UDP-glucuronic Acid Pyrophosphatase Research Articles

Metabolism of the amino acid β-pyrazol-1-ylalanine and its parent base pyrazole

β-Pyrazol-1-yl- dl-alanine, an uncommon amino acid from plants of the Cucurbitaceae, was fed to mice. Although pyrazole is known to affect the liver enzymes UDP-glucose dehydrogenase, UDP-glucoronyl transferase and UDP-glucuronic acid pyrophosphatase, and also depresses their liver glycogen concentrations, β-pyrazol-1-ylalanine had no such effects. β-Pyrazol-1-ylalanine could not be detected in the liver of the experimental animals but was present in the urine. No other change in urinary amino acid content was observed. Studies with [ 14C]-β-pyrazol-1-yl- dl-alanine showed the administered amino acid was excreted over a 4-day period, 93% of the compound supplied was recovered. Similar recoveries were obtained with the l-enantiomer from cucumber seed. The metabolic inertness of β-pyrazol-1-ylalanine was also apparent in experiments involving subcutaneous injection of this compound. Administration of pyrazole confirmed an earlier report of resultant increased activity of liver UDP-glucose dehydrogenase and UDP-glucuronyl transferase, and of the depression of activity of liver UDP-glucuronic acid pyrophosphatase. A concomitant 40% decrease in liver glycogen content was seen. The urine contained a novel metabolite, identified as a peptide conjugate of a pyrazole derivative. Mass spectrometry and p.m.r. spectroscopy indicate that this derivative is 3,4,4-trimethyl-5-pyrazolone. The amino acid constituents are aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine and leucine. The urine of mice receiving pyrazole contained less free glycine and alanine than controls. From the results, it is concluded that pyrazole is not a catabolite of dietary β-pyrazol-1-ylalanine but to the contrary, the amino acid is essentially excreted unchanged. Formation of 3,4,4-trimethyl-5-pyrazolone from pyrazole would imply C-methylation, a process that has not been previously observed in a mammalian detoxication context.

Effect of several xenobiotics on the activities of enzymes affecting ascorbic acid synthesis in rats.

The dietary addition of several xenobiotics, such as PCB, DDT, aminopyrine, chloretone, BHT and BHA, caused significant increases in the ascorbic acid in urine and liver of rats. The administration of all types of xenobiotics used in the present experiments increased the activity of hepatic UDP-glucose dehydrogenase (1.3-2.8-fold), and the administration of PCB, DDT, BHT or BHA significantly increased the activity of hepatic UDP-glucuronyl transferase (2.2-13.1-fold). The activity of beta-glucuronidase was slightly increased with feeding of PCB, DDT, chloretone or aminopyrine. However, the activity of hepatic UDP-glucuronic acid pyrophosphatase, the conversion of D-glucuronic acid or D-glucuronolactone into L-ascorbic acid and the activity of hepatic L-gulonolactone oxidase did not increase with the administration of PCB or DDT. It is suggested that the increases in the activities of UDP-glucose dehydrogenase and UDP-glucuronyl transferase would have a major role in the stimulation of ascorbic acid synthesis in xenobiotic treated rats.

UDP-glucuronosyltransferase activity in rat-and hairless mouse skin-microsomes.

1. Cutaneous UDP-glucuronosyltransferase activity (E.C.2.4.1.17) was demonstrated in rat- and hairless mouse-skin microsomes using 1-naphthol as substrate. 2. Addition of the detergent Brij 35 increased the activity by approximately twofold in both species. 3. Inhibitor studies demonstrated that under the assay conditions used any UDP-glucuronic acid pyrophosphatase or beta-glucuronidase present did not interfere with the conjugation reaction. 4. Substrate inhibition was observed in hairless mouse-skin preparations and biphasic response to increasing naphthol concentration was seen in rat-skin microsomes. 5. The apparent Km values were considerably lower than those reported for liver. The sp. activity (per mg microsomal protein) in unactivated rat-skin microsomes was about 50% of that reported in unactivated rat-liver microsomes. 6. Pretreatment with 3-methylcholanthrene resulted in a small increase in cutaneous UDP-glucuronosyltransferase activities in both species.

Responses of the D-glucuronic acid pathway in rat tissues to treatment with tetrachlorodibenzodioxin.

1. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was administered to rats to study its effects on the enzyme activities of the D-glucuronic acid pathway in the liver, small intestine and kidney. 2. The UDP-glucuronosyl transferase activity of male albino rats given TCDD (80 mug/kg, one dose, i.p.) 6 days before killing was significantly increased in all tissues examined, and UDP-glucuronic acid pyrophosphatase activity was markedly decreased in the liver. D-Glucuronolactone and L-gulonate dehydrogenase activities in the liver and small intestine were slightly decreased after TCDD treatment. 3. The activities of UDP-glucose dehydrogenase and beta-glucuronidase were unchanged. 4. The 24 h urinary excretion of L-ascorbic acid was enhanced 8-fold, although no difference was detected in the excretion of D-glucaric acid between the control and experimental animals. 5. These results suggest an increased capacity for glucuronide conjugation after treatment with TCDD. 6. The lack of increase in the urinary excretion of D-glucaric acid further challenges its use as a reliable indicator of enhanced drug metabolism.

