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N Blanckaert

Publications and source records attributed to N Blanckaert.

At least 55 records · Page 3Linked to original sources
PubMed1988

Topology and regulation of bilirubin UDP-glucuronyltransferase in sealed native microsomes from rat liver.

Bilirubin UDP-glucuronyltransferase displays marked latency in native microsomes. To examine whether this latency correlates with structural integrity of the microsomal vesicles and reflects lumenal orientation of the enzyme's catalytic center, we analyzed the relationship between transferase activity and the degree of expression of mannose (Man)-6-phosphatase, which is a marker enzyme of the cisternal face of the ER membrane. Using detergent, sonication, or the pore-forming Staphylococcus aureus alpha-toxin to breach the microsomal membrane permeability barrier, we found that after each of these pretreatments a remarkably close direct relationship existed between latency changes for bilirubin UDP-glucuronyltransferase and Man-6-phosphatase. This finding suggested that the transferase may have the same transverse topology as the phosphohydrolase. We also compared the effects of membrane-impermeant proteinases on bilirubin UDP-glucuronyltransferase activity in native and disrupted microsomes. Whereas the unspecific proteinase nagarse markedly inactivated (to less than 30% of activities in controls) the transferase in disrupted microsomes, treatment with the proteinase had little effect on transferase activity in sealed microsomal vesicles. The results suggest that the active site of bilirubin UDP-glucuronyltransferase is on the lumenal face of the endoplasmic reticulum membrane. It was also found that activation of transferase activity by UDP N-acetylglucosamine, which is the presumed allosteric effector of UDP-glucuronyltransferase, was markedly altered by relatively small changes in structural integrity of the microsomes and totally abolished when latency of Man-6-P hydrolysis fell below approximately 80%. Collectively, these findings demonstrate that the microsomal membrane permeability barrier is a major determinant of expression of microsomal UDP-glucuronyltransferase activity and that quantitative assessment of integrity of the microsomes is essential for studying kinetic properties and regulation of microsomal UDP-glucuronyltransferase.

Animals↗
PubMed1988

Carrier-mediated translocation of uridine diphosphate glucose into the lumen of endoplasmic reticulum-derived vesicles from rat liver.

Radiolabeled UDPGlc incubated with rough endoplasmic reticulum (RER)-derived microsomes from rat liver became associated with the vesicles. This microsomal uptake of nucleotide sugar was time and temperature dependent. Analysis of the molecular species containing radiolabel revealed that initial uptake represented entry of predominantly intact UDPGlc in the microsomes. Conclusive evidence for proper translocation of UDPGlc across the microsomal membrane into the intravesicular space was obtained by demonstrating that UDPGlc was transported into an osmotically sensitive compartment. Microsomal uptake of UDPGlc exhibited features characteristic of carrier-mediated transport including saturation, specificity, and countertransport. Inhibition and trans-stimulation studies showed that other uridine-containing nucleotide sugars and 5'-UMP were substrates of the postulated microsomal carrier system for UDPGlc, while cytosine- or guanosine-containing nucleotides and non-5'-uridine monophosphates were, at best, very poor substrates. UDPGlc translocation activities were lower in smooth microsomal fractions than in the RER-derived vesicles, indicating that contamination with Golgi membranes could not be responsible for microsomal transport of UDPGlc. Our findings suggest that rat liver endoplasmic reticulum possesses a carrier system mediating proper translocation of UDPGlc and 5'-uridine-substituted structural analogues across the membrane.

Animals↗
PubMed1987

On the binding of bilirubin and its structural analogues to hepatic microsomal bilirubin UDPglucuronyltransferase.

