Corticosteroid Biosynthesis Revisited: No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism.

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A Metabolomic Strategy to Screen the Prototype Components and Metabolites ofShuang-Huang-Lian Injectionin Human Serum by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry

Journal of Analytical Methods in Chemistry ◽

10.1155/2014/241505 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 4

Author(s):

Mingxing Guo ◽

Baosheng Zhao ◽

Haiyu Liu ◽

Li Zhang ◽

Long Peng ◽

...

Keyword(s):

Human Serum ◽

Human Body ◽

Matrix Effects ◽

Ultra Performance Liquid Chromatography ◽

Acute Respiratory Tract Infection ◽

Chemical Compositions ◽

Drug Induced ◽

Flight Mass Spectrometry ◽

Chinese Patent ◽

Metabolic Reactions

Shuang-huang-lian injection(SHLI) is a famous Chinese patent medicine, which has been wildly used in clinic to treat acute respiratory tract infection, pneumonia, influenza, and so forth. Despite the widespread clinical application, the prototype components and metabolites ofSHLIhave not been fully elucidated, especially in human body. To discover and screen the constituents or metabolites of Chinese medicine in biofluids tends to be more and more difficult due to the complexity of chemical compositions, metabolic reactions and matrix effects. In this work, a metabolomic strategy to comprehensively elucidate the prototype components and metabolites ofSHLIin human serum conducted by UPLC-Q-TOF/MS was developed. Orthogonal partial least squared discriminant analysis (OPLS-DA) was applied to distinguish the exogenous, namely, drug-induced constituents, from endogenous in human serum. In the S-plot, 35 drug-induced constituents were found, including 23 prototype compounds and 12 metabolites which indicated thatSHLIin human body mainly caused phase II metabolite reactions. It was concluded that the metabolomic strategy for identification of herbal constituents and metabolites in biological samples was successfully developed. This identification and structural elucidation of the chemical compounds provided essential data for further pharmacological and pharmacokinetics study ofSHLI.

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Stimulation of 24R,25-dihydroxyvitamin D3 synthesis by metabolites of vitamin D3

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1983.245.4.e359 ◽

1983 ◽

Vol 245 (4) ◽

pp. E359-E364 ◽

Cited By ~ 1

Author(s):

G. S. Reddy ◽

G. Jones ◽

S. W. Kooh ◽

D. Fraser ◽

H. F. DeLuca

Keyword(s):

Vitamin D ◽

Vitamin D3 ◽

The Other ◽

Hydroxyl Group ◽

Vitamin D Metabolites ◽

Structural Requirements ◽

Dihydroxyvitamin D3 ◽

Hydroxylated Metabolites ◽

Perfusion Period ◽

Stimulation Of

Previously we have shown that the isolated perfused kidney from vitamin D-deficient rats converts [3H]25(OH)D3 into [3H]1 alpha,25(OH)2D3. When certain vitamin D metabolites were added to perfusate the same kidney began to synthesize [3H]24R,25(OH)2D3. In this study we investigated the structural requirements of the vitamin D molecule necessary to stimulate synthesis of [3H]24R,25(OH)2D3 in a 1-hydroxylating kidney. Kidneys were perfused with tracer [3H]25(OH)D3 (450 pM) alone and in the presence of a variety of hydroxylated metabolites and fluorinated analogues of vitamin D3 at concentrations of 450 pM to 25 microM. Tracer [3H]25(OH)D3 alone resulted in synthesis of only [3H]1 alpha,25(OH)2D3 during the 6-h perfusion period. 25-Hydroxylated metabolites [25(OH)D3, 25 nM; 1 alpha,25(OH)2D3, 25 nM; 24R,25(OH)2D3, 25 nM; 24(F)2,25(OH)D3, 50 nM] stimulated [3H]24R,25(OH)2D3 production at 2 h of perfusion. On the other hand, analogues without the 25-hydroxyl group [D3; 1 alpha(OH)D3; 25(F)D3; 1 alpha(OH),25(F)D3; 1 alpha(F)D3; 1 beta(F)D3]; did not stimulate [3H]24R,25(OH)2D3 synthesis. We conclude that the 25-hydroxyl group is an essential determinant of 24-hydroxylation.

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Exploring the Metabolism of Loxoprofen in Liver Microsomes: The Role of Cytochrome P450 and UDP-Glucuronosyltransferase in Its Biotransformation

Pharmaceutics ◽

10.3390/pharmaceutics10030112 ◽

2018 ◽

Vol 10 (3) ◽

pp. 112 ◽

Cited By ~ 6

Author(s):

Riya Shrestha ◽

Pil Cho ◽

Sanjita Paudel ◽

Aarajana Shrestha ◽

Mi Kang ◽

...