Hepatic hydroxylation and glucuronidation in the rat after subacute pyrazole treatment

Male Wistar rats were given pyrazole subcutaneously at a dose of 100 mg/ kg daily, for 4 days, and the in vitro activities of drug metabolizing enzymes and the concentrations of copper, zinc and magnesium were measured in the liver. During the course of treatment, the animals failed to thrive at the rate of the controls. Although protein content of microsomes was unchanged, the phospholipid content increased. Pyrazole administration significantly decreased the activity of NADPH-cytochrome c reductase, while it did not affect the O-demethylation of p-nitroanisole and the microsomal content of cytochrome P-450. The activities of UDPglucose dehydrogenase, UDPglucuronosyltransferase and l-gulonate dehydrogenase were markedly enhanced. On the contrary, pyrazole administration produced a decrease in the activities of UDPglucuronic acid pyrophosphatase, β-glucuronidase and d-glucuronolactone dehydrogenase. The pentobarbital sleeping-time was prolonged after pyrazole treatment for 4 days but the animals showed a decreased spontaneous locomotor activity already on the third day of treatment. This suggests that a depression of the central nervous system was present, which was due to pyrazole itself. The total hepatic content of copper was significantly higher in pyrazole-treated animals than in the control group. The concentrations of zinc and magnesium were increased in the hepatic cytosolic fraction. These results indicate that pyrazole affects the enzymes involved both in hydroxylation and in glucuronidation reactions. In view of the unspecificity of the compound in affecting various enzyme systems, its use in studies on ethanol metabolism in vivo does not seem warranted.

UDPglucuronic acid pyrophosphatase assay with the aid of alkaline phosphatase.

A simple and sensitive method has been described for the determination of UDPglucuronic acid pyrophosphatase activity. Pyrophosphatase-free alkaline phosphatase preparation is added to the reaction mixture in order to hydrolyze the phosphate esters (UMP and alpha-D-glucuronic acid 1-phosphate) produced by pyrophosphatase. The inorganic phosphate liberated is measured by a modification of Fiske and SubbaRow's method. The phosphatase coupled method is time saving, easy to perform and accurate. It can also be used for pyrophosphatase assays with other nucleotide substrates like UDPglucose, UDP-N-acetylglucosamine, NAD+, NADH, NADP+ and NADPH.

Modifications of drug metabolism by disulfiram and diethyldithiocarbamate II. d-glucuronic acid pathway

Hepatic enzymes connected with the formation and metabolism of free d-glucuronic acid were affected in rats after treatment with disulfiram or diethyldithiocarbamate (300 mg/kg, intragastrically, per day, 4 X). The activities of UDPglucose dehydrogenase, UDPglucuronic acid pyrophosphatase, UDPglucuronosyltransferase and l-gulonate dehydrogenase were enhanced, while those of glucose-6-phosphate dehydrogenase, β-glucuronidase and d-glucuronolactone dehydrogenase were inhibited. These changes were more pronounced with disulfiram than diethyldithiocarbamate. Treatment with phenobarbital (80 mg/kg, i.p., per day, 4 X) enhanced UDP glucuronosyltransferase, but brought about different effects on the other enzymes. Concurrent administration of phenobarbital with disulfiram or diethyldithiocarbamate led to potentiation or antagonism of the primary effects of each compound when given alone. The results suggest that activation of the d-glucuronic acid pathway may proceed in various ways, and that it is not necessarily followed by a simultaneous induction of the microsomal mixed-function oxygenase activity.

The hepatic glucuronate pathway in the rat after treatment with 3,3',5-triiodothyronine and phenobarbital.

Abstract The hepatic D‐glucuronic acid pathway was studied in male rats after administration of 3, 3′, 5‐triidothyronine (20 μg intraperitoneally daily, for three weeks). Some of the key enzymes of the pathway showed decreased activities during hyperthyroidism. D‐glucuronolactone dehydrogenase, UDPglucuronosyltransferase and UDPglucuronic acid pyrophosphatase were not affected, whilst the activity of β‐glucuronidase was significantly increased. D‐Glucaric acid excretion in urine was enhanced and that of L‐ascorbic acid was decreased. Phenobarbital administered to hyperthyroid and euthyroid animals (80 mg/kg intraperitoneally, daily, for 3 days) did not markedly affect the differences in the enzyme activities between the two groups. UDP‐Glucuronosyltransferase and D‐glucuronolactone dehydrogenase activities were induced, and in the case of UDPglucuronosyltransferase the induction was more pronounced during hyperthyroidism. D‐Glucaric acid excretion in the urine was increased by phenobarbital treatment both in hyperthyroid and normal animals. L‐Ascorbic acid excretion was also increased, but the increase was greater in euthyroid than in hyperthyroid animals. The results suggest that hyperthyroidism favours the metabolism of free D‐glucuronic acid towards the formation of D‐glucaric acid. Treatment with phenobarbital, however, also enhances to some extent the biosynthesis of L‐ascorbic acid.

