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Steroidal 17-carbonyl reduction is crucial to the production of natural bioactive steroid medicines, and boldenone (BD) is one of the important C-17-hydroxylated steroids. Although efforts have been made to produce BD through biotransformation, the challenges of the complex transformation process, high substrate costs, and low catalytic efficiencies have yet to be mastered. Phytosterol (PS) is the most widely accepted substrate for the production of steroid medicines due to its similar foundational structure and ubiquitous sources. 17β-Hydroxysteroid dehydrogenase (17βHSD) and its native electron donor play significant roles in the 17β-carbonyl reduction reaction of steroids. In this study, we bridged 17βHSD with a cofactor regeneration strategy in Mycobacterium neoaurum to establish a one-step biocatalytic carbonyl reduction strategy for the efficient biosynthesis of BD from PS for the first time. After investigating different intracellular electron transfer strategies, we rationally designed the engineered strain with the coexpression of 17 β hsd and the glucose-6-phosphate dehydrogenase (G6PDH) gene in M. neoaurum . With the establishment of an intracellular cofactor regeneration strategy, the ratio of [NADPH]/[NADP + ] was maintained at a relatively high level, the yield of BD increased from 17% (in MNR M3M- ayr1 S.c ) to 78% (in MNR M3M- ayr1 & g6p with glucose supplementation), and the productivity was increased by 6.5-fold. Furthermore, under optimal glucose supplementation conditions, the yield of BD reached 82%, which is the highest yield reported for transformation from PS in one step. This study demonstrated an excellent strategy for the production of many other valuable carbonyl reduction steroidal products from natural inexpensive raw materials. IMPORTANCE Steroid C-17-carbonyl reduction is one of the important transformations for the production of valuable steroidal medicines or intermediates for the further synthesis of steroidal medicines, but it remains a challenge through either chemical or biological synthesis. Phytosterol can be obtained from low-cost residues of waste natural materials, and it is preferred as the economical and applicable substrate for steroid medicine production by Mycobacterium. This study explored a green and efficient one-step biocatalytic carbonyl reduction strategy for the direct conversion of phytosterol to C-17-hydroxylated steroids by bridging 17β-hydroxysteroid dehydrogenase with a cofactor regeneration strategy in Mycobacterium neoaurum. This work has practical value for the production of many valuable hydroxylated steroids from natural inexpensive raw materials.

The CYP106A subfamily hydroxylates steroids, diterpenes, and triterpenes in a regioselective and stereoselective manner, which is a challenging task for synthetic chemistry. The well-studied CYP106A2 enzyme, from the Bacillus megaterium strain ATCC 13368, is a highly promising candidate for the pharmaceutical industry. It shares 63 % amino acid sequence identity with CYP106A1 from B. megaterium DSM319, which was recently characterized. A focused steroid library was screened with both CYP106A1 and CYP106A2. Out of the 23 tested steroids, 19 were successfully converted by both enzymes during in vitro and in vivo reactions. Thirteen new substrates were identified for CYP106A1, while the substrate spectrum of CYP106A2 was extended by seven new members. Finally, six chosen steroids were further studied on a preparative scale employing a recombinant B. megaterium MS941 whole-cell system, yielding sufficient amounts of product for structure characterization by nuclear magnetic resonance. The hydroxylase activity was confirmed at positons 6β, 7β, 9α, and 15β. In addition, the CYP106A subfamily showed unprecedented 11-oxidase activity, converting 11β-hydroxysteroids to their 11-keto derivatives. This novel reaction and the diverse hydroxylation positions on pharmaceutically relevant compounds underline the role of the CYP106A subfamily in drug development and production.

Predicting drug kinetics and dynamics in an individual patient is a worthy goal and has some chance of success for drugs subject to marked pharmacogenetic differences. However, one should not expect any prediction success for drugs primarily metabolized by the major cytochrome P450 enzyme, CYP3A4, whether one uses an exogenous drug or an endogenous metabolic process, such as the oxidation of cortisol.