Hepatic glucuronidation of the asymmetrical natural bilirubin molecule results in formation of two different positional isomers, bilirubin C-8 monoglucuronide and bilirubin C-12 monoglucuronide. In view of the existence of multiple isoforms of UDPglucuronyltransferase, which is the microsomal enzyme system responsible for bilirubin esterification, we performed kinetic analysis of microsomal glucuronidation of bilirubin and a number of its structural congeners to determine whether synthesis of the two monoglucuronide isomers involved two distinct substrate-binding sites or reflected two different modes of binding to a single catalytic site. Both isomers were found in all tested species (man, rat, guinea pig, sheep), but there were marked species differences in the C-8/C-12 ratio of monoglucuronide found in bile or formed by liver microsomes. Correspondence between in vivo and in vitro results for such regioselectivity of glucuronidation was excellent in each species. On the basis of our results of kinetic analysis of bilirubin esterification at variable pigment substrate concentrations and inhibition studies with alternative substrates, we postulate that both natural monoglucuronide isomers are synthesized at a single binding site. Possible mechanisms responsible for the markedly regioselective esterification of bilirubin by rat and sheep liver were investigated by study of glucuronidation of selected structural analogues of the pigment. Our results do not support explanations of regioselectivity of bilirubin glucuronidation in terms of (i) preferential binding of either the C-8- or C-12-containing dipyrrolic half of the asymmetrical bilirubin molecule or (ii) enantioselective complexation of bilirubin UDPglucuronyltransferase to one of the two chirality enantiomers of intramolecularly hydrogen-bonded bilirubin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Endogenous esterification of bilirubin by liver microsomes. Evidence for an intramicrosomal pool of UDP-glucose and lumenal orientation of bilirubin UDP-glycosyltransferase.

Conjugation of natural bilirubin (BR) depends on a hepatic microsomal UDP-glycosyltransferase using UDP-Glc, UDP-xylose, and predominantly UDP-GlcA. We found that esterification of BR occurred when washed intact microsomes derived from rat or guinea pig liver were incubated with BR in the absence of added UDP-sugar. This endogenous esterification was shown to lead predominantly to formation of the two positional isomers of BR monoglucoside and displayed the same regioselectivity as found for the BR monoglucosides formed by microsomes incubated with a saturating concentration of added UDP-Glc. This finding and absence of endogenous esterification in liver microsomes from mutant rats lacking BR UDP-glycosyltransferase activities demonstrated that endogenous esterification depended on UDP-glycosyltransferase and indicated, therefore, that UDP-Glc was present in the intact microsomal vesicles. With UDP-Glc added to the extramicrosomal incubation medium, BR glucosidation was markedly enhanced when the membrane permeability barrier was disrupted by pretreatment of the microsomes with detergent, sonication, or Staphylococcus aureus alpha-toxin. In contrast, such membrane disruption resulted in abolishment of endogenous esterification of BR, and a direct relationship was found between impairment of endogenous esterification and degree of vesicle disruption, suggesting that the UDP-Glc on which endogenous esterification depended was present in the lumenal space of the microsomes. Kinetic evidence and absence of an effect of increasing the microsomal concentration of dolichol-P-Glc (Dol-P-Glc) on endogenous esterification excluded direct or indirect involvement of Dol-P-Glc in the endogenous esterification reaction. Preincubation of intact microsomes with UDP-Glc or UDP-xylose at 37 degrees C, but not at 0 degrees C, led to expansion of the microsomal UDP-sugar pool on which endogenous esterification depended, suggesting that both UDP-sugars can enter the microsomal vesicles by a temperature-dependent mechanism. In contrast to these findings, no increase of BR esterification was detected when the microsomes had been preincubated at 37 degrees C with UDP-GlcA. We conclude that native, intact microsomes contain a lumenal pool of endogenous UDP-Glc and that BR UDP-glucosyltransferase and UDP-xylosyltransferase, by virtue of a lumenal orientation, have direct access to the postulated intramicrosomal pool of nucleotide sugar.

Animals↗
PubMed1987

Relationships between serum bilirubins and production and conjugation of bilirubin. Studies in Gilbert's syndrome, Crigler-Najjar disease, hemolytic disorders, and rat models.

