UDP-glucuronic Acid Levels Research Articles

Two UDP-glucuronic acid decarboxylases involved in the biosynthesis of a bacterial exopolysaccharide in Paenibacillus elgii.

Xylose is described as a component of bacterial exopolysaccharides in only a limited number of bacterial strains. A bacterial strain, Paenibacillus elgii, B69 was shown to be efficient in producing a xylose-containing exopolysaccharide. Sequence analysis was performed to identify the genes encoding the uridine diphosphate (UDP)-glucuronic acid decarboxylase required for the synthesis of UDP-xylose, the precursor of the exopolysaccharide. Two sequences, designated as Peuxs1 and Peuxs2, were found as the candidate genes for such enzymes. The activities of the UDP-glucuronic acid decarboxylases were proven by heterologous expression and real-time nuclear magnetic resonance analysis. The intracellular activity and effect of these genes on the synthesis of exopolysaccharide were further investigated by developing a thymidylate synthase based knockout system. This system was used to substitute the conventional antibiotic resistance gene system in P. elgii, a natural multi-antibiotic resistant strain. Results of intracellular nucleotide sugar analysis showed that the intracellular UDP-xylose and UDP-glucuronic acid levels were affected in Peuxs1 or Peuxs2 knockout strains. The knockout of either Peuxs1 or Peuxs2 reduced the polysaccharide production and changed the monosaccharide ratio. No polysaccharide was found in the Peuxs1/Peuxs2 double knockout strain. Our results show that P. elgii can be efficient in forming UDP-xylose, which is then used for the synthesis of xylose-containing exopolysaccharide.

The Effect of Carbapenem Antibiotics on Plasma Concentrations of Valproic Acid

To critically evaluate the mechanisms of the interaction between valproic acid and carbapenem antibiotics. A PubMed search (January 1971-June 2009) was performed to identify literature on the interaction between valproic acid and carbapenem antibiotics. Additional references were identified through review of bibliographies of identified articles. Data on the mechanisms of the interaction between valproic acid and carbapenem antibiotics were extracted from identified references that were published in English. Valproic acid plasma concentrations fall markedly during concomitant administration with carbapenem antibiotics due to a combination of absorption, distribution, and metabolism mechanisms. Carbapenems appear to inhibit the intestinal transporter of valproic acid, thereby reducing absorption of orally administered valproic acid. In vivo experiments in rats demonstrate a 57% reduction in absorption of orally administered valproic acid in the presence of imipenem. Follow-up studies in Caco-2 cells suggest that the inhibition probably occurs at the basolateral membrane. In addition, enterohepatic recycling of valproic acid may be diminished due to carbapenem activity against gut flora producing beta-glucuronidase. When rabbits and rats were given intravenous valproic acid-glucuronide, the glucuronide metabolite of valproic acid, 50-90% of the conversion back into valproic acid was inhibited in the presence of a carbapenem. An increase in erythrocyte distribution of valproic acid has also been observed in the presence of carbapenems. After intravenous administration of a carbapenem and valproic acid, valproic acid plasma concentrations fell in the presence of a carbapenem, yet whole blood concentrations of valproic acid did not change significantly. Follow-up studies suggest that the mechanism of this distribution shift is that multidrug resistance proteins on adenosine triphosphate-binding cassette transporters on erythrocyte membranes are inhibited by carbapenems. Thus, valproic acid is not effluxed out of the erythrocytes. Finally, carbapenems may enhance glucuronidation of valproic acid by increasing UDP-glucuronic acid levels. In rats, UDP-glucuronic acid levels increased by 1.7-fold in the presence of panipenem, which was proportionate to the increase in valproic acid-glucuronide formation. Published data demonstrate a serious and complex interaction between valproic acid and carbapenem antibiotics. Coadministration should be avoided, but if no other antibiotic therapies exist, it is imperative to monitor valproic acid concentrations more frequently. Clinicians should anticipate higher doses of valproic acid to maintain therapeutic serum concentrations during coadministration and subsequent dose reductions upon discontinuation of the carbapenem antibiotic.