Comparison of the effect of phenobarbital on the D-glucuronic acid pathway in euthyroid and hypothyroid rats.

Abstract: Hypothyroidism was produced in male rats by thiouracil (50 mg/kg daily, i. g., for 1 month), in order to study the thyroid control of the D‐glucuronic acid pathway in the liver. The activities of UDPglucose dehydrogenase, UDPglucuronosyltransferase, UDPglucuronic acid pyrophosphatase, D‐glucuronolactone dehydrogenase and 3‐hydroxyacid dehydrogenase were significantly enhanced by hypothyroidism. In contrast, β‐glucuronidase activity was decreased while that of L‐gulonolactone dehydrogenase was not affected. The excretion of L‐ascorbic acid in the urine was unchanged and that of D‐glucaric acid was decreased. Phenobarbital administration (80 mg/kg daily, intraperitoneally, for 3 days) induced UDPglucuronosyltransferase and D‐glucuronolactone dehydrogenase activities both in euthyroid and hypothyroid animals. The excretion of L‐ascorbic acid in the urine failed to rise after phenobarbital treatment in the hypothyroid state, although it increased in animals with normal thyroid function. D‐Glucaric acid excretion in the urine was increased by phenobarbital to the same extent in hypothyroid and euthyroid animals. The results suggest that hypothyroidism accelerates the flow through the D‐glucuronic acid pathway and perhaps that further metabolism of free D‐glucuronic acid takes place mainly via the L‐xylulose route, due to the highly increased activity of 3‐hydroxyacid dehydrogenase. Phenobarbital administration mainly brings about the same effects on both euthyroid and hypothyroid animals. The exception is the lack of increase in the urinary excretion of L‐ascorbic acid during hypothyroidism.

Hepatic udpglucuronosyltransferase: Partial purification after 3-methylcholanthrene pretreatment of the rats

1. 1. Rats were pretreated with 3-methylcholanthrene in order to increase the activity of hepatic UDPglucuronosyltransferase (g9-nitrophenol). UDPglucuronosyltransferase was solubilized by trypsin digestion and digitonin treatment. Seventy to ninety per cent of the activity was recovered in the soluble fraction after centrifugation at 105,000g for 60 minutes. 2. 2. The enzyme was further purified by Sepharose 4 B gel filtration, after which the increase in the specific activity of the pooled preparation was more than 100-fold. 3. 3. The gel filtrated preparation was rather stable, maintaining about 70% of its activity after 6 days storage at 0–4°C. 4. 4. UDPGlucuronic acid pyrophosphatase activity was found to be about one-hundredth of that in native rat liver microsomes. 5. 5. The phospholipid peak was located in the middle of fractions having UDPglucuronosyltransferase activity.

The effect of steroids and nucleotidesoon solubilized bilirubin uridine diphosphate-glucuronyltransferase.

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis-Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The K(m) for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca(2+) were inhibitory in the absence of Mg(2+) but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3beta-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.

Alterations in tissue levels of uridine diphosphate glucose dehydrogenase, uridine diphosphate glucuronic acid pyrophosphatase and glucuronyl transferase induced by substances influencing the production of ascorbic acid

Chemical agents known to enhance the production of l-ascorbic acid in animals were studied in vivo to determine their effects on tissue levels of three enzymes which may be concerned with the biosynthesis of the vitamin. The agents were found to have differential effects on the enzymes tested. Barbital and Chloretone, but not other enhancing agents, increase the activity of UDPG dehydrogenase in liver extracts. This alternation in activity is prevented by ethionine and by hypophysectomy. The guinea-pig enzyme was similarly affected by the two drugs, although ascorbic acid is not produced. Carcinogenic hydrocarbons and aminopyrine increase the activity of glucuronyl transferase in liver. The hepatic level of UDP-glucuronic acid pyrophosphatase, on the other hand, does not respond appreciably to the enhancing agents, although ethionine and barbital administered together elevate this enzyme in liver without stimulating ascorbic acid excretion. In vitro, barbiturates have an inhibitory action on the pyrophosphatase. The alterations in enzyme activities are discussed in relation to the possibility that ascorbic acid is synthesized via a pathway involving glucuronyl transferase rather than UDP-glucuronic acid pyrophosphatase.