Steroid glucuronyltransferases of rat liver. Properties of oestrone and testosterone glucuronyltransferases and the effect of ovariectomy, castration and administration of steroids on the enzymes.
ABSTRACT: 1. Microsomal preparations from rat liver, kidney and intestine were tested for UDP-glucuronyltransferase activity by using oestrone, oestradiol-17 beta, oestriol, testosterone, cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone as substrates. The microsomal preparation from the liver glucuronidated oestrone, oestradiol-17 beta and testosterone. 2. The specific activity of the enzyme was significantly higher in livers from female rats than in those from male rats. 3. Testosterone was actively glucuronidated by both sexes. Cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone were not glucuronidated by any of the three tissues. 4. The non-ionic detergent Lubrol WX activates liver microsomal UDP-glucuronyltransferase 2-3-fold with oestrone and testosterone as substrates. 5. Oestrone glucuronyltransferase was inhibited by oestradiol-17 beta, predominantly competitively and by testosterone non-competitively. Bilirubin was a non-competitive inhibitor of oestrone glucuronidation. p-Nitrophenol had no effect. 6. Oestrone glucuronyltransferase could not be stimulated by either acute or prolonged treatment of animals with phenobarbital, whereas a single dose of 3-methylcholanthrene led to a moderate stimulation. 7. Ovariectomy leads to a 56% decrease in oestrone glucuronyltransferase activity; administration of oestradiol-17 beta induces the enzyme to normal activity after 12 days, and after 15 days the activity is twice the control value. Actinomycin D and cycloheximide block the oestradiol-17 beta-induced increase in enzyme activity. 8. Castration has no effect on the activity of testosterone glucuronyltransferase, nor does administration of testosterone influence enzyme activity. The results provide strong evidence for the existence of multiple steroid glucuronyltransferases in the liver of the rat.
Project description:1. Reconstitution of UDP-glucuronyltransferase preparations with phosphataidylcholine liposomes facilitated the purification of testosterone UDP-glucuronyltransferase. 2. Transferase activity towards testosterone co-purifies with that towards 4-nitrophenol. 3. UDP-glucuronyltransferase activity towards oestrone was separated from that towards testosterone. 4. These results suggest that testosterone and 4-nitrophenol may be glucuronidated by a different form of UDP-glucuronyltransferase from the one glucuronidating oestrone.
Project description:Aim, materials & methods: Urinary cortisol profile has the potential as a diagnostic biomarker. We therefore developed a stable-isotope dilution ultraperformance chromatography multistage MS-based method to quantify cortisol and 16 metabolites in human urines. Results & conclusion: The LOD for cortisol and its metabolites ranges from 0.02 to 5.81 pg/?l urine. The inter- and intraday variations were 3.7-12.9% and 3.5-15.6%, respectively. Among the metabolites analyzed, significant person-to-person heterogeneity was observed, demonstrating the need for comprehensive metabolite profiling in diagnosis. Nevertheless, the glucuronides of dihydrocortisol, dihydrocortisone, tetrahydrocortisol, allo-tetrahydrocortisol and tetrahydrocortisone are the major ones. The sum of the glucuronidated and free forms constitute >93% of the metabolites analyzed, which is termed as total cortisol equivalent. Total cortisol equivalent may serve as a surrogate of cortisol secretion. Clinical trial registration number: NCT02500472.
Project description:The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.
Project description:Detailed studies on the hydrolysis of p-acetylphenyl sulphate and oestrone sulphate by rat liver preparations strongly indicate that arylsulphatase C and oestrogen sulphatase are the same enzyme. Liver is the richest source of both enzymes, which have identical intracellular distributions, being localized mainly in the microsomal fraction. Low oestrogen sulphatase and arylsulphatase C activities were present in foetal liver and these increased at a similar rate after birth. The activities of the enzymes in an ethionine-induced hepatoma were similarly low. Results of heat inactivation, mixed-substrate and competitive-inhibition experiments employing liver microsomal fractions were also consistent with one enzyme being involved. Oestradiol-17beta 3-sulphate was also hydrolysed by microsomal preparations and activity towards both this substrate and oestrone sulphate was inhibited by oestrone and oestradiol-17beta. The physiological significance of this inhibition is discussed.
Project description:We previously showed that in perinatal rhesus monkeys hepatic UDP-glucuronyltransferase activities appear to develop differentially in two clusters, analogous to those of the rat. We demonstrate here that hepatic UDP-glucuronyltransferase activities differ between the rat and the rhesus monkey in their response to glucocorticoids. Treatment of pregnant rhesus monkeys with dexamethasone during late gestation increases UDP-glucuronyltransferase activities towards bilirubin, oestradiol and testosterone in the foetal-liver microsomal fraction to 25, 4 and 4 times their low control values respectively. Analogous dexamethasone treatment in rat fails to increase these activities significantly in the foetal liver. These findings suggest that maternal glucocorticoid therapy in late gestation could greatly increase the newborn primate's capacity to glucuronidate bilirubin.
