C-21- and 6-hydroxylation of progesterone by rabbit liver subcellular fractions

1977 ◽  
Vol 55 (6) ◽  
pp. 602-608 ◽  
Author(s):  
Arun C. Dey ◽  
Ian R. Senciall
Keyword(s):  
Microsomal Fraction ◽  
Liver Microsomes ◽  
Subcellular Fractions ◽  
Rabbit Liver ◽  
Microsomal Metabolism ◽  
Maximal Yield ◽  
First Time ◽  
Hydroxy Metabolites

In vitro C-21-hydroxylation of [3H]progesterone (P) has been demonstrated for the first time with rabbit liver microsomes and mitochondria. Deoxycorticosterone (DOC) was rigorously characterized as a metabolite of both mitochondrial and microsomal metabolism, whereas 6α-hydroxy DOC and 6α-hydroxy P were only identified as microsomal metabolites. 6β-Hydroxy metabolites were also detected but were of less quantitative significance. Formation of 6α-hydroxy P and 6α-hydroxy DOC increased steadily between 5 and 120 min of incubation with the microsomal fraction, whereas DOC increased up to 30 min of incubation and then declined. Maximal yield of DOC was 25.9 and 22.5 pmol/mg protein with the mitochondrial and microsomal fractions, respectively.

scholarly journals Differential effect of L-thyroxine on phospholipid biosynthesis in mitochondria and microsomal fraction

1980 ◽  
Vol 186 (1) ◽  
pp. 127-133 ◽  
Author(s):  
J M Pasquini ◽  
I Faryna de Raveglia ◽  
N Capitman ◽  
E F Soto
Keyword(s):  
Differential Effect ◽  
Microsomal Fraction ◽  
Liver Mitochondria ◽  
Subcellular Fractions ◽  
Control Groups ◽  
Adult Rats ◽  
And Control ◽  
Isolated Liver ◽  
Normal Controls

1. The action of L-thyroxine on the incorporation of radioactive choline or CDP-choline into phosphatidylcholine in vitro was explored in liver and brain microsomal fraction and mitochondria obtained from young adult rats. 2. In liver mitochondria isolated from animals treated with L-thyroxine (40 mg/kg body wt. during 6 days), the incorporation of both radioactive precursors into phosphatidylcholine was significantly decreased compared with normal controls, whereas in the total homogenate and in the microsomal fraction the incorporation was similar in the experimental and control groups. In subcellular fractions isolated from brain, the incorporation of precursors was similar in L-thyroxine-treated and normal animals. 3. Liver mitochondria isolated from normal animals incubated in vitro with CDP-choline, in the presence of different concentrations of L-thyroxine, showed also a marked decrease in the incorporation of label into phosphatidylcholine, whereas no significant changes were found in the total homogenate and in the microsomal fraction compared with control experiments. 4. The differential effect of L-thyroxine on the incorporation of radioactive precursors into phosphatidylcholine of isolated liver subcellular fractions gives further support to the hypothesis that liver mitochondria can independently synthesize part of their own phospholipids. 5. Possible mechanisms of the action of the hormone at the mitochondrial level are discussed.

scholarly journals The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary

1971 ◽  
Vol 123 (2) ◽  
pp. 143-152 ◽  
Author(s):  
A. P. F. Flint ◽  
D. T. Armstrong
Keyword(s):  
Microsomal Fraction ◽  
Specific Radioactivity ◽  
Subcellular Fractions ◽  
Side Chain ◽  
Electron Microscopic ◽  
Side Chain Cleavage ◽  
Chain Cleavage ◽  
Cleavage Enzyme ◽  
Rat Ovary

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-14C]acetate in vitro for various times. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [14C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./μmol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./μmol. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20α-hydroxypregn-4-en-3-one was 6150d.p.m./μmol. 3. By using glutamate dehydrogenase and cytochrome (a+a3) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-14C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [14C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.

scholarly journals The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro

1977 ◽  
Vol 166 (3) ◽  
pp. 429-435 ◽  
Author(s):  
G. Subba Rao ◽  
Leonard N. Norcia ◽  
Joanne Pieringer ◽  
Ronald A. Pieringer
Keyword(s):  
Enzyme Activity ◽  
Rat Brain ◽  
Enzyme Preparation ◽  
Microsomal Fraction ◽  
Inhibitory Effect ◽  
Subcellular Fractions ◽  
Triton X ◽  
Derivatives Of

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3′-phosphate 5′-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3′-phosphate 5′-sulphatophosphate–galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3′-phosphate 5′-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg2+ strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent Km for galactosyldiacylglycerol is 200μm, and that for cerebroside is 45μm. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.

scholarly journals In vitro formation of metabolic-intermediate cytochrome P450 complexes in rabbit liver microsomes by tiamulin and various macrolides

2003 ◽  
Vol 34 (4) ◽  
pp. 405-411 ◽  
Author(s):  
Monica Carletti ◽  
Federica Gusson ◽  
Anna Zaghini ◽  
Mauro Dacasto ◽  
Luigi Marvasi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Liver Microsomes ◽  
Rabbit Liver ◽  
Metabolic Intermediate

