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Biomedical subjects

N Blanckaert

Publications and source records attributed to N Blanckaert.

At least 37 records · Page 2Linked to original sources
PubMed1996

Uridine diphosphoxylose enhances hepatic microsomal UDP-glucuronosyltransferase activity by stimulating transport of UDP-glucuronic acid across the endoplasmic reticulum membrane.

The UDP-glucuronosyltransferase (UGT) system fulfils a pivotal role in the biotransformation of potentially toxic endogenous and exogenous compounds. Here we report that the activity of UGT in rat liver is stimulated by UDP-xylose. This stimulation was found in native microsomal vesicles as well as in the intact endoplasmic reticulum (ER) membrane, as studied in permeabilized hepatocytes, indicating the potential physiological importance of UDP-xylose in the regulation of UGT. We present evidence that UDP-xylose enhances UGT activity by stimulation of (i) the uptake of UDP-glucuronic acid across the ER membrane and (ii) the elimination of the UDP and/or UMP reaction product out of the ER lumen. UDP-xyloe produced a marked trans-stimulation of microsomal UDP-glucuronic acid uptake when it was present within the lumen of the ER. When UDP-xylose was presented at the cytosolic side of the ER, it acted as a weak inhibitor of UDP-glucuronic acid uptake. Likewise, cytosolic UDP-glucuronic acid strongly trans-stimulated efflux of intravesicular UDP-xylose, whereas cytosolic UDP-xylose was inefficient in trans-stimulating efflux of UDP-glucuronic acid. Microsomal UDP-xylose influx was markedly stimulated by UMP and UDP. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP or UDP, indicating that UMP and UDP exeted their effect on UDP-xylose uptake by trans-stimulation from the luminal side of the ER membrane.

Animals↗
PubMed1995

Mechanism of stimulation of microsomal UDP-glucuronosyltransferase by UDP-N-acetylglucosamine.

We propose the existence in rat liver endoplasmic reticulum (ER) of two asymmetric carrier systems. One system couples UDP-N-acetylglucosamine (UDPGlcNAc) transport to UDP-glucuronic acid (UDPGlcA) transport. When UDPGlcNAc was presented at the cytosolic side of the ER, it then acted as a weak inhibitor of UDPGlcA uptake. By contrast, UDPGlcNAc produced a forceful trans-stimulation of microsomal UDPGlcA uptake when it was present within the lumen of the ER. Likewise, cytosolic UDPGlcA strongly trans-stimulated efflux of intravesicular UDPGlcNAc, whereas cytosolic UDPGlcNAc was ineffective in trans-stimulating efflux of UDPGlcA. A second asymmetric carrier system couples UDPGlcNAc transport to UMP transport. Microsomal UDPGlcNAc influx was markedly stimulated by UMP present inside the microsomes. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP, indicating that UMP exerted its effect on UDPGlcNAc uptake by trans-stimulation from the lumenal side of the ER membrane. Contrariwise, extravesicular UMP only minimally trans-stimulated efflux of intramicrosomal UDPGlcNAc. It is widely accepted that UDPGlcNAc acts as a physiological activator of hepatic glucuronidation, but the mechanism of this effect has remained elusive. Based on our findings, we propose a model in which the interaction of two asymmetric transport pathways, i.e. UDPGlcA influx coupled to UDPGlcNAc efflux and UDPGlcNAc influx coupled to UMP efflux, combined with intravesicular metabolism of UDPGlcA, forms a mechanism that leads to stimulation of glucuronidation by UDPGlcNAc.

Animals↗
PubMed1995

Bile pigment composition and bilirubin esterification in the developing chick.