The pattern of serum bilirubins was determined in serum of humans and rats with unconjugated hyperbilirubinemia due to increased pigment load or defective hepatic conjugation. Bilirubin ester conjugates were present in all serum samples tested and were identified as bilirubin 1-O-acyl glucuronides. In Gilbert's syndrome, the concentration of total conjugates was comparable to the values in healthy control subjects. Because the concentration of unconjugated pigment was increased, the fraction of conjugated relative to total bilirubins was markedly decreased. Sera from patients with Crigler-Najjar disease differed from those with Gilbert's syndrome by the higher unconjugated bilirubin levels and the undetectability of diconjugated bilirubins. A striking finding was that in hemolytic disease, the concentration of both monoconjugates and diconjugates was enhanced in parallel with the increase of unconjugated pigment. Therefore, the fraction of conjugated relative to total bilirubins remained within the normal range. As in Gilbert's syndrome, heterozygote R/APfd-j/+ rats with impaired hepatic bilirubin conjugation exhibit an increased unconjugated bilirubin level in serum, whereas the concentration of total conjugates was comparable to the values in normal rats. In serum of normal rats loaded intraperitoneally with unconjugated bilirubin, both unconjugated and mono- and diconjugated bilirubins were increased in parallel so that the ratio of unconjugated to esterified pigment remained unaffected. Decreased hepatic conjugation or increased bilirubin load was associated with a lower percentage of diconjugates relative to total conjugates both in human and rat serum. The present results are consistent with a compartmental model in which there is bidirectional transfer across the sinusoidal membrane for unconjugated bilirubin as well as for the bilirubin glucuronides. Because typical patterns of serum bilirubins are found in Gilbert's syndrome and patients with hemolytic hyperbilirubinemia, determination of esterified bilirubins in serum is of value to study the pathophysiology and the differential diagnosis of unconjugated hyperbilirubinemia.

Anemia, Hemolytic↗
PubMed1986

Assay of mannose-6-phosphatase in untreated and detergent-disrupted rat-liver microsomes for assessment of integrity of microsomal preparations.

An accurate, precise, and convenient procedure was developed for measurement of the latency of the low-Km mannose-6-phosphatase activity for the purpose of assessment of the membrane permeability barrier in microsomes. This approach is based on previous work of Arion et al. [J. Biol. Chem. (1976) 251, 4901-4907] and consists of measurement of mannose-6-phosphatase activity in the untreated microsomal fraction and in the corresponding microsomes that are fully disrupted in order to eliminate the membrane permeability barrier. Complete disruption of rat liver microsomes was achieved by incubation for 60 min at 0 degree C in the presence of 4 mM zwitterionic detergent 3-[(3-cholamido-propyl)dimethyl-ammonio]-2-hydroxy-1-propane sulphonate (Chapso). That the microsomal membrane permeability barrier was eliminated under those conditions was suggested by the fact that the enzyme activation (up to 50-fold) produced by this pretreatment was at least as large as the effect of any other previously reported disruptive procedure. Disruption of the microsomes by Chapso or by ultrasonication markedly enhanced the thermolability of the mannose-6-phosphatase activity. In addition, exposure of the microsomes to high concentrations of Chapso produced enzyme inactivation that could be partially reversed by dilution of the detergent prior to assaying the enzymic activity. Investigation of these enzyme inactivation phenomena under various incubation conditions for disruption of the microsomes by Chapso and for subsequent assay of mannose-6-phosphatase activity in the presence of Chapso enabled us to define conditions under which instability of the enzyme was undetectable. Using these optimized procedures for disruption of microsomes and assay of hexose-6-P phosphohydrolase, we found that the low-Km mannose-6-phosphatase activity of untreated rat liver microsomes consistently was less than 5% of the total enzyme activity in the fully disrupted microsomes. Accurate and precise assay of the structural latency of mannose-6-phosphatase in membrane preparations must be performed under well-controlled conditions, with special attention to the marked thermolability of the enzyme in the presence of detergent, and is a prerequisite for using this approach for the purpose of assessing intactness of microsomal preparations.

