Bilirubin UDP-glucuronyltransferase Activity Research Articles

Antipyrine clearance, aminopyrine N-demethylase, and bilirubin UDP-glucuronyl transferase activity in patients with amoebic liver abscess.

Three indices of drug metabolism, antipyrine clearance in vivo, and aminopyrine N-demethylase and bilirubin UDP-glucuronyl transferase activity in liver biopsies, were studied in fifteen patients with amoebic liver abscess (with and without jaundice). The mean (+/- S.E.) antipyrine half-life in patients with jaundice was 21.64 (+/- 1.52) h and in patients without jaundice was 19.36 (+/- 0.93) h. It was significantly prolonged in both groups of patients as compared to controls (11.63 +/- 0.86 h). It showed a good correlation with serum albumin (p less than 0.01), prothrombin time index (p less than 0.01), and aminopyrine N-demethylase (p less than 0.05). Aminopyrine N-demethylase was found to be decreased in patients without jaundice but no significant change could be observed in patients with jaundice. Bilirubin UDP-glucuronyl transferase showed no significant change in either of the groups.

1100 THE ARRHENIUS PLOT OF HEPATIC MICROSOMAL GLUCOSE-6-PHOSPHATASE ACTIVITY OF THE CONGENITALLY JAUNDICED (GUNN) RAT

The failure of the Gunn rat to conjugate bilirubin results from either defective (or absent) bilirubin UDP-glucuronyl transferase or from a defect in the membrane environment of that enzyme. The latter possibility was investigated by constructing Arrhenius plots from 38-8°C of microsomal glucose-6-phosphatase activity of Gunn (jj,n=6), Wistar (JJ,n=9) and heterozygote (Jj,n=8) rats. This enzyme was studied because the Gunn rat is not defective in glucose-6-phosphatase, and this enzyme is tightly bound to the microsome. The plots of jj, Jj and JJ were identical in the following ways: a) the specific activity at 37°C was 2.51 μmol hydrolyzed/mg protein/10 min indicating no jj defect in enzyme activity; b) there was a change in slope at 35°C probably indicating a change in protein structure; c) the energy of activation was 12800 cal/mol. However, a clear difference was observed in the following way: JJ demonstrated a discontinuous plot indicating a lipid phase change at 11°C, but the plot of jj was a continuous straight line to 8°C. Jj exhibited an intermediate discontinuity at 8°C. This shift in the phase change to colder temperatures indicates a difference in membrane fluidity among groups. The proposed explanation for the deficient activity of bilirubin UDP-glucuronyl transferase in the Gunn rat is a difference in membrane fluidity which results in an anomalous environment for the enzyme.

Influence of drugs on the bilirubin UDP-glucuronyltransferase activity and the concentration of Y and Z acceptor proteins in rat liver.

The aim of this paper is to report on the influence of phenobarbital, the combination of phenobarbital/nikethamide, the racemate and (+) isomer of methylphenobarbital on the activity of bilirubin UDP-glucuronyltransferase and the concentration of intrahepatic Y and Z protein in rats. The activity of bilirubin DP-glucuronyltransferase increased after the treatment in all groups. Pretreatment with phenobarbital/nikethamide resulted in the highest enzyme activity, using wet weight as reference. (+) Methylphenobarbital isomer as well as the racemate showed effects similar to those of phenobarbital. The concentration of hepatic Y acceptor protein increased in all pretreated groups by about 30%. The drugs did not increase the concentration of Z protein. The (+) isomer of methylphenobarbital seems better suited as an inductive drug in the prophylaxis of hyperbilirubinemia and in the treatment of nonconjugated hyperbilirubinemia than phenobarbital or phenobarbital/nikethamide since it has no sedative effect.