The characteristics of the microsomal hydroxylation of tolbutamide

1991 ◽  
Vol 69 (3) ◽  
pp. 400-405 ◽  
Author(s):  
Pierre M. Bélanger ◽  
Serge St-Hilaire
Keyword(s):  
Carbon Monoxide ◽  
Liver Microsomes ◽  
Hepatic Metabolism ◽  
Species Variation ◽  
Rat Liver Microsomes ◽  
Hydroxylase Activity ◽  
Microsomal Metabolism ◽  
Microsomal Hydroxylation ◽  
Cytochrome P 450

The in vitro metabolism of tolbutamide to the hydroxymethyl derivative was studied using hepatic microsomal homogenates. The hydroxymethyl metabolite was quantitated by HPLC. The hepatic microsomal hydroxylase was completely inhibited by carbon monoxide and was NADPH dependent. Metyrapone, α-naphthoflavone, phenelzine, mercuric chloride, and nitrogen significantly inhibited the reaction indicating the involvement of the cytochrome P-450 monooxygenase. Species variation showed that the order of hepatic microsomal activity was rat > rabbit >> guinea pig >> mouse and hamster. The reaction increased with time up to 40 min and followed Michaelis–Menten kinetics in rat liver microsomes with apparent Km and Vmax values of 224.4 μM and 359.9 pmol∙mg−1∙min−1, respectively. The reaction was induced by phenobarbital but was depressed after pretreatment with 3-methylcholanthrene and isosafrole. However, expression of the hydroxylase activity per nanomoles of cytochrome P-450 showed that the activity was much higher in liver microsomes of isosafrole pretreated rats. These results indicate the involvement of different isozymes of cytochrome P-450 in the microsomal hydroxylation of tolbutamide.Key words: tolbutamide metabolism, tolbutamide hydroxylation, microsomal hydroxylation, microsomal metabolism of tolbutamide, hepatic metabolism of tolbutamide.

scholarly journals Species Differences in Microsomal Metabolism of Xanthine-Derived A1 Adenosine Receptor Ligands

2021 ◽  
Vol 14 (3) ◽  
pp. 277
Author(s):  
Daniela Schneider ◽  
Dirk Bier ◽  
Marcus Holschbach ◽  
Andreas Bauer ◽  
Bernd Neumaier
Keyword(s):  
Adenosine Receptor ◽  
Molecular Mechanisms ◽  
Animal Species ◽  
Species Differences ◽  
Liver Microsomes ◽  
Suitable Model ◽  
Microsomal Metabolism ◽  
A1 Adenosine Receptor ◽  
Metabolite Profiles

Tracer development for positron emission tomography (PET) requires thorough evaluation of pharmacokinetics, metabolism, and dosimetry of candidate radioligands in preclinical animal studies. Since variations in pharmacokinetics and metabolism of a compound occur in different species, careful selection of a suitable model species is mandatory to obtain valid data. This study focuses on species differences in the in vitro metabolism of three xanthine-derived ligands for the A1 adenosine receptor (A1AR), which, in their 18F-labeled form, can be used to image A1AR via PET. In vitro intrinsic clearance and metabolite profiles of 8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX), an established A1AR-ligand, and two novel analogs, 8-cyclobutyl-3-(3-fluoropropyl)-1-propylxanthine (CBX) and 3-(3-fluoropropyl)-8-(1-methylcyclobutyl)-1-propylxanthine (MCBX), were determined in liver microsomes from humans and preclinical animal species. Molecular mechanisms leading to significant differences between human and animal metabolite profiles were also examined. The results revealed significant species differences regarding qualitative and quantitative aspects of microsomal metabolism. None of the tested animal species fully matched human microsomal metabolism of the three A1AR ligands. In conclusion, preclinical evaluation of xanthine-derived A1AR ligands should employ at least two animal species, preferably rodent and dog, to predict in vivo behavior in humans. Surprisingly, rhesus macaques appear unsuitable due to large differences in metabolic activity towards the test compounds.

scholarly journals Screening and Identification of the Main Metabolites of Schisantherin a In Vivo and In Vitro by Using UHPLC-Q-TOF-MS/MS

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 258 ◽  
Author(s):  
Wei Feng ◽  
Ling-Yu Zhou ◽  
Rui-Feng Mu ◽  
Le Gao ◽  
Bing-Yuan Xu ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
High Performance ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Sprague Dawley ◽  
First Time ◽  
Schisantherin A ◽  
Tof Ms

Schisantherin A is an active ingredient originating from Schisandra chinensis (Turcz.) which has hepatoprotective and anti-oxidation activities. In this study, in vitro metabolisms investigated on rat liver microsomes (RLMs) and in vivo metabolisms explored on male Sprague Dawley rats of Schisantherin A were tested, respectively. The metabolites of Schisantherin A were identified using ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). Based on the method, 60 metabolites were successfully identified and structurally characterized including 48 phase-I and 12 phase-II metabolites. Among the metabolites, 45 metabolites were reported for the first time. Moreover, 56 and eight metabolites were detected in urine and bile and 19 metabolites were identified in rats’ plasma. It demonstrated that hepatic and extra-hepatic metabolic pathways were both involved in Schisantherin A biotransformation in rats. Five in vitro metabolites were structurally characterized for the first time. The results indicated that the metabolic pathways mainly include oxidation, reduction, methylation, and conjugation with glucuronide, taurine, glucose, and glutathione groups. This study provides a practical strategy for rapidly screening and identifying metabolites, and the results provide basic data for future pharmacological and toxicology studies of Schisantherin A and other lignin ingredients.

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