Ammonium Hydroxide↗
PubMed1986

Unconjugated and conjugated bilirubin pigments during perinatal development. I. Studies on rat serum and intestine.

Unconjugated, mono- and diconjugated bilirubin was measured in rat plasma and intestine during perinatal development. Bilirubin monoconjugates were present in the intestines of 18-day-old fetuses, while diconjugates appeared on the 20th day. The concentration of conjugates increased rapidly, reaching 312 mumol/g of intestinal tissue 1 day after birth. The absolute concentration of unconjugated bilirubin increased in parallel. On the 18th day of fetal life, about 80% of the pigment in the intestine was unconjugated, while conjugates largely predominated from the 20th day onwards. Conjugation and/or excretion of bilirubin in the intestine seems therefore to develop rapidly during the last days of life. The plasma concentration of total bilirubin in 18-day-old fetuses was about 2-3 times higher than in adult serum, and it increased slightly during the following days. 2-7% of the pigment was conjugated. Transplacental clearance of bilirubin seems therefore to be deficient in this species. On the 1st day of extrauterine life, bilirubin concentration increased sharply in plasma, reaching 14.76 mumol/l, 38% in the conjugated form. This finding suggests that, unlike humans, an excretory defect is predominant in the neonatal rat.

Animals↗
PubMed1986

Formation of bilirubin conjugates in human newborns.

Bilirubin conjugates in the serum of newborn human infants were investigated using the alkaline methanolysis-high-performance liquid chromatography method, a specific and sensitive method for measurement of unconjugated bilirubin and bilirubin mono- and diester conjugates. Serum samples were analyzed from 13 premature infants, 11 full term newborns, 22 healthy adults, seven pregnant women at term and their corresponding infants cord blood at delivery, 46 cord blood specimens obtained at unselected deliveries, three cord bloods from infants with maternal-fetal blood group incompatibility, and two cord bloods from infants with intrauterine hypoxia. Bilirubin conjugates were not detectable in the healthy adults, maternal blood, or in the cord blood specimens except from infants with blood group incompatibility or intrauterine hypoxia. The two isomeric monoconjugates of bilirubin appeared in serum during the first 24 to 48 postnatal h in both premature and full term infants, followed by the diconjugate on the 3rd day. Conjugated esters accounted for 2 to 5% of the total bilirubin, with the diconjugate constituting 21% of total conjugated pigment (day 3). In all instances, the unconjugated serum bilirubin concentration had increased to at least 2 mg/dl in the course of physiologic neonatal hyperbilirubinemia before bilirubin conjugates became detectable. Both premature and full term human infants displayed the identical pattern of bilirubin conjugation in serum.

Adult↗
PubMed1986

The congenic normal R/APfd and jaundiced R/APfd-j/j rat strains: a new animal model of hereditary non-haemolytic unconjugated hyperbilirubinaemia due to defective bilirubin conjugation.

In this paper the production of the R/APfd-j/j strain which is congenic with the R/APfd strain is reported. The R/APfd-j/j completely lacks hepatic bilirubin UDP-glucuronyltransferase activity, as do our GUNNXR/Pfd-j/j rat strain and various other stocks of GUNN rats (j/j) described in the literature. Our recombinant inbred strain GUNNXR/Pfd-j/j was produced from non-inbred GUNN (j/j) rats. This GUNNXR/Pfd-j/j rat was used as a donor of the jaundice gene j, the R/APfd rat serving as the recipient. After eight backcross-intercross cycles (16 generations) the R/APfd-j/j strain was obtained which is congenic with the R/APfd strain. Congenicity was demonstrated by various techniques including transplantation of skin tissue, strain-specific tumour cells and hepatocytes, the mixed lymphocyte reaction, and comparison of biochemical markers. The potential of the novel inbred strain of jaundiced rat, R/APfd-j/j, and the corresponding control strain R/APfd for biochemical and clinical studies of bilirubin metabolism are briefly discussed.

