. An unlabelled internal standard, testosterone acetate, was added to plasma samples. Following preliminary purification by thin-layer chromatography, progesterone in pregnancy plasma was evaluated as the free steroid with a flame ionization detector. The hormone in the plasma of women during the menstrual cycle and of cycling domestic animals was determined as the 3-enol ester heptafluorobutyrate by electron capture detection. The validity of testosterone acetate as an internal standard was proved by simultaneous processing of [7α- 3 H]progesterone and [4- 14 C]testosterone acetate and the determination of the isotope ratio in samples. The results of control experiments and normal values are also presented.

Hereditary angioneurotic edema (HAE) is a complement-related clinical disorder with a deficiency of the C1 esterase inhibitor protein. Eight patients with severe attacks of the disease were treated with the adrenal “androgen” dehydroepiandrosterone sulphate (DS). Steroid therapy for 3–28 months resulted in dramatic improvement in their clinical state and a moderate increase in the serum concentration of C1 inhibitor. There was a significant increase in the serum level of either unconjugated dehydroepiandrosterone (D) or of DS during treatment.

A gas chromatographic method has been presented for the determination of unconjugated dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone in human ovarian tissue. The procedure utilized radioactive tracers added to homogenate for correcting methodological loss, preliminary separation of steroids by thinlayer chromatography, acetylation, rechromatography on chromatoplate and gas chromatography on 3% SE-30 or 1% XE-60 columns with flame ionisation detection of steroids by using internal standards. Results of control experiments and representative clinical findings on normal and polycystic ovaries are reported.

An increased secretion of gonadotropic hormone was found in hyperthyroid males despite the high basal serum total testosterone and oestradiol levels. This suggests that hyperthyroidism affects the responsiveness of the hypothalamic-pituitary axis to sexual steroid hormones. While in the hyperthyroid patients the elevation of the serum testosterone level in response to chorionic gonadotropin was lower than normally, the LH and FSH responses to LRH were increased. The results indicate that male hyperthyroidism is associated with a loss of responsiveness of the Leydig-cells to adequate stimuli, to which the pituitary reacts by a compensatory increase in its LH-secretion and by an increased reactivity to LRH. It is suggested that in addition to the direct effect of the increased thyroid hormone levels, a secondary elevation of the oestradiol concentration plays a major part in the alterations in question.

A method has been presented for the quantitative determination of 21-deoxy-N-(N'-methyl-piperazinyl)-prednisolone (Depersolone), a water soluble corticosteroid, and one of its metabolites, prednisolone, in human urine. Following administration of 30 or 90 mg Depersolone i.v., the dynamic appearance of this hormone and the prednisolone metabolite, their biological half-lives and the effect of the synthetic corticosteroid on the excretion of grouped and individual 17-ketosteroids were determined. Results of experiments revealed a dose dependent excretion of the unchanged hormone and a biological half-life 12 to 23 h in control subjects. Prednisolone as a metabolite seems to play a minor role in Depersolone action. The excretion rate of Depersolone was greater and its half-life less than normal in hepatopathies. The dynamic appearance of the hormone was found slow in the urine of patients with prolonged corticosteroid therapy.

A 2-to 16-fold increase in the number of glucocorticoid receptors of circulating lymphocytes was observed in 17 septic patients as compared to 10 control patients with no inflammatory disease. The number of receptors was low in 3 septic patients who did not respond to antibiotic and glucocorticoid therapy and died. Intravenous administration of 100 mg of cortisol reduced the number of receptors available to the assay by about 30% in both septic and control patients.

The plasma cholic acid, chenodesoxycholic acid and desoxycholic acid levels were studied by spectrofluoremetry in 153 cases. The values of 67 controls with no evidence of hepatobiliary or intestinal disease were compared with those of 86 patients with liver and biliary tract disease. The fasting values failed to provide more diagnostic information than did conventional laboratory assays. Plasma bile acid concentrations exceeding 2.5 mu mol/l are conclusive of liver or biliary disease. A cholic acid/chenodesoxycholic acid quotient higher than 1.0 is a sign of cholestasis. Estimation of bile acids after food intake was found more informative. The plasma cholic acid- and chenodesoxycholic acid levels underwent a considerable increase 1 to 2 hours after meals. A more marked increase of chenodesoxycholic acid than of cholic acid (the ratio of the two being in excess of 1.0) is indicative of cholestasis and is most marked in primary biliary cirrhosis.

Female patients with "intrinsic" bronchial asthma without corticosteroid therapy for at least 3 months revealed low blood concentration of total protein-unbound + protein-bound) and free (biologically active, protein-unbound) cortisol, dehydroepiandrosterone and dehydroepiandrosterone sulphate, as also a low urinary excretion of dehydroepiandrosterone metabolites. The results suggest that bronchial asthma is associated with hypoadrenia due to an impaired production not only of cortisol, but also of adrenocortical androgens resulting in an insufficient hormone supply of the target organs. These seem noteworthy, since recent observations showed relationships between the immune responsiveness and sex hormones.