Glucuronidation of bilirubin and the occurrence of pigment gallstones in patients with chronic haemolytic diseases

A group of thirty-seven patients with increased haem catabolism has been studied to gain insight in their bilirubin conjugating capacity. Bilirubin UDP-glucuronyl transferase activity (GlcATa) in the liver and bilirubin monoconjugates in bile were measured and the hepatic bilirubin clearance was calculated from the radio-chromium-survival data. In the present group, 41% of the patients clearly had a deficiency in bilirubin conjugation similar to what is classically found in Gilbert's syndrome. The association may facilitate detection of these patients as serum bilirubin levels were higher (65.8 microM +/- 19) (m +/- 1 SD) in the fifteen patients with associated Gilbert's syndrome versus thirteen having only haemolysis (43.6 microM +/- 15). A fair correlation was found between the percentage of monoconjugates in bile and the GlcATa levels in the liver as well as with the calculated hepatic bilirubin clearance, although some discrepancies exist. Using these determinations, a clearcut separation from normal values was not obtained, suggesting at least in the present group of patients that Gilbert' syndrome represents only one end of a continuum of bilirubin conjugation rates and not a separate entity. Pigment stones in the gall-bladder were documented in 51% of the patients and usually at an early age. There was no relationship towards sex, serum bilirubin, GlcATa in liver, total bilirubin or monoconjugates in bile. Age played some role as well as the type of haemolysis as all patients with congenital dyserythropoiesis (n = 4) or acquired haemolysis (n = 3) had lithiasis. Moderate chronic cholecystitis was present, whereas an accumulation of iron and bile pigment was evident in the liver.

Stimulation by secretin of bilirubin UDP-glycosyltransferase activities and of cytochrome P-450 concentration in rat liver.

The activity of bilirubin UDP-glucuronyltransferase in liver tissue was increased 1.5-fold after 90 min of secretion administration (4 i.u./h per kg body wt.) in anaesthetized Wistar rats biopsied half-hourly over a period of 2 h. In unanaesthetized R/A Wistar rats, activities of liver enzymes were assayed after administration secretin for 1 h. Bilirubin UDP-glycosyltransferase activities and cytochrome P-450 concentration were increased, but p-nitrophenol UDP-glucuronyltransferase and UDP-glucose dehydrogenase activities remained unchanged.

Chronic persistent hepatitis and unconjugated hyperbilirubinemia

The authors studied 12 patients with chronic persistent hepatitis and persistent or intermittent mild unconjugated hyperbilirubinemia. Maximum serum total bilirubin concentration ranged from 2.1 to 3.6 mg/dl. Hemolysis was not evident. Hepatic bilirubin UDP-glucuronyltransferase activity assayed in each patient ranged from 0.16 to 0.39 U (mean ± SEM = 0.27 ± 0.02) compared to 0.68–1.99 (1.35 ± 0.08) in 23 normals, 0.78–2.28 (1.41 ± 0.05) in 53 patients with acute hepatitis, 0.34–1.74 (0.81 ± 0.09) in 16 patients with anicteric chronic persistent hepatitis, and 0–0.62 (0.24 ± 0.03) in 33 patients with Gilbert's syndrome. The mean UDP-glucuronyltransferase activity was significantly lower in anicteric chronic persistent hepatitis compared to normals, but higher than in Gilbert's syndrome. The incidence of unconjugated hyperbilirubinemia among first degree relatives was 0:32 in icteric chronic persistent hepatitis compared to 24:85 (28%) in Gilbert's syndrome. These results show that the likely cause for the unconjugated hyperbilirubinemia associated with chronic persistent hepatitis is an acquired depression of hepatic bilirubin UDP-glucuronyltransferase activity. The data suggest that the enzyme defect is related to chronic persistent hepatitis.

SERUM INHIBITORY ACTIVITY TO GLUCURONYL TRANSFERASE IN PYLORIC STENOSIS

The unconjugated hyperbilirubinemia noted in infants with pyloric stenosis (PS) Is believed secondary to a reduction in hepatic bilirubin UDP-glucuronyl transferase (GT) activity. In order to determine if a circulating inhibitor to GT is present and related to the reduced activity and the jaundice, 60 infants with PS were studied immediately prior to surgery. Bilirubins were determined in all infants and 42 sera, 43 gastric aspirate specimens and 29 urine samples were assayed for a possible inhibitor of GT. The mean total serum bilirubin concentration was 1.6mg/dl and the unconjugated fraction was 1.2mg/dl with 35 patients having values in excess of 1.1mg/dl. The 72 urine and gastric aspirate specimens revealed no inhibitory activity while 28 of the 42 sera demonstrated significant (> 20%) inhibitory activity with a mean of 39% inhibition of GT using a standardized in vitro assay. 12 age matched healthy infants served as controls and demonstrated no serum inhibitory activity. Of the 28 infants with significant serum inhibition only 10 had elevated serum bilirubin concentrations while 14 patients had hyperbilirubinemia without serum inhibitory activity present. These data confirm the frequent appearance of jaundice, establish the presence of significant serum inhibitory activity against GT but show no relationship between a circulating inhibitor of GT and hyper-bilirubinemia in infants with PS.