Animals↗
PubMed1986

Measurement of bilirubin-protein conjugates in serum and application to human and rat sera.

Evidence suggests that the esterified bilirubins in serum of patients with conjugated hyperbilirubinemia can react with serum protein, predominantly albumin, producing bilirubin-protein conjugates. To specifically measure this pigment fraction, we now have developed an assay that requires neither sample preincubation nor special equipment and is based on (1) selective removal of the bilirubins that are reversibly bound to serum protein by using organic solvent extraction, and (2) subsequent measurement of the bilirubin-protein conjugates in the denatured protein pellet by a diazo method. The accuracy of the bilirubin-protein conjugate assay was verified by determining the individual recoveries of the various pigment fractions known to occur in patient serum samples. Near-complete removal (approximately 97%) of unconjugated bilirubin and its various monoesterified and diesterified bilirubins from the serum proteins was demonstrated, with quantitative recovery of total serum protein (greater than 99%) and of bilirubin-protein conjugates (greater than or equal to 95%; tested with purified bilirubin-albumin conjugate). The assay has a demonstrated linearity for bilirubin-protein conjugate concentrations between 3 mumol/L and 170 mumol/L, is precise (total and within-day coefficient of variation, assessed over a 12-month period, less than or equal to 5%), and is essentially free of interference by hemoglobin (for hemoglobin concentrations up to 5.0 gm/L). A fair linear correlation (r = 0.975) was found between the bilirubin-protein conjugate assay results and the values for diazo-positive pigment in serum that is not accounted for by unconjugated bilirubin and its esters. Bilirubin-protein conjugates were found only in sera of patients and rats with conjugated hyperbilirubinemia, not in those with unconjugated hyperbilirubinemia.

Animals↗
PubMed1985

Treatment of enzyme deficiency by hepatocyte transplantation in rats.

The inbred, homozygous Gunn rat exhibits unconjugated hyperbilirubinemia due to a hereditary absolute deficiency of bilirubin UDP-glucuronyltransferase activity. The mechanism of action of hepatocyte transplantation (HTX) in the treatment of enzyme deficiency has been investigated in this study. Gunn rats underwent HTX by the injection of isolated hepatocytes from a nondeficient donor rat (Wistar) into the spleen. A transient, statistically significant decrease in total plasma bilirubin (TB) levels was observed. Gunn rats receiving Gunn hepatocytes did not show such a decrease. Histological examination 2-3 months post-HTX of the recipient spleens showed the absence of grafted hepatocytes in the first group and graft survival in the second. Bile specimens from sublethal irradiated Gunn rats, collected 6 days after HTX with viable Wistar hepatocytes, all contained bilirubin mono- and diglucuronides. Control groups consisting of Gunn rats receiving nonviable Wistar hepatocytes or Gunn hepatocytes, and sham-operated Gunn rats did not excrete bilirubin glucuronides in bile. It was also demonstrated that bilirubin UDP-glucuronyltransferase activity, which appeared in Gunn rats after HTX with Wistar hepatocytes, was only transient. It is concluded that the decrease of TB in the Gunn rat after HTX with nondeficient hepatocytes is explained by the appearance of the enzyme, which was absent in the recipient animal. Viable, nondeficient hepatocytes are required for the elicited bilirubin conjugation. Rejection of the transplanted hepatocytes abolishes this effect.

Animals↗
PubMed1985

An unusual case of Crigler-Najjar disease in the adult. Classification into types I and II revisited.