Effects of glucosamine sulfate on intracellular UDP-hexosamine and UDP-glucuronic acid levels in bovine primary chondrocytes

To analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes. Chondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using (35)S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis. Levels of UDP-HexN and UDP-GlcA were unchanged at 10microM concentration of GS in low-glucose DMEM, while addition of 1mM GlcN or GS in low-glucose DMEM for 10min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30min after addition of 1mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100microM or 1mM of GS for 24h. Physiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels.

GALACTOSAMINE PREVENTS ETHINYLESTRADIOL-INDUCED CHOLESTASIS

Ethinylestradiol (EE) induces intrahepatic cholestasis in experimental animals being its derivative, ethinylestradiol 17beta-glucuronide, a presumed mediator of this effect. To test whether glucuronidation is a relevant step in the pathogenesis of cholestasis induced by EE (5 mg/kg b.wt. s.c. for 5 consecutive days), the effect of simultaneous administration of galactosamine (200 mg/kg b.wt. i.p.) on biliary secretory function was studied. A single injection of this same dose of galactosamine was able to decrease hepatic UDP-glucuronic acid (UDP-GA) levels by 85% and excretion of EE-17beta-glucuronide after administration of a tracer dose of [3H]EE by 40%. Uridine (0.9 g/kg b.wt. i.p.) coadministration reverted the effect of galactosamine on hepatic UDP-GA levels and restored the excretion of [3H]EE-17beta-glucuronide. When administered for 5 days, galactosamine itself did not alter any of the serum markers of liver injury studied (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase) or biliary secretory function. When coadministered with EE, galactosamine partially prevented the impairment induced by this estrogen in total bile flow, the bile-salt-independent fraction of bile flow, basal bile salt secretion, and the secretory rate maximum of tauroursodeoxycholate. Uridine coadministration partially prevented galactosamine from exerting its anticholestatic effects. In conclusion, galactosamine administration partially prevented EE-induced cholestasis by a mechanism involving decreased UDP-GA availability for subsequent formation of EE 17beta-glucuronide. The evidence thus supports the hypothesis that EE 17beta-glucuronide is involved in the pathogenesis of EE cholestasis.

Topography of glycosylation and UDP-xylose production

In order to define the location and organization of the numerous reactions involved in polysaccharide assembly during synthesis of proteoglycans and glycoproteins, the topography of some of the glycosylation reactions in chondroitin sulfate synthesis was examined using a relatively new technique for generating permeable cells. Permeable chondrocytes were shown to directly take up nucleotide sugar precursors and incorporate them into chondroitin sulfate proteoglycan (CSPG), allowing specific labeling at each step in chondroitin sulfate synthesis. Subcellular fractionation following labeling with UDP-[14C]xylose, UDP-[14C]galactose, UDP-[14C]glucuronic acid, or [35S]PAPS localized the labeled CSPG to the compartment where each glycosylation reaction occurred. From these experiments it appears that xylose addition begins in the endoplasmic reticulum and continues in the Golgi apparatus where galactose, glucuronic acid, and sulfate are added. This conclusion was confirmed by direct visualization of xylose incorporation using electron microscopic autoradiography (Vertel, B. M., Walters, L. M., Flay, N., Kearns, A. E., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11105-11112). Further examination of xylose addition showed that permeable chondrocytes can utilize both exogenous UDP-xylose transported into the lumen and UDP-xylose generated from UDP-glucuronic acid within the lumen. The enzyme responsible for this reaction, UDP-glucuronate carboxy-lyase, co-localized with xylosyltransferase activity in subcellular fractions. Orientation toward the lumen in subcellular compartments was determined by trypsin sensitivity in the permeable chondrocytes. Therefore, we conclude that UDP-xylose can be produced in the lumen of the compartment where it is utilized in CSPG synthesis, obviating the need for a direct transport mechanism for this nucleotide sugar and providing close regulation of UDP-xylose and UDP-glucuronic acid levels.