Perinatal Development of Bilirubin UDP-Glycosyltransferase Activities in Rat Liver

Bilirubin UDP-glucuronyltransferase and UDP-xylosyltransferase activity could already be demonstrated in rat liver from day 19 of fetal life onwards (4 days before birth). Bilirubin-glucuronide was present in hepatocytes of 21-day-old fetal liver strongly suggesting that the enzyme detected in vitro was really active in vivo. This further supports the theory that secretion from the cell is also immature. The known deficiency of UDP-glucose dehydrogenase in fetal and neonatal rat liver could lead to decreased intracellular concentrations of UDP-glucuronic acid and possibly to increased concentrations of UDP-glucose. However, no glucosides were present in the fetal hepatocytes.

Bilirubin metabolism in the spiny dogfish, Squalus Acanthias, and the small skate, Raja Erinacea

1. 1. The main bilirubin conjugate in bile of spiny dogfish ( Squalus Acanthias) and small skate ( Raja Erinacea) is bilirubin monoglucuronide. 2. 2. Microsomal preparations from dogfish and small skate liver have similar bilirubin UDPglucuronyltransferase (UDPGT) activity and catalyze the conjugation of bilirubin with glucose from UDPglucose. 3. 3. The activity of bilirubin glucosidation (UDPG1T) was 0.5 times UDPGT activity in dogfish and 0.15 times in skate liver microsomes. 4. 4. Sodium cholate increased UDPGT and UDPG1T activities in dogfish and skate liver microsomal preparations only minimally, but the detergent markedly increased thermolability of UDPGT in skate liver microsomes.

Chronic Hyperbilirubinemia Associated with Hepatic Uptake and Storage Impairment: A new syndrome resembling that of the mutant Southdown sheep

A 19-year-old female complained of moderate jaundice since early childhood, without any other clinical abnormality. Apart from the hyperbilirubinemia (which comprised around 50% of conjugated bilirubin), usual hepatic function tests were normal. Oral cholecystogram showed a delayed opacification of a normal gallbladder. Liver histology and hepatic bilirubin UDP-glucuronyltransferase activity were normal. The plasma disappearance rates of bromosulfophthalein, dibromosulfopthalein, rose bengal, and indocyanine green were dramatically reduced. There was a marked impairment of the relative storage capacities of bromosulfophthalein and dibromosulfophthalein; this reduction was associated with, and thought to be the cause of, some decrease of the biliary transport maximum. This new form of chronic benign hyperbilirubinemia seems therefore primarily due to a defect of hepatic uptake and storage, and closely resembles the disease observed in the mutant Southdown sheep. Since it is not yet possible to quantify the Y- and Z-proteins from a needle biopsy of human liver, their role in the pathogenesis of the present disease remains speculative.

Bilirubin UDP-glucuronyltransferase activity of wistar rat kidney.

Wistar rat kidneys have been shown to possess a bilirubin glucuronyltransferase (BGT) activity capable of conjugating about 3/5 of the total pool of unconjugated bilirubin within 48 h of being grafted to Gunn rat hosts. Bilirubin conjugated by the kidney is taken up by the liver and excreted in the bile. Except when the bile duct is ligated, no conjugated bilirubin appears in the plasma or urine. Renal BGT activity is about 1/20th of the hepatic activity on a weight basis in Wistar rats. The Gunn rat's hyperbilirubinemia probably causes an induction of the renal enzyme since its activity doubles in 48 h.

On the mechanism of polychlorinated biphenyl-induced hypobilirubinaemia

Treatment of normal rats with a polychlorinated biphenyl mixture or DDT increased liver microsomal protein concentration. Hepatic microsomal bilirubin UDP-glucuronyltransferase activity was elevated in the DDT-treated, but not in the polychlorinated biphenyl-treated rats. In contrast polychlorinated biphenyl, but not DDT, reduced serum bilirubin concentrations in jaundiced Gunn rats, as did sulphadiazine; sulphonamides compete with bilirubin for binding sites on plasma albumin. Polychlorinated biphenyls appear to lower serum bilirubin concentration by reducing the binding of bilirubin to plasma protein.