Introduction: In spite of significant changes in the management policies of intersexuality, clinical evidence show that not all pubertal or adult individuals live according to the assigned sex during infancy., Aim: The purpose of this study was to analyze the clinical management of an individual diagnosed as a female pseudohermaphrodite with congenital adrenal hyperplasia (CAH) simple virilizing form four decades ago but who currently lives as a monogamous heterosexual male., Methods: We studied the clinical files spanning from 1965 to 1991 of an intersex individual. In addition, we conducted a magnetic resonance imaging (MRI) study of the abdominoplevic cavity and a series of interviews using the oral history method., Main Outcome Measures: Our analysis is based on the clinical evidence that led to the CAH diagnosis in the 1960s in light of recent clinical testing to confirm such diagnosis., Results: Analysis of reported values for 17-ketosteroids, 17-hydroxycorticosteroids, from 24-hour urine samples during an 8-year period showed poor adrenal suppression in spite of adherence to treatment. A recent MRI study confirmed the presence of hyperplastic adrenal glands as well as the presence of a prepubertal uterus. Semistructured interviews with the individual confirmed a life history consistent with a male gender identity., Conclusions: Although the American Academy of Pediatrics recommends that XX intersex individuals with CAH should be assigned to the female sex, this practice harms some individuals as they may self-identify as males. In the absence of comorbid psychiatric factors, the discrepancy between infant sex assignment and gender identity later in life underlines the need for a reexamination of current standards of care for individuals diagnosed with CAH.

Hydrocortisone, cortexolone, hydrocortisone-17-butyrate, and budesonide were oxidized into alpha-ketoaldehydes by air exposure in the presence of Cu(OAc)(2). When free hydroxyl functions were present at position 17, hydrocortisone and cortexolone, the formed oxidation products, were identified as hemiacetal dimeric structures involving the free hydroxyl functions at position 17 and the newly formed aldehydes at position 21. Dimeric structures were established by using 1H913C0 correlations (HSQC and HMBC) and 1H-1H correlations (COSY and ROESY). The hemiacetal function was further confirmed by reaction of the dimer formed from hydrocortisone with two equivalents of 3-methyl-2-benzotriazolinone hydrazine (MTBH), giving quantitatively two equivalents of the 3-methyl-2-benzotriazolinone hydrazone of 21-dehydrohydrocortisone. When no free hydroxyl function was present as in the case of hydrocortisone-17-butyrate and budesonide, the expected alpha-ketoaldehydes were obtained.

The psychological condition of astronauts is an important factor for ensuring mission success in limited space. Head-down tilting bed rest is a well-accepted method by which to simulate an acute stage of human adaptation to the weightless state in space flight. In our previous studies, the enhancement of depressive and neurotic levels occurred during a 20-day horizontal bed rest. In this study, we attempted to examine the depressive and neurotic levels, the mood status, and behavioral tendency of the subjects and to analyze the changes of 24-hour urinary excretion of 17-hydroxycorticosteroid-glucronides (17-OHCS) for an indicator of changes in the endocrine system due to physical and psychological stress during a 20-day 6-degrees head-down tilting bed rest (BR).

We attempted to determine compounds in human urine which, differing from cortisol (17-OHCS), show high values in healthy individuals, decrease with failing health, clearly decline with advancing disease and finally reach very low values in severe disease. We have eventually established that 17-ketosteroid sulfate conjugates (17-KS-S) are the compounds we were searching for. Hans Selye regarded stress as the rate of wear and tear and 17-OHCS as its indicator, but we considered that, differing from inanimate objects, living organisms consume energy to cope with stress and "repair" "wear and tear" of the tissue and "recover" its function. This concept led us to determine the organism's requirement for two mechanisms: "Wear and tear" which could be represented by the secretion of 17-OHCS (Selye), and "repair and recovery", which could be determined by the amounts of 17-KS-S derived from dehydroepiandrosterone (DHEA)-sulfate, a product of the adrenal cortex, which enhances neuronal survival, longterm memory, maintains the function of peripheral tissues, and stimulates immune system (17-KS mainly consists of 17-KS-S and 17-KS glucuronides 17-KS-G, the latter derived from cortisol, DHEA and testosterone). We hold that stress response is a series of biological process, beginning from CRH.ACTH and catecholamines, cortisol (17-OHCS), followed by insulin, acting as an anabolic agent and finally DHEA (17-KS-S) leads the tissues to repair by utilizing produced energy. Balanced changes of hormones, such as 17-KS-S, 17-OHCS, cortisol, catecholamines and insulin are seen in healthy individuals under healthy lifestyles and disruption of the balance brings apparent reduction in 17-KS-S. From this standpoint, we wish to develop research in interrelations between biologically antagonistic 17-KS-S and 17-OHCS, focusing particularly at 17-KS-S, which represents a contact point for mind and body, the healthy state of which is kept by appropriate sleeping and exercise programs on the basis of adequate food intake. The above three factors, 17-KS-S, 17-OHCS, and 17-KS-G, are expressed in creatinine ratio (mg/g creatinine).