Project description:The hepatic microsomal enzyme UDP-glucuronyltransferase undergoes a complex developmental pattern in which enzyme activity is first detectable on the 18th day of gestation in rats. Prepubertal activities are similar for males and females. However, postpubertal sexual differentiation of enzyme activity occurs in which male activities are twice those of females. Neonatal administration of testosterone propionate or diethylstilboestrol to intact animals resulted in lowered UDP-glucuronyltransferase activity in liver microsomal fractions of adult male rats, whereas no changes were observed in the adult females and prepubertal male and female animals. Neonatal administration of testosterone propionate and diethylstilboestrol adversely affected male reproductive-tract development as evidenced by decreased weights of testes, seminal vesicles and ventral prostate. Diethylstilboestrol also markedly decreased spermatogenesis. Hypophysectomy of adult male rats resulted in negative modulation of microsomal UDP-glucuronyltransferase and prevented the sexual differentiation of enzyme activity. In contrast hypophysectomy had no effect on female UDP-glucuronyltransferase activity. A pituitary transplant under the kidney capsule was not capable of reversing the enzyme effects of hypophysectomy, therefore suggesting that the male pituitary factor(s) responsible for positive modulation of UDP-glucuronyltransferase might be under hypothalamic control in the form of a releasing factor. Neonatal testosterone propionate and diethylstilboestrol administration apparently interfered with the normal sequence of postpubertal UDP-glucuronyltransferase sexual differentiation.
Project description:A reliable procedure for the assay of liver microsomal 16 alpha-hydroxylation of oestrone 3-sulphate has been developed for the guinea pig. It is based on the rapid, quantitative separation of oestradiol and oestriol by Sephadex LH-20 columns after the chemical reduction and enzymic hydrolysis of the incubation products. Microsomal preparations and incubation conditions that optimized 16 alpha-hydroxylation of oestrone 3-sulphate were employed. Under these circumstances, reduction of the substrate at C-17 and hydrolysis of the sulphate were minimized. Conditions were established that yielded reaction linearity with respect to time and microsomal concentration. This hydroxylation had an absolute requirement for NADPH, which could not be satisfied by NADH. Apparent Km values for oestrone 3-sulphate and NADPH, under the conditions used, were 14 microM and 0.17 mM respectively. 16 alpha-Hydroxylase activity was present in the liver microsomal fraction from heavily pigmented, female English Shorthaired guinea pigs. Much lower activity was detected in mature pigmented males and albino females. No activity could be demonstrated in mature, albino males.
Project description:1. The following compounds were glucuronidated in the presence of UDP-glucuronic acid and a microsomal preparation made from guinea-pig liver: (14)C-labelled 3-O-methyladrenaline, 3-O-methylnoradrenaline, 3-methoxytyramine and 4-hydroxy-3-methoxyphenethanol, as well as unlabelled harmalol and harmol. 2. [(14)C]Homovanillic (4-hydroxy-3-methoxyphenylacetic) acid was not a substrate for the microsomal glucuronyltransferase. 3. The K(m) values for harmalol and harmol were 0.69x10(-4)m and 0.50x10(-4)m respectively. 4. The K(m) values for UDP-glucuronic acid, in the presence of (14)C-labelled 3-O-methylnoradrenaline, harmalol and harmol as aglycones, were 0.57x10(-4)m, 0.44x10(-4)m and 2.20x10(-4)m respectively. 5. Mg(2+) added at 2.5-10mm activated glucuronyltransferase, with harmalol as substrate. Concentrations above 10mm inhibited the enzymic activity. 6. The overall, or net, transglucuronidating activity of microsomal preparations of the liver, with harmalol as substrate, was greatest for guinea pig, and very much lower for rabbit, mouse and rat.
Project description:UDP-glucuronyltransferase activity of neonatal-chick liver or phenobarbital-treated chick-embryo liver catalysed the glucuronidation of 1-naphthol, 4-nitrophenol and 2-aminophenol. Only low transferase activity towards testosterone was detected, and activity towards bilirubin was not detectable. Liver microsomal transferase activity towards the three phenols was increased approx. 20-50-fold by phenobarbital treatment of chick embryos or by transfer of liver cells into tissue culture. A single form of UDP-glucuronyltransferase, which appears to catalyse the glucuronidation of these three phenols, was purified to near homogeneity from phenobarbital-treated chick-embryo liver microsomal fraction for the first time. The use of this purified enzyme as a standard protein facilitated the identification of this protein in chick-embryo liver microsomal fraction. Further, the accumulation of this microsomal protein was observed following phenobarbital treatment of chick embryos and during tissue culture of chick-embryo liver cells. The value of this model system for the study of the induction of UDP-glucuronyltransferase by drugs and hormones is discussed.
Project description:Hepatic UDP-glucuronyltransferase activity was resolved into two fractions, one exhibiting oestrone glucuronyltransferase activity and the other exhibiting p-nitrophenol glucuronyltransferase activity. Hydroxyapatite-column chromatography removed greater than 95% of the phospholipids from both preparations. The partially purified delipidated enzymes were essentially devoid of catalytic activity, but activities were restored by the addition of phospholipids or phosphatidylcholine mixtures containing various saturated and unsaturated fatty acids. Both oestrone and p-nitrophenol glucuronyl-transferase activities were reconstituted to similar degrees with the phosphatidylcholine mixtures. When purified phospholipids were tested, phosphatidylcholine and lysophosphatidylcholine were most effective in restoring activity, whereas phosphatidylethanolamine was the least effective. These results further suggest that oestrone and p-nitrophenol UDP-glucuronyltransferases are dependent on phospholipids for their activity.