Circadian variation of hepatic UDP-glucuronic acid and the glucuronidation of xenobiotics in mice

A circadian cycle in hepatic UDP-glucuronic acid (UDP-GA) concentration was observed in mice that was essentially the reverse of those seen for hepatic glycogen and UDP-glucose. That is, hepatic UDP-GA levels were highest during the fasting period and lowest during the feeding period. However, there was no significant difference between the half-lives or the apparent rates of glucuronidation for either acetaminophen or salicylamide at 8 a.m. and 5 p.m. Therefore, the previously-reported circadian variation in acetaminophen toxicity is probably due to circadian variation in hepatic glutathione levels rather than in hepatic glucuronidation capacity.

Role of nucleotide pyrophosphatase in enflurane-induced reduction of UDP-glucuronic acid concentration in mouse liver

Because hepatic UDP-glucuronic acid levels decrease upon exposure to volatile anesthetics, the present study was designed to determine the mechanism by which enflurane decreases UDP-glucuronic acid in mice by measuring the concentrations of intermediates and the activities of enzymes in the UDP-glucuronic acid pathway. UDP-glucuronic acid concentrations were decreased by 40% in both male and female mice after 10 min of enflurane-induced narcosis. Concentration of UDP-glucose and the activities of diethylstilbestrol UDP-glucuronosyltransferase and UDP-glucose dehydrogenase were not affected by enflurane treatment. In contrast, nucleotide pyrophophatase activity was increased approximately 50% in both sexes. Thus, the decrease in hepatic UDP-glucuronic acid upon exposure of mice to enflurane is probably due to increased degradation by nucleotide pyrophosphatase.

Mechanism of decreased acetaminophen glucuronidation in the fasted rat

The mechanism by which an acute fast decreases the glucuronidation of hepatotoxic doses of acetaminophen in the rat was examined. Fasting did not depress the level of the enzyme, glucuronyl transferase, or the basal level of the co-substrate, UDP-glucuronic acid (UDPGA). Administration of a hepatotoxic dose of acetaminophen rapidly depleted UDPGA levels in both fed and fasted rats to the same nadir. Fed and fasted rats differed in that the rate of repletion of UDPGA levels was markedly slower in fasted rats. The total hepatic levels of UDP-glucose dehydrogenase and its cofactor, NAD +, were not decreased by fasting. In fasted rats, hepatic levels of the UDPGA precursor, UDP-glucose, were approximately 60% those of fed rats both before and after a hepatotoxic dose of acetaminophen. In fed rats, acetaminophen induced a marked depletion of hepatic glycogen levels and a dramatic increase in blood glucose levels. Acetaminophen induced a similar marked increase in blood glucose levels in fasted rats in spite of the fact that they lacked hepatic glycogen. It is concluded that the fastinginduced decrease in the glucuronidation of hepatotoxic doses of acetaminophen results from decreased production of UDPGA. The decreased synthetic capacity for UDPGA does not appear to be due to the inability of the liver to produce glucose units perse, but rather to the fasting-induced altered activities of the enzymes of carbohydrate metabolism which, in turn, alter the fate of glucose-6-phosphate derived from gluconeogenesis.

Diminished rates of glucuronidation and sulfation in perfused rat liver after chronic ethanol administration