Noninbred male guinea-pigs (b. mass 250-300 g) were kept for 35 days on diets in which 50% of standard proteins were replaced for cow milk protein (CMP) 20/80 and its enzymatic hydrolyzate (EH) 20/80 obtained by ultrafiltration. CMP 20/80 and EH 20/80 have been examined for the effect on proteolytic activity of gastric mucosa, small intestinal activity of trypsin and chymotrypsin, hepatic cytochrome P-450 and metabolism of excreted 17-OCS. The animals given EH 20/80 exhibited enhanced total proteolytic activity of trypsin and chymotrypsin, higher total levels of cytochromes P-450 and P-450B in the liver, increased urine excretion of 17-OCS, reduced amount of polar forms in all the glucocorticoid fractions. Potential mechanisms securing the action of enzymatic hydrolyzates on the systems responsible for nonspecific resistance to food allergens are suggested for discussion.

The effect of compound and minor prescriptions of Radix Aconiti Lateralis Preparata, Rhizoma Zingiberis and Cortex Cinnamomi on sympathetic nervous system, adrenal gland, TSH and LH of rats was studied. Results showed that both prescriptions made the heart rate quicken, estrous cycle shorten, improved the adrenal cortex hormone and promote TSH and LH synthesis and secretion. The two Heat-nature products (Radix Aconiti Praeparata and Rhizoma Zingiberis) may be used instead of all the three products to make a Heat Syndrome animal model.

Two groups of noninbred male guinea pigs (initial bw 250-300 g) received test rations in which 50% of standard ration protein were substituted for milk protein (P 20/80) and its enzymatic hydrolysate (H 20/80). The influence of P 20/80 and H 20/80 on some parameters of the gastro-intestinal tract (GIT) barrier function and 17-OCS metabolism was studied. The action of H 20/80 as compared to P 20/80 was expressed in the intensified excretion of free fraction of 17-OCS and glucuronides. The content of hydroxylated glucocorticoids increased in both fractions. At the same time elevated total proteolytic activity in the gastric mucosa and a tendency to decreased GIT permeability for chicken ovalbumin macromolecules were observed in animals that received H 20/80. It has been suggested that enzymatic hydrolysates of food proteins influence endocrine regulation of metabolic processes.

In 85 randomly chosen employees of the coal tar distillation department working at least 5 years, exposed to toxic substances not exceeding MAC except tar substances and benzo(a)pyrene, whose concentrations were high, blood level of cortisol and 17 OHCS and 17 KS excretion in urine were examined. In the most exposed group cortisol level decreased and the 17 OHCS excretion augmented.

To clarify the indication of surgery in incidentally discovered asymptomatic adrenal masses, we analyzed 386 Japanese cases, 379 cases reported in Japan during the past 25 years (from 1964 to 1988) and 7 cases from our own experience. From a total of 460 patients, we carefully selected 379 patients satisfying our criterion of the absence of symptoms and signs suggestive of active hormone over-secretion as described in each case report. From the Japanese series, there was a high incidence of pheochromocytoma patients (20 of 37 patients) who had no symptoms and signs but had high plasma or urine catecholamines. Scintigraphy with 131I-meta-iodo-benzyl-guanidine was useful in the diagnosis of pheochromocytoma. For the other asymptomatic adrenal tumors, except for myelolipoma and adrenal cyst, differential diagnosis between malignant and benign adrenal lesions by imaging procedures such as whole body computed tomography (CT), ultrasonography (US), adrenocortical scintigraphy, and angiography was not always possible. In addition, among the 109 patients with cortical tumors whose hormonal data were reported, no clear-cut differentiation of malignant tumor from benign by means of these data could be obtained. Since 1980 whole body CT scanner and high resolution US scanner have become widely available, and there have been 283 cases of asymptomatic adrenal tumors who satisfied our criterion. Cortical carcinomas smaller than 3 cm and 6 cm in diameter account for 3.8% and 6.6%, respectively, of the total of 101 cases of cortical carcinoma, cortical adenoma, ganglioneuroma, and hemangioma during this period. The size of the smallest cortical carcinoma with metastasis was 2 cm in diameter in this series. Pre-operatively, an adrenocortical carcinoma 2.8 cm in diameter in our patient could not be diagnosed as such by imaging techniques and measurement of plasma hormones. These findings suggest that an adrenal mass larger than 3 cm should be removed and a patient with a smaller cortical tumor should be carefully followed up.

