Efficient One-Step Biocatalytic Multienzyme Cascade Strategy for Direct Conversion of Phytosterol to C-17-Hydroxylated Steroids.

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. 17ß-Hydroxysteroid dehydrogenase (17ßHSD) and its native electron donor play significant roles in the 17ß-carbonyl reduction reaction of steroids. In this study, we bridged 17ßHSD 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 17ßhsd 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-ayr1S.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 17ß-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.

Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium - identification of a novel 11-oxidase activity.

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 6ß, 7ß, 9α, and 15ß. In addition, the CYP106A subfamily showed unprecedented 11-oxidase activity, converting 11ß-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.

A Holy Grail of clinical pharmacology: prediction of drug pharmacokinetics and pharmacodynamics in the individual patient.

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.

Male gender identity in an XX individual with congenital adrenal hyperplasia.

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.

Air oxidation of 17-hydroxycorticosteroids catalyzed by cupric acetate: formation of hemiacetal dimers.

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.

Evaluation of psychological effects due to bed rest.

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).

[Stress and anticortisols--17-ketosteroid sulfate conjugate as a biomarker in tissue repair and recovery].

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).

[Effect of milk proteins and their enzymatic hydrolyzates on non-specific resistance of some systems to food allergens]. / Vliianie belkov moloka i poluchennykh na ikh osnove fermentativnykh gidrolizatov na sostoianie nekotorykh sistem, obespechivaiushchikh nespetsificheskuiu rezistentnost k pishchevym allergiziruiushchim vozdeistviiam.

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.

[Effect of compound and minor prescription of heat-nature products radix Aconiti lateralis preparata, rhizoma Zingiberis and cortex Cinnamomi on the sympathetic nervous system, adrenal gland, TSH and LH].

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.

[The effect of cow's milk and its hydrolysates on the metabolism of 17-hydroxycorticosteroids and the functional state of the gastrointestinal tract in guinea pigs]. / Vliianie belkov korov'ego moloka i ikh gidrolizatov na metabolizm 17-oksikortikosteroidov i funktsional'noe sostoianie zheludochno-kishechnogo trakta u morskikh svinok.

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.

[Effect of working conditions in the coke-processing department on the secretory function of the adrenal cortex]. / Wplyw warunków pracy w wydziale weglopochodnych koksowni na wydzielanie kory nadnerczy.

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.

Asymptomatic adrenal tumor; 386 cases in Japan including our 7 cases.

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.

Electron microscopic findings in benign deoxycorticosterone and 11-deoxycortisol-producing adrenal tumor.

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.

[A case of adrenal black adenoma associated with Cushing's syndrome].

A 36-year-old female was admitted to Asahi General Hospital because of hypertension. Laboratory findings showed elevated levels of urine 17 (11)-hydroxycorticosteroid and blood cortisol. Computerized tomography and ultrasound sonography demonstrated a round tumor at the suprarenal region. The black tumor, 5 X 3 X 2 cm, was removed by left adrenalectomy. Microscopic findings revealed adrenal adenoma consisting of compact cells containing numerous pigmented granules. Postoperative course was uneventful. Hormonal condition was recovered 11 months after operation.

The effect of antipyrine and rifampin on the metabolism of diazepam.

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.

Steroid 21-hydroxylation by human preovulatory follicles from stimulated cycles: a mass spectrometrical study of deoxycorticosterone, 21-hydroxypregnenolone and 11-deoxycortisol in follicular fluid.

A highly specific technique based on gas chromatography-mass spectrometry associated with stable isotope dilution was applied to the analysis of follicular fluid aspirated from preovulatory follicles of women under ovarian stimulation prior to in vitro fertilization. Deoxycorticosterone, 21-hydroxypregnenolone and 11-deoxycortisol have been identified and quantified in the nanomolar concentration range. Significant positive correlations were found between these 21-hydroxy-steroids and their immediate precursors, thus indicating a probable common cellular origin. Corticosterone was tentatively identified and cortisol was evidenced at concentrations lower than peripheral plasma levels. The occurrence in human follicular fluid of cortisol, together with different high concentration intermediates, constitutes evidence for ovarian intra-follicular 21-hydroxylase activity, and probably also for 11 beta-hydroxylation enzyme activity.