Keyword(s):

Cytochrome P450 ◽

Active Metabolite ◽

Experimental Studies ◽

Liver Microsomes ◽

Cytochrome P450s ◽

Hydroxylated Metabolites ◽

Udp Glucuronosyltransferase ◽

Cyp Isoforms ◽

Propionic Acid Derivative

Loxoprofen, a propionic acid derivative, non-steroidal anti-inflammatory drug (NSAID) is a prodrug that is reduced to its active metabolite, trans-alcohol form (Trans-OH) by carbonyl reductase enzyme in the liver. Previous studies demonstrated the hydroxylation and glucuronidation of loxoprofen. However, the specific enzymes catalyzing its metabolism have yet to be identified. In the present study, we investigated metabolic enzymes, such as cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), which are involved in the metabolism of loxoprofen. Eight microsomal metabolites of loxoprofen were identified, including two alcohol metabolites (M1 and M2), two mono-hydroxylated metabolites (M3 and M4), and four glucuronide conjugates (M5, M6, M7, and M8). Based on the results for the formation of metabolites when incubated in dexamethasone-induced microsomes, incubation with ketoconazole, and human recombinant cDNA-expressed cytochrome P450s, we identified CYP3A4 and CYP3A5 as the major CYP isoforms involved in the hydroxylation of loxoprofen (M3 and M4). Moreover, we identified that UGT2B7 is the major UGT isoform catalyzing the glucuronidation of loxoprofen and its alcoholic metabolites. Further experimental studies should be carried out to determine the potency and toxicity of these identified metabolites of loxoprofen, in order to fully understand of mechanism of loxoprofen toxicity.

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Metabolomic Interactions: An Insight into Health and Disease

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i39a32142 ◽

2021 ◽

pp. 61-67

Author(s):

Maheswara Reddy Mallu ◽

Shaik Mohammad Anjum ◽

Sai Sri Samyutha Katravulapalli ◽

Sri Sai Priya Avuthu ◽

Koteswara Reddy Gujjula ◽

...

Keyword(s):

Metabolic Engineering ◽

Human Body ◽

Metabolic Pathways ◽

Biological Processes ◽

Therapeutic Treatment ◽

The Past ◽

Metabolic Functions ◽

Health And Disease ◽

Metabolic Reactions ◽

Insight Into

Over the past decade, metabolic engineering has emerged as an active and distinct discipline characterized by its over-arching emphasis on integration. In practice, metabolic engineering is the directed improvement of cellular properties through the application of modern genetic methods. The concept of metabolic regulations deals with the varied and innumerable metabolic pathways that are present in the human body. A combination of such metabolic reactions paves the way to the proper functioning of different physiological and biological processes. Dealing with the adversities of a disease, engineering of novel metabolic pathways showcases the potential of metabolic engineering and its application in the therapeutic treatment of diseases. A proper and deeper understanding of the metabolic functions in the human body can be known from simulated yeast models. This review gives a brief understanding about the interactions between the molecular set of metabolome and its complexity.

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Mechanisms of OCT4 on 3,5,3'-tri-iodothyronine and FSH-induced granulosa cell development in female mice

Endocrinology ◽

10.1210/endocr/bqab183 ◽

2021 ◽

Author(s):

Qiaozhi Wang ◽

Yilin Yao ◽

Xiaoshu Ma ◽

Baoqiang Fu ◽

Ningxin Li ◽

...

Keyword(s):

Granulosa Cells ◽

Apoptotic Cell ◽

Cell Development ◽

Cellular Growth ◽

Akt Signaling Pathway ◽

Steroid Biosynthesis ◽

Oct4 Expression ◽

Regulatory Processes ◽

Key Enzyme ◽

Transcription Factor 4

Abstract Octamer-binding transcription factor 4 (OCT4) regulates the pluripotency of stem cells and also plays important roles in granulosa cells growth, which is regulated by follicle-stimulating hormone (FSH). Thyroid hormone (TH) is important for the development and maturation of follicles and the maintenance of various endocrine functions. Although 3,5,3′-triiodothyronine (T3) enhances the effects of FSH on the regulation of the growth of granulosa cells and development of follicles, it is unclear whether and how TH combines with FSH to regulate OCT4 expression in granulosa cells during the preantral to early antral transition stage. Our results showed that T3 enhanced FSH-induced OCT4 expression. However, T3/FSH-induced cellular growth was reduced by OCT4 siRNA. OCT4 knockdown significantly increased the number of apoptotic cell. Moreover, T3 combined with FSH to increase ERβ expression, but did not significantly affect ERα expression. ERβ knockdown dramatically decreased T3/FSH-induced OCT4 expression and cell development and increased cell apoptosis. The PI3K/Akt pathway was involved in hormones inducing OCT4 and ERβ expressions. Furthermore, the hormones regulating OCT4 and ERβ expressions were regulated by cytochrome P450 lanosterol 14a-demethylase (CYP51), a key enzyme in sterol and steroid biosynthesis. T3 and FSH cotreatment potentiated cellular development by upregulating OCT4 expression, which is mediated by CYP51 and ERβ. These regulatory processes are mediated by the PI3K/Akt signaling pathway. These findings suggest that OCT4 mediates the T3 and FSH-induced development of follicles.