The ultrastructural characteristics of a benign adrenal adenoma that produces deoxycorticosterone and 11-deoxycortisol were examined by transmission electron microscopy. Both large, round mitochondria with a few cristae in the peripheral portion and spherical or oval mitochondria with sacrotubular cristae were observed in adenoma cells. The development of agranular or granular endoplasmic reticulum varied from cell to cell. In some cells, many vacuoles and collagenous fibers were also seen. Thus, the adrenal tumor we examined was composed of the cells derived from the fascicular zone, which are associated with Cushing's syndrome, and the cells of the glomerular zone observed in association with primary aldosteronism.

Experiments were performed on dogs with hypothalamic lesions as well as on intact and hypophysectomized dogs. Adrenal venous blood samples were collected and analyzed for 17-hydroxycorticosteroids (17-OHCS). In intact dogs, a marked increase in adrenal 17-OHCS secretion was observed after an i.v. injection of histamine and following an i.v. infusion of cyanide. Adrenal 17-OHCS secretion in response to histamine was markedly reduced but not completely abolished by hypophysectomy and was partially impaired by anterior median eminence lesions and by posterior median eminence lesions, but was not significantly impaired by supramammillary lesions. Adrenal 17-OHCS secretion in response to cyanide was completely abolished by hypophysectomy, markedly reduced by posterior median eminence lesions and partially impaired by anterior median eminence lesions and by supramammillary lesions. Thus, a partial dissociation of the adrenocortical secretory responses to histamine and cyanide was observed in dogs with hypothalamic lesions, suggesting that the mechanism and the pathway to the anterior pituitary involved in the adrenocortical secretory response to histamine and cyanide are somewhat different.

The results of biochemical examinations of the hormonal function of the adrenal cortex in 22 patients with Parkinson's disease are presented as seen prior and following stereotaxic operations on the ventro-lateral nucleus of the thalamus. It was found that parkinsonics have a decreased androgenic, glucocorticoid and mineralocorticoid function of the adrenal cortex. Long history and growing severity of the disease manifest themselves in a distinct inhibition of the adrenal cortex function. The importance of the changes in the functional state of the hypothalamo-hypophyseal system in the revealed disorders in the hormonal activity is emphasized. Along with a beneficial clinical effect surgery brings about an improvement of the functional state of the adrenal cortex.

Recent investigations have furnished a complete analysis of the hemodynamic events accompanying whole-body immersion. About 700 ml of blood are translocated into the intrathoracic circulation, and heart volume increases by 180 +/- 62 ml. These changes are followed by an increase in stroke volume and cardiac output of over 30%. At the same time a reflex reduction of total peripheral resistance and venous tone occurs. Renin and aldosterone activity are reduced while the 17-hydroxycorticosteroid is not affected. Treatment of the subject with DOCA attenuates but does not extinguish the excess sodium excretion of immersion. This finding strengthens the arguments in favor of an unknown factor enhancing sodium excretion. Finally, the relative activation of the three factors that serve volume control, the excretory function of the kidney, capillary filtration pressure, and the thirst mechanism, is discussed.

This paper has been designed to review some current concepts about biochemistry, pathophysiology and clinics of androgen-binding proteins, both TeBG and cellular receptors. Some important experimental models to clarify the interactions between androgens and target cells are mentioned. Further, major clinical applications in which binding parameters were or will be of great importance are discussed in details.