Unconjugated hyperbilirubinaemia in achalasia.

Moderate unconjugated hyperbilirubinaemia decreasing after pneumatic dilatation of the gastrooesophageal sphincter, so permitting a normal amount of food to be taken was observed in two patients with achalasia. Liver biopsy was performed, and hepatic digitonin-activated bilirubin UDP-glucuronyltransferase activity was decreased in both, as is usually found in patients with Gilbert's syndrome. In the patients examined the slight and variable hyperbilirubinaemia associated with Gilbert's syndrome seemed thus to have been aggravated because of the decreased food intake due to achalasia. This situation may be compared to the jaundice sometimes found in neonates with pyloric stenosis or other types of obstruction of the upper gastrointestinal tract. Observations in rats and man favour a complex mechanism for fasting-induced hyperbilirubinaemia.

Effect of various steroids on the hepatic glucuronidation and biliary excretion of bilirubin.

In rats, treatment with spironolactone or pregnenolone-16α-carbonitrile (PCN) accelerates bile flow and enhances hepatic microsomal bilirubin–UDP glucuronyltransferase activity (EC 2.4.1.17) and the biliary transport maximum (Tm) of bilirubin. The distribution of ethylanthranilate azo-pigments in fractions separated by thin-layer chromatography also indicates increased glucuronidation of bilirubin. These findings explain the mechanism of enhanced serum bilirubin disappearance in rats treated with the steroids.

The formation of bilirubin mono- and diglucuronide by rat liver microsomal fractions

The product of the in vitro reaction catalyzed by bilirubin UDPglucuronyl-transferase has been studied with an improved chromatographic procedure for the separation and measurement of the diazo pigments. The source of enzyme activity was total microsomes and the smooth and rough microsomal subfractions after dialysis against EDTA. Biliburin monoglucuronide was the only conjugate formed by smooth microsomes while total and rough microsomes formed bilirubin mono and diglucuronide. These findings are compatible with the presence in rough microsomes of a factor(s), not present in smooth microsomes, necessary for the formation of bilirubin diglucuronide. The activity of bilirubin UDPglucuronyltransferase in rough microsomes was higher than in the smooth fraction.

Augmentation of bilirubin UDP glucuronyltransferase activity in rat liver homogenates by glutethimide. 1.

This study demonstrates that glutethimide is as effective as phenobarbital in increasing the specific activity of hepatic bilirubin UDP glucuronyltransferase activity in Sprague–Dawley rats. The data also indicate that the effect is dose-dependent and can be elicited by as little as 4 days of therapy. Both glutethimide and phenobarbital increase enzyme activity to normal levels in heterozygous Gunn rats, but fail to stimulate any activity in homozygous Gunn rats, who presumably lack the genetic capacity to synthesize this enzyme.

The effect of growth hormone, thyroxine, and testosterone on the ontogeny of bilirubin UDPglucuronyltransferase in mouse liver

The activity of bilirubin UDPglucuronyltransferase is low in the livers of newborn mice. Within a week after birth the activity of the enzyme rises to the adult level in mice which are dwarfs because of a deficiency of growth hormone and a partial deficiency of thyroid stimulating hormone, but surpasses the adult value by about 60% in normal mice. Neither growth hormone nor thyroxine is effective in elevating enzyme activity in the dwarfs. Within two weeks after birth the activity of the enzyme in normal mice declines to that in the dwarfs. At maturity a marked decrease in bilirubin UDPglucuronyltransferase activity occurs in normal males. Testosterone appears to be responsible for this decrease.

Bilirubin excretion in rats with normal and impaired bilirubin conjugation: effect of phenobarbital.