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Discovery of a potent cyclooxygenase-2 inhibitor, S4, through docking-based pharmacophore screening, in vivo and in vitro estimations

Molecular BioSystems ◽

10.1039/c6mb00229c ◽

2016 ◽

Vol 12 (8) ◽

pp. 2541-2551 ◽

Cited By ~ 1

Author(s):

Tien-Sheng Tseng ◽

Show-Mei Chuang ◽

Nai-Wan Hsiao ◽

Yi-Wen Chen ◽

Yu-Ching Lee ◽

...

Keyword(s):

Human Body ◽

Cyclooxygenase 2 ◽

Prostaglandin Production ◽

Key Enzyme ◽

Pharmacological Target ◽

Pharmacophore Screening ◽

Anti Inflammatory

Cyclooxygenase (COX; EC: 1.14.99.1), the key enzyme in prostaglandin production in the human body, is a major pharmacological target for developing anti-inflammatory agents.

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Microstructural comparison of synthetic bioceramics with natural bioceramics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084491 ◽

1991 ◽

Vol 49 ◽

pp. 38-39

Author(s):

Shulin Wen ◽

Jingwei Feng ◽

A. Krajewski ◽

A. Ravaglioli

Keyword(s):

Recent Work ◽

Human Body ◽

Human Tissues ◽

Main Constituent ◽

Laboratory Furnace ◽

Chinese Adult ◽

Human Enamel ◽

Biological Compatibility ◽

Synthetic Hydroxyapatite ◽

Synthetic Work

Hydroxyapatite bioceramics has attracted many material scientists as it is the main constituent of the bone and the teeth in human body. The synthesis of the bioceramics has been performed for years. Nowadays, the synthetic work is not only focused on the hydroapatite but also on the fluorapatite and chlorapatite bioceramics since later materials have also biological compatibility with human tissues; and they may also be very promising for clinic purpose. However, in comparison of the synthetic bioceramics with natural one on microstructure, a great differences were observed according to our previous results. We have investigated these differences further in this work since they are very important to appraise the synthetic bioceramics for their clinic application.The synthetic hydroxyapatite and chlorapatite were prepared according to A. Krajewski and A. Ravaglioli and their recent work. The briquettes from different hydroxyapatite or chlorapatite powders were fired in a laboratory furnace at the temperature of 900-1300°C. The samples of human enamel selected for the comparison with synthetic bioceramics were from Chinese adult teeth.

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Study on erythrocytes by SEM photogrammetry (SEMP) technique

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161187 ◽

1990 ◽

Vol 48 (3) ◽

pp. 724-725

Author(s):

Tong Wensheng ◽

Lu Lianhuang ◽

Zhang Zhijun

Keyword(s):

Quantitative Analysis ◽

Cell Membrane ◽

Clinical Diagnosis ◽

Human Body ◽

Blood Cells ◽

Three Dimensional ◽

Normal Person ◽

Blood Disease ◽

Computation Technique ◽

Important Basis

This is a combined study of two diffirent branches, photogrammetry and morphology of blood cells. The three dimensional quantitative analysis of erythrocytes using SEMP technique, electron computation technique and photogrammetry theory has made it possible to push the study of mophology of blood cells from LM, TEM, SEM to a higher stage, that of SEM P. A new path has been broken for deeply study of morphology of blood cells.In medical view, the abnormality of the quality and quantity of erythrocytes is one of the important changes of blood disease. It shows the abnormal blood—making function of the human body. Therefore, the study of the change of shape on erythrocytes is the indispensable and important basis of reference in the clinical diagnosis and research of blood disease.The erythrocytes of one normal person, three PNH Patients and one AA patient were used in this experiment. This research determines the following items: Height;Length of two axes (long and short), ratio; Crevice in depth and width of cell membrane; Circumference of erythrocytes; Isoline map of erythrocytes; Section map of erythrocytes.

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Artificial cells containing sustainable energy conversion engines

Emerging Topics in Life Sciences ◽

10.1042/etls20190103 ◽

2019 ◽

Vol 3 (5) ◽

pp. 573-578 ◽

Cited By ~ 3

Author(s):

Kwanwoo Shin

Keyword(s):

Energy Conversion ◽

Nicotinamide Adenine Dinucleotide ◽

Sustainable Energy ◽

Living Cells ◽

Energy Transduction ◽

Artificial Cells ◽

Energy Conversion Process ◽

Metabolic Reactions ◽

Metabolic Activities ◽

Reduced Nicotinamide Adenine Dinucleotide

Living cells naturally maintain a variety of metabolic reactions via energy conversion mechanisms that are coupled to proton transfer across cell membranes, thereby producing energy-rich compounds. Until now, researchers have been unable to maintain continuous biochemical reactions in artificially engineered cells, mainly due to the lack of mechanisms that generate energy-rich resources, such as adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH). If these metabolic activities in artificial cells are to be sustained, reliable energy transduction strategies must be realized. In this perspective, this article discusses the development of an artificially engineered cell containing a sustainable energy conversion process.

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