The authors report a case of Steinert's disease in a woman and discuss the endocrine profile of this disease after giving an account of the criteria of diagnosis. Disorders of gonad function are mild in women, primary testicular atrophy is very frequent in man with reduction in 17-ketosteroids and testosterone. Thyroid function was normal but, in a few cases, a low fixation curve was found (our case) corrected by TSH stimulation. The frequency of cataract emphasizes the interest of this sign for detection. Diabetes, associated with hyperinsulinism, seemed more frequent than in a population without Steinert's disease. The pathogenesis of these endocrine disorders appears secondary and is ill explained if one considers it as a single disease. Better knowledge, no doubt linked to progress in biochemistry of normal and myopathic muscle, will help to explain the pathogenesis.

Eleven patients presenting with acute necrotizing ulcerative gingivitis (ANUG) collected 24-hour urine samples during and after the course of their ANUG. The Porter-Silber technique was used to determine the 17-hydroxycorticosteroid concent of the urine, a physiological measure of stress. All of the patients had significantly higher levels of 17-hydroxycorticosteroid during the course of their ANUG than after it had resolved.

A combination of several in vivo parameters has been applied in male healthy volunteers to test the suitability of these parameters to indicate enzyme induction in man: Urinary excretion of D-glucaric acid and 6 beta-hydroxycortisol, activity of serum gamma-glut amyltranspeptidase, and pharmacokinetics of aminopyrine respond significantly to phenobarbital treatment. Glucaric acid excretion is enhanced about 7-fold. Its response to induction overcomes the large individual and inter-individual variations which exist in the untreated state for glucaric acid excretion and the other parameters applied, as well. Total body clearance of aminopyrine as obtained after an oral test dose increases more than twofold from 251 to 551 ml/min upon phenobarbital treatment. This arises from increases in both the elimination constant and the apparent volume of distribution, as well. Urinary excretion of aminoantipyrine during 24 hr is about doubled, whereas the elimination of acetyl-aminotipyrine is not much affected. 6 beta-hydroxycortisol excretion in urine and activity of serum gamma-glutamyltranspeptidase are significantly augmented to about 150% of control values. Half life times of phenobarbital measured after termination of treatment are in normal range, suggesting no self-induction of phenobarbital metabolism. Because of the complexity of drug metabolizing enzymes only a combination of different parameters reliably indicates alterations in this enzyme system by inducing agents.

This biologically oriented review attempts to complement earlier and more psychological performance based reviews of total sleep deprivation. Also, the effects of total sleep deprivation are interpreted, here, from a function of human sleep perspective, rather than from the more usual stress viewpoint. It would appear that total sleep deprivation does not produce any major changes in biochemical and physiological measures of somatic functioning. Such a conclusion would not seem to support an hypothesis that human sleep is for body restitution. Although there are equivocal results amongst several of the more psychophysiological measures, EEG does show a pronounced change. This latter finding, together with many reports of psychological performance detriment, would appear to sustain a CNS or cerebral restitutional role for human sleep. However, before definitive conclusions can be drawn from the biochemical and physiological findings of total sleep deprivation, the following qualifications of experimental methodology are made: (1) Apart from sleep loss, many studies have provided unnatural regimes which may have ameliorated possible effects of total sleep deprivation. (2) Most studies are of relatively short duration and may not have been sufficiently long for effects to develop. (3) Measures are often limited in range and depth of analysis. (4) Subjects are mostly fit, young adult, intelligent males and there is little constitutional variability. (5) Reported changes of statistical significance may be of no physiological significance and changes of possible physiological significance may be obscured by statistical procedures. (6) Intervening behavioural variables, such as novelty and anticipation of the sleep deprivation situation, may confound real effects.

The hypothalamic-pituitary-adrenal axis was investigated in 15 asthmatic children treated with inhaled beclomethasone dipropionate (mean 490 micrograms/day) and 11 asthmatic control subjects receiving no corticosteroid therapy. Measurements of 24-h urinary free cortisol and 17 hydroxy corticosteroids, serum cortisol, response to ACTH, and the oral metyrapone test showed no significant difference between the two groups. All the patients' results were within normal limits, and carbohydrate metabolism, as shown by blood glucose and hemoglobin A1c, was not affected by beclomethasone therapy. Thus, in the above dose, inhaled beclomethasone does not cause suppression of the hypothalamic-pituitary-adrenal axis.