Rates of glucuronidation and sulfation of 7-hydroxycoumarin were studied in perfused livers from normal chow-fed rats, or in livers from rats that had been fed liquid control or ethanol-containing diets. During infusion of 100 μM 7-hydroxycoumarin, rates of glucuronidation were similar in livers from chow-fed or control diet rats, but were 34% less in livers from ethanol-fed rats. These rates of glucuronidation in perfused livers could not be explained by changes of UDP-glucuronyltransferase activity, which was highest in hepatic musomes from ethanol-treated rats and lowest in musomes from chow-fed rats. The low rates of glucuronidation in livers from ethanol-treated rats were correlated with low hepatic concentrations of UDP-glucuronic acid, which were less than 70% of the levels measured in the other treatment groups. However, the diminished UDP-glucuronic acid levels could not be explained by alterations in adenine nucleotides, NAD +/NADH ratios, glycogen, UDP-glucose, or activity of UDP-glucose dehydrogenase. Rates of sulfation declined during prolonged 7-hydroxycoumarin infusion in livers from ethanol-treated rats, but not in livers from rats that had received the control diet. Similarly, hepatic concentrations of adenosine-3'-phosphate 5'-sulfatophosphate (PAPS) also decreased with time only in livers from ethanol-treated rats. Thus, chronic ethanol feeding impairs glucuronidation and sulfation in perfused livers as a result of diminished availability of the required cofactors for these conjugation pathways.

Determination of tissue UDP-glucuronic acid levels by high-pressure liquid chromatography

A new and sensitive method to measure UDP-glucuronic acid extracted from as little as 25 mg wet weight tissue has been developed. This procedure employs high-pressure liquid chromatography and liquid scintillation spectrophotometry to measure p-[ 14C]nitrophenylglucuronide generated enzymatically from p-[ 14C]nitrophenol and UDP-glucuronic acid. The reaction was catalyzed by UDP-glucuronyltransferase obtained from rat liver microsomes. The tissue levels of UDP-glucuronic acid assayed were 2 to 20 μmol/100 g wet wt, which are well below the levels detectable by the widely used spectrophotometric method.

Dependence of glucuronidation rate on UDP-glucuronic acid levels in isolated hepatocytes

Increasing evidence suggests that variability in hepatic glucuronidation is a critical determinant of mammalian species-specific sensitivity to the Fusarium mycotoxin deoxynivalenol (DON). Concurrently, ongoing work regarding the effects of DON on fish has led to broad classifications concerning species-specific sensitivity (e.g. rainbow trout are highly sensitive to DON and channel catfish are highly tolerant to DON). This study was designed to determine if sensitivity to DON in fish is related to inherent differences in UDP-glucuronosyltransferase (UDPGT) activity and to evaluate if increased dietary digestible starch content could be an effective strategy to increase glucuronidation capacity. Rainbow trout or Nile tilapia (initial average body weight = 21.9 and 8.1 g/fish, respectively) were fed two series (12 or 24% digestible starch) of three diets containing graded levels of naturally occurring DON (0.1, 0.7 and 1.3 ppm) for 10 weeks. Significant linear decreases in weight gain, thermal-unit growth coefficient (TGC, P ≤ .05), feed efficiency (P < .001), whole body crude protein (CP) content (P < .001), retained nitrogen (RN, P ≤ .05) and nitrogen retention efficiency (NRE, P < .01) were associated with increasing levels of DON in rainbow trout regardless of the dietary digestible starch content. There were no significant effects of DON on any of these parameters in Nile tilapia (P > .05) with the exception of a quadratic increase in NRE (P < .01) in fish fed the diets containing 24% digestible starch. The effect of DON on growth performance and nutrient utilization was not altered by the digestible starch content of the diet (P > .05, DON × digestible starch). A significant quadratic decrease (P ≤ .05) in UDPGT activity was associated with increasing levels of DON in rainbow trout fed the diet containing 12% digestible starch, whereas trout and tilapia fed the diet containing 24% digestible starch experienced significant quadratic or linear increases (P ≤ .05) in UDPGT activity, respectively. However, UDPGT activity was approximately 10-fold higher in rainbow trout than in Nile tilapia. This suggests that UDPGT activity is not responsible for the observed species-specific sensitivity to DON or it is not an appropriate biomarker of DON glucuronidation. A significantly higher relative fold expression of CYP1A in tilapia compared to trout (P = .0001) may offer potential opportunity for future study. We demonstrate for the first time that Nile tilapia are unaffected by practically relevant levels of DON which are detrimental to rainbow trout. This study also extends the current knowledge of DON metabolism and represents an important contribution to the development of efficacious nutritional mycotoxin mitigation strategies.