Unconjugated, sulpho- and glucurono-conjugated androgen hormone metabolites have been determined in the urine of patients with rheumatoid arthritis. An increase in the excretory rate of unconjugated 5 beta-reduced 17-ketosteroids and a decrease in that of 17-ketosteroid conjugates, especially in dehydroepiandrosterone sulphate and in the sum of dehydroepiandrosterone, etiocholanolone and androsterone glucuronoside were observed. In contrast to unconjugated metabolites, there was less significant change in the 5 beta-metabolite conjugates in urine. Corticosteroid treatment resulted in an additional decrease of metabolite excretion by patients. Further study is necessary to determine the causative factors in the altered steroid pattern observed in this severe, non-endocrine disease.

An enzyme immunoassay for the measurement of 17-hydroxyprogesterone (OHP) is described. The antigens, OHP-3-CMO-BSA and 11-deoxycortisol-21-hemisuccinate-BSA have been used to produce antisera in rabbits. The enzyme marker was horseradish peroxidase. Bound/free separation was achieved by the double antibody/polyethylene glycol method. The method was used for plasma samples, capillary blood dried on paper disc and for saliva. The normal values for OHP in newborns and children were determined. This method makes possible a selected screening program for congenital adrenal hyperplasia in the newborns.

The effect of ACTH and prolactin on the synthesis of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) was studied in cell suspensions of "normal" and tumorous (adenoma) human adrenal cortex. A stimulation of DHEA and no response of DHEAS production by ACTH in "normal" adrenocortical cell suspension was observed. However ACTH stimulated both DHEA and DHEAS synthesis in tumorous adrenocortical cells. Prolactin did not influence either the basal or the ACTH stimulated DHEA and DHEAS production of adrenocortical cells irrespective of their origin. Our results are compatible with the concept that the biosynthesis of DHEA is under ACTH control, while other factor(s) regulate(s) the sulfate pathway of DHEA secretion under normal conditions. In tumorous adrenocortical cells DHEA may be regulated--at least partly--by ACTH. Prolactin seems to have no direct effect on DHEA and DHEAS synthesis. It is postulated that the relationship between serum prolactin and DHEAS (or DHEA) levels observed by several authors might be an extraadrenal effect of prolactin on adrenal androgens.

Serum testosterone (T), dihydrotestosterone (DT) and androstendione (AD) concentration were determined in male patients with pancreatic cancer (n = 14), chronic pancreatitis (n = 26), other malignancies of the gastrointestinal tract (n = 19) and in control subjects (n = 16). The T level was decreased in patients with pancreatic cancer relative to other groups of patients but the difference was not statistically significant. The T/DT ratio was significantly lower in pancreatic cancer (2.5 +/- 1.0) compared to chronic pancreatitis (9.6 +/- 2.3, p 0.01). The specificity of the ratio is 84%, while the sensitivity was found to be 92%. The discriminative value was found to be 4. The T/DT ratio seems to be a valuable marker in the differentiation of pancreatic cancer from chronic pancreatitis.

Dehydroepiandrosterone (D), D sulphate (DS), testosterone (T) and oestradiol (OE2) were determined by radioimmunoassay; cortisol (F) by a spectrofluorimetric method in the serum of 54 female patients with SLE. The values were evaluated in two age groups (32 patients with active ovarian function and 22 in menopause). The serum cortisol, T and DS levels were decreased, and there was no difference in that of free (unconjugated) D and OE2 concentrations compared to normal female subjects.

The individual protein fractions, together with the unbound (biologically active) and specifically globulin-bound 11-hydroxysteroids, were determined in the plasma and synovial fluid of patients with rheumatoid arthritis (RA). The values were compared with those measured in control subjects and in patients with severe osteoarthrosis (OA). Hypoalabuminaemia and hyperglobulinaemia, as well as an increase in the unbound and a decrease in the specifically bound corticosteroids were found in the plasma of the RA patients. The protein and corticosteroid levels were lower, and the level of the protein-bound corticosteroid fraction higher, in the synovial fluid than in the plasma. Significant differences between the RA and OA patients in respect of these parameters were also observed. The findings indicate that the altered hormone pattern is not a nonspecific result of the permanent stress situation to which RA patients are subjected. They point out the existence of a fairly specific but still unknown mechanism that compensates the high level of unbound corticosteroids, thus accounting for the absence of any clinical signs of hypercortisonism.

The role of pituitary hormones in the regulation of adrenal androgen (AA) secretion is not well understood. In the present study the plasma dehydroepiandrosterone suolphate (DHEAS) concentrations of two groups of hypopituitary children with normal and low prolactin levels were compared

Changes in the oestrogen and progesterone level in the blood and ovarian tissue of mature carps from fish farms were examined using gas chromatographic method, before and after spawning as well as under wintering. Due to the high individual variation the observations cannot be regarded as quantitative. The highest oestrogen level was found before spawning, both in the ovaries and the blood, this level was somewhat lower after spawning and did not show any further change under wintering. The resting progesterone level had a low value, both in the ovaries and the blood, but it increased after spawning. The oestrogen concentration of blood was somewhat higher in the latter. Effect of pituitary extracts on changes of the sex hormone concentration in blood and ovary of wild carps was investigated too. The results of the hormone determinations, changes in body weights and "coefficients of maturity" are presented.