1 The elimination and metabolism of diazepam in man was investigated following the induction of the liver microsomal enzyme system by antipyrine. 2 Seven healthy volunteers were given 1200 mg antipyrine as an inducing agent for a period of 14 days. Before and after the induction period the elimination of diazepam and desmethyldiazepam was measured in the plasma by gaschromatography. As parameters of liver microsomal enzyme activity, antipyrine elimination and gamma-glutamyl-transpeptidase in the plasma, D-glucaric acid and 6-beta-hydroxycortisol urinary excretion were measured on both occasions. 3 Following the induction period most parameters of microsomal enzyme activity measured were significantly changed indicating an increase of the microsomal enzyme system. The elimination of diazepam was significantly altered having a half-life of 37 h before and 18 h afterwards combined with a significant increase in total body clearance after the induction period, although the volume of distribution remained unaltered. The formation of the main metabolite N-desmethyldiazepam was not changed, but its elimination was increased having a half-life of 139 or 58 h respectively. 4 The elimination of unchanged diazepam and desmethyldiazepam is significantly increased by the induction of the liver microsomal enzyme system using antipyrine as an inducing agent in healthy volunteers, which might be important under certain clinical conditions.

An adrenal tumor was discovered fortuitously in a patient with no clinical features of Cushing's syndrome. On adrenal imaging, there was good uptake in the nodule but no visualization of the contralateral adrenal. The latter was seen, however, in a second scan performed under ACTH treatment. In the hormone assessment, basal cortisol and 17-hydroxycorticoids were normal and cortisol diurnal variation was near normal, but a dexamethasone suppression test and ACTH responses to metyrapone and insulin hypoglycemia were abnormal. Eight months after excision of a spongiocytic-type adenoma, the remaining adrenal was visible on scintigram and the hormonal tests were normal. This pattern suggests that the clinical Cushing's syndrome but enough to suppress partially ACTH and, consequently, visualization of the contralateral gland.

The research activity of the physiological laboratories (Medical and Hygienic Institute, Cluj-Napoca, Roumania) in the area of stress studies is described from three aspects: a) Methods of stress provocation and assessment; b) The effects of hormones, especially of anterior pituitary, on the adaptation to physical load; c) Nervous and endocrine factors of stress (physical exertion, fatigue, high pressure, anoxia).

Based on the conceptions of the relationship between energy and adaptation-immunological systems a number of hormone-metabolic indices were compared with immune reactivity as evidenced by skin tests and rosette-formation reaction. It is shown that lowering of immunological indices in oncological patients is associated with a decreased sensitivity of dexametasone suppression of cortisol, an increased concentration of 11-oxycorticosteroids in blood, a higher level of triglycerides, cholestrol, beta-lipoproteins and less glucose tolerance. The use of phenformin during 3 months in patients with atherosclerosis resulted in a considerable increase of the index of phytohemagglutinin stimulated blasttransformation of lymphocytes and rosette-formation reaction. This effect was associated with normalization of lipid metabolism indices.

The review of the reported data indicates that endocrine glands react to the constant magnetic field (CMF). The paper presents information about the phasic pattern of hypophyseal-adrenal reaction and CMF effects on the sympathoadrenal system and thyroid. The review refers to many publications demonstrating high sensitivity to testes of the CMF. The paper discusses potential therapeutic applications of the CMF with emphasis on the fact that most morphological data available need biochemical support.

The elimination of diazepam and antipyrine and the urinary excretion of their metabolites were investigated in 21 healthy volunteers before and after 7 days of administration of antipyrine, 1200 mg, and rifampin, 600 or 1200 mg. After administration of antipyrine and rifampin in two doses, antipyrine total body clearance increased by 53% and 60% or 98%, respectively. The clearance to metabolite showed a preferential induction of the norantipyrine pathway with different proportions after antipyrine and rifampin; rifampin, 1200 mg, also enhanced the 4-hydroxyantipyrine pathway further. After antipyrine, diazepam total body clearance was increased by 102%, affecting all metabolic pathways to a similar extent. After rifampin in both doses, diazepam total body clearance rose equally to 300% and desmethyl- and 3-hydroxydiazepam metabolic clearance to 400%. Therefore rifampin preferentially affects norantipyrine or desmethyl- and 3-hydroxydiazepam metabolic formation, suggesting induction of different (iso)zymes of cytochrome P-450.