We present the case of a 25-year-old man with Crigler-Najjar disease who had since birth a marked unconjugated hyperbilirubinemia without bilirubin overproduction, without any neurological involvement and in whom phenobarbital administration failed to produce any effect. Analysis of his biliary bile pigments on two occasions showed (i) a decrease excretion of bilirubin, as indirectly suggested by a high ratio of biliary bile acids over total bilirubin; (ii) an increase in unconjugated bilirubin IX alpha quantitated by thin-layer chromatography (TLC) following alkaline methanolysis and by direct extraction and TLC of the tetrapyrroles; (iii) a high proportion of bilirubin monoconjugates whereas the excretion of diconjugates was very low. Classification of the present patient into Crigler-Najjar disease type I or II was not possible. The most striking and practical difference among the various cases of Crigler-Najjar disease remains the response to phenobarbital. Among cases of Crigler-Najjar disease which respond to enzyme induction and Gilbert's syndrome, the continuous spectrum suggests a common defect.

Adult↗
PubMed1985

Solid-phase extraction and liquid chromatography for improved assay of cyclosporine in whole blood or plasma.

In this simple, precise, accurate, and specific isocratic liquid chromatographic procedure for determining cyclosporine, the cyclosporine is extracted from 1 mL of whole blood or from plasma, with 500 micrograms of cyclosporin D added per liter as internal standard, by elution from a Bond-ElutTM C18 extraction column with 300 microL of a mixture of ethanol and tetrahydrofuran. A 100-microL aliquot of the eluate, injected onto a cyano-phase analytical column, is eluted with a mixture of acetonitrile and pH 7.0 phosphate buffer at a flow rate of 1.0 mL/min and at 50 degrees C. Detection is at 210 nm. The chromatography is complete in less than 14.0 min. The method can measure less than 10.0 micrograms/L. Analytical recovery of cyclosporine added to whole blood ranged from 99 to 109% for concentrations up to 2000 micrograms/L. Between-run CVs ranged from 6.4 to 6.6%. None of numerous drugs and steroids tested interfered. Results by radioimmunoassay exceeded by 20 to 350% those measured by the present method.

Chromatography, Liquid↗
PubMed1984

Radioassay of UDP-glucuronyltransferase-catalysed formation of bilirubin monoglucuronides and bilirubin diglucuronide in liver microsomes.

A radioassay for specific determination of the rates of UDP-glucuronic acid-dependent conversion of bilirubin into the two isomeric (C-8, C-12) bilirubin monoglucuronides and bilirubin diglucuronide is described and illustrated by its application to rat liver microsomes. The method is based on measurement of the relative amounts of radiolabel in unesterified bilirubin and its mono- and di-esterified reaction products after incubation with [14C]bilirubin as substrate. This analysis is performed by the alkaline-methanolysis procedure, combined with one of two t.l.c. systems developed in order to enhance the sensitivity, accuracy and precision of the radioassay. Results for rates of total bilirubin glucuronide formation obtained with the new assay and the standard enzyme assay based on the ethyl anthranilate diazo-method were identical. However, the sensitivity of the latter technique is approx. 10-fold lower than that of the radioassay.

Animals↗
PubMed1984

Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase.

Highly purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver, when reconstituted with Gunn-rat liver microsomes (microsomal fraction), was able to catalyse the conversion of unesterified bilirubin into both bilirubin monoglucuronide and diglucuronide. Under zero-order kinetic conditions for monoglucuronide formation, the fraction of bilirubin diglucuronide formed by incubation of bilirubin with the reconstituted highly purified transferase accounted for 18% of total bilirubin glucuronides, which was only slightly lower than the fraction of diglucuronides (23% of total bilirubin glucuronides) formed by incubation with hepatic microsomes in the presence of UDP-N-acetylglucosamine or Lubrol. The reconstituted purified enzyme also catalysed the UDP-glucuronic acid-dependent conversion of bilirubin monoglucuronide into diglucuronide and, when bilirubin was incubated with UDP-glucose or UDP-xylose, the formation of bilirubin glucosides and xylosides respectively. These results suggest that a single microsomal bilirubin UDP-glycosyltransferase may be responsible for the formation of bilirubin mono- and di-glycosides.

Animals↗