Elevated serum bile acid levels were observed in overweight subjects as compared to those with an ideal weight. Fasting for 14 days resulted in a gradual decrease to the normal level of serum cholic, chenodeoxycholic and deoxycholic acid concentrations. There was no change in the serum bile acid level during a two days refeeding period. The findings suggest that, in addition to the beneficial weight reduction, normalization of bile acid homeostasis can be achieved by fasting in obesity.

In the present work the correlation between the immunological parameters and plasma androgen hormone levels in rheumatoid arthritis (RA) was examined. Female patients were divided in groups according to their age and immunologic state. Plasma dehydroepiandrosterone (D), D sulphate (DS), and testosterone (T) levels were determined by radioimmunological methods. The values were related first to those of normal controls. The T and DS levels were significantly decreased in all patient groups examined, the D level was low only in "Rf+ active" state aged 18 to 45 years. Furthermore, it was examined whether the decreased hormone levels were in relationship either with the autoantibody (Rf) formation or with the activity of the disease. According to the observations the T levels were influenced neither by the autoantibody formation nor by the activity of the disease. The DS and D levels appeared to correlate both with the autoantibody formation and the activity of the disease, particularly in young patients, being significantly low in the "Rf+ active" state. From these we concluded that the condition of the immune system has an influence on the sex hormone and/or adrenal androgen function.

Dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone concentration was determined by our sensitive gas-liquid chromatographic method in ovarian tissues obtained from surgery of patients without hirsutism and with Stein-Leventhal syndrome. The steroids, except testosterone, were detectable in all ovaries studied. Dehydroepiandrosterone and androstenedione, regarded as preandrogens, were present in an increased amount in almost all patients with polycycstic ovaries. Gas chromatographic evidence was obtained for the presence of testosterone in two of the cases. The delta4/3betaOH ratio reflecting 3beta-hydroxysteroid dehydrogenase activity was decreased only in same patients with the Stein-Leventhal syndrome suggesting that the impaired function of this enzyme is not an obligatory feature of polycystic ovaries. Concentration of pregnenolone and progesterone measured in a part of cases varied in a great range although the determination was caried out before luteal phase. Simultaneous determination of hormones in both ovarian tissues revealed an active and an inactive period of the gland in the given time, since a great difference of hormone concentration in bilateral ovarian tissues were observed. A comparison of hormone content in ovaries and the urinary excretion of metabolites showed poor correlation between the two parameters of hormone production.

The dynamic excretion of aldadiene, the principal metabolite of spironolactone with biological activity, was studied in normal subjects and patients with liver cirrhosis following administration of spironolactone. Aldadiene appeared in the three-days urine collection in around 3% of the dose. There was no significant difference in the biological half-life, calculated from the urinary pattern, and in the rate of excretion in patients with hepatopathy and hospitalized controls. Healthy volunteers without hospitalization exhibited the most rapid elimination of the metabolite. An increase of spironolactone dose enhanced the per cent rate of aldadiene excretion, while the biological half-life did not change. Spironolactone decreased the rate of dehydroepiandrosterone and androsterone excretion even after a single dose. Determination of total 17-ketosteroids is of no value on evaluation of adrenal function during spironolactone therapy, due to a Zimmermann-positivity of spironolactone metabolites.

[4-14C]-Dehydroepiandrosterone and [7 alpha-3H]-Dehydroepiandrosterone sulphate were injected simultaneously to normal and obese female subjects. The percentage recovery of 14C and 3H radioactivies in dehydroepiandrosterone sulphate, androsterone sulphate, etiocholanolone sulphate, androsterone glucuronoside and etiocholanolone glucuronoside was determined in the day-to-day urine collections for 72 hr. Results showed a normal total 3H recovery and a poor 14C recovery in urinary conjugates of obese patients. The rate of appearance of 3H activity was not identical in the individual metabolites of normal subjects, and it was not normal in obesity. Overweight subjects exhibited an acceleration in [7 alpha-3H]-Dehydroepiandrosterone sulphate metabolism to androsterone glucuronoside. The observation regarding the rate of appearance of urinary conjugates bearing 14C isotope correlate with our previous finding in which a glandular overproduction of free dehydroepiandrosterone was found and an uptake of this steroid by the adipose tissue was suggested. Our results showed that the poor recovery of 14C radioactivity in urine of obese female subjects was not an aspecific consequence of illness.

Female patients with rheumatoid arthritis (RA) in Steinbrocker's II and III rating scale have been examined. They were without steroid treatment at least six months before observation. Plasma protein picture showed hypoalbuminaemia and hyperglobulinaemia. There was no difference relative to controls either in the total (free + protein bound) plasma dehydroepiandrosterone (D) level, or in its distribution with plasma proteins. In the age group of 18 to 45 years, a statistically significant decrease have been observed in the total plasma dehydroepiandrosterone sulphate (DS) level without any change in its distribution in protein binding. Furthermore, low androsterone sulphate (AS) levels were found irrespective of the age of patients. The results gave further information on the pathomechanism resulting in an abnormal androgen hormone pattern of blood and a low metabolite excretion, observed previously in patients with RA:.