The effect of phenobarbital on bilirubin excretion was studied in rats with different capacities for bilirubin conjugation. Drug treatment induced substantial increases in bilirubin UDP-glucuronyl transferase activity in the liver of both normal and heterozygous Gunn rats, but not homozygous Gunn rats in which enzyme activity is completely absent. However, enhancement of bilirubin excretion in vivo was observed only in heterozygous Gunn rats. In these animals the maximum capacity to excrete bilirubin into bile (T(max)), like the activity of the conjugating enzyme, was half normal; phenobarbital caused an increase in T(max) to levels characteristic of normal animals, with a twofold rise in the excretion of conjugated pigment. This appeared to be largely unrelated to enhancement of bile flow, and there was no stimulation of alternate pathways of bilirubin excretion. Conjugated bilirubin was consistently recovered from the plasma and urine of both untreated normal and heterozygous Gunn rats infused with unconjugated pigment. The quantities thus recovered comprised a similar fraction of the total pigment conjugated in both types of animal. Moreover, there were linear correlations between T(max) and both the rate of bile flow and the activity of the conjugating enzyme over the range of values represented by control rats of both types. These findings suggest that the process by which conjugated bilirubin is secreted into the bile is closely related to conjugation and limits the final excretory rate at different levels of pigment excretion. The phenobarbital effect uniquely observed in heterozygous Gunn rats appears to be mediated primarily by enhancement of the limited capacity for bilirubin conjugation with an associated rise in functional secretory capacity.

Bilirubin Uridine Diphospho-glucuronyltransferase in Rat Liver Microsomes: Genetic Variation and Maturation

Extract: Optimal conditions for the in vitro assay of bilirubin uridine diphospho- (UDP) glu-curonyltransferase activity in rat liver microsomes are described. Solvent partitioning was used to separate the conjugated from nonconjugated bilirubin, thus avoiding dependency on the rate of coupling with diazotized sulfanilic acid for the distinction between bilirubin and its conjugated form. The inclusion of uridine diphospho-N-ace-tylglucosamine (UDPNAG) in the reaction mixture permitted the rate of conjugation of bilirubin by fresh rat liver homogenates and microsomes to occur at greater saturation of the available enzyme with the substrates bilirubin and UDP-glucuronic acid. Liver microsomes, isolated in 0.15 M KC1, increased their activity for bilirubin conjugation and decreased their dependency on UDPNAG during the first 10 days of storage at — 15°. Chromatographic separation of the azo pigments of the conjugated bilirubin gave evidence to suggest that bilirubin monoglucuronide was the initial product and bilirubin diglucuronide appeared in increasing amounts in more prolonged incubations. These results suggested that bilirubin monoglucuronide can be intermediate to the formation of bilirubin diglucuronide. Bilirubin UDP-glucuronyltrans-ferase activity in hepatic microsomes of adult homozygous Gunn rats was not demonstrable. In microsomes of heterozygous Gunn rats and normal Wistar and Sprague-Dawley rats bilirubin UDP-glucuronyltransferase activity was found to be 31.0 and 58.0 μg bilirubin conjugated/mg microsomal N/30 min, respectively. Measurements in developing rats indicated that the maturation in enzyme activity occurred by at least two distinct means: increase of specific activity of the microsomes, and an increase in the content of microsomes per gram of liver (Table IV). Speculation: A method for quantitative measurement of bilirubin UDP-glucuronyltransferase activity applicable to samples obtained by needle biopsy has been needed. The method described in this report meets this need and may permit more precise differential diagnosis of retention jaundice of infancy and childhood and especially the “physiologic” jaundice of the newborn.

Glucuronidation of bilirubin and the occurrence of pigment gallstones in patients with chronic haemolytic diseases

Abstract. A group of thirty-seven patients with increased haem catabolism has been studied to gain insight in their bilirubin conjugating capacity. Bilirubin UDP-glucuronyl transferase activity (GlcATa) in the liver and bilirubin monoconjugates in bile were measured and the hepatic bilirubin clearance was calculated from the radio-chromium-survival data. In the present group, 41% of the patients clearly had a deficiency in bilirubin conjugation similar to what is classically found in Gilbert's syndrome. The association may facilitate detection of these patients as serum bilirubin levels were higher (65-8 μM ± 19) (m ± 1 SD) in the fifteen patients with associated Gilbert's syndrome versus thirteen having only haemolysis (43-6 μ± 15). A fair correlation was found between the percentage of monoconjugates in bile and the GlcATa levels in the liver as well as with the calculated hepatic bilirubin clearance, although some discrepancies exist. Using these determinations, a clearcut separation from normal values was not obtained, suggesting at least in the present group of patients that Gilbert's syndrome represents only one end of a continuum of bilirubin conjugation rates and not a separate entity.