In vivo and in vitro bile pigment metabolism was studied in embryos, chicks, and mature White Leghorn hens. Biliverdin and bilirubin pigments were present in bile of embryos from the earliest stage examined (14 d). Bilirubin accounted for 2.5-11.5% of total bile pigments, with the higher percentages in the early embryo. Biliverdin was exclusively in unesterified form. In contrast, bilirubin was almost entirely (> 98%) in esterified form. Glucosides consistently predominated over glucuronides and xylosides (6:3:1). In serum, bilirubin and biliverdin were undetectable at all embryonic stages and after hatching. Bilirubin UDP-glycosyltransferase activities with UDP-glucuronic acid, UDP-glucose, and UDP-xylose were detectable in chick embryo liver and averaged 67, 72, and 102%, respectively, of the corresponding adult mean values, without significant change throughout development. We conclude that multiple bilirubin esterification systems mature early in the avian embryo. This is in marked contrast to the development of bilirubin metabolism in the mammalian fetus in which detoxication of this potentially toxic pigment is achieved by placental clearance, and bilirubin esterification matures only after delivery. These contrasting developmental patterns are consistent with an adaptive response to the different requirements in the avian embryo and mammalian fetus for self-protective detoxification of unconjugated bilirubin.

Animals↗
PubMed1995

Elimination of paraprotein interference in determination of plasma inorganic phosphate by ammonium molybdate method.

Phosphate concentrations were determined in 52 cases of paraproteinemia. The unmodified acidic ammonium molybdate method produced 19% spuriously high results. The false increase of phosphate concentration was attributable to formation of precipitate in the reaction mixture. The precipitate was formed by interaction between immunoglobulins and the unmodified acidic ammonium molybdate reagent. The magnitude of interference bore no relation to the type, concentrations, or isoelectric point of the paraproteins or to the presence or absence of free light chains. Diluting the sample to approximately 40 g/L total protein reduced but did not always eliminate the interference. In some cases paraprotein concentration as low as 8 g/L falsely increased plasma phosphate results. Apparently, only IgG and IgM but not IgA paraproteins produced the interference. Deproteination by ultrafiltration or by treatment with trichloroacetic acid removed the interference. The Kodak slide method and the new modified Boehringer Mannheim phosphate test were found to be interference-free. However, in some cases the latter new formulation is sensitive to substantial changes in ionic concentration of the reaction mixture.

Ammonium Sulfate↗
PubMed1994

Carrier-mediated transport of intact UDP-glucuronic acid into the lumen of endoplasmic-reticulum-derived vesicles from rat liver.

Uptake and metabolism of UDP-glucuronic acid (UDPGlcA) by rough-endoplasmic-reticulum (RER)-derived vesicles was studied. Analysis of the molecular species, double-labelling experiments and trans-stimulation experiments revealed that initial uptake represented entry into microsomes of predominantly intact UDPGlcA, concomitant with rapid hydrolysis of the internalized nucleotide sugar. The uptake constituted effective translocation from the medium into the lumen of the vesicles. Thus the amount of vesicle-associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space. Permeabilized microsomes were unable to retain UDPGlcA. The microsomal uptake of UDPGlcA met the criteria of bidirectional carrier-mediated translocation. Transport was time- and temperature-dependent, saturable, selective, capable of trans-stimulation, and operational against a concentration gradient. Microsomal uptake was inhibited by N-ethylmaleimide that was presented at the cytosolic side of the endoplasmic-reticulum (ER) membrane. Uptake studies performed in membrane preparations that were highly enriched in RER, smooth ER or Golgi revealed that UDPGlcA was taken up by the ER as well as by the Golgi apparatus. Our findings demonstrate the existence in rat liver ER of a carrier system mediating proper translocation of intact UDPGlcA across the membrane.

Animals↗
PubMed1994

Functional characterization of carrier-mediated transport of uridine diphosphate N-acetylglucosamine across the endoplasmic reticulum membrane.

Uptake and metabolism of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) by rough-endoplasmic-reticulum (rER)-derived vesicles was studied. Analysis of the molecular species, double-label experiments, cis-inhibition and trans-stimulation experiments revealed that uptake represented entry of intact UDP-GlcNAc into the microsomal lumen. The amount of vesicle-associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space, and permeabilized microsomes were unable to retain UDP-GlcNAc. These findings indicate that uptake constituted effective translocation from the medium into the lumen of the vesicles. The microsomal uptake of UDP-GlcNAc met the criteria of bidirectional, carrier-mediated translocation. Transport was time and temperature dependent, saturable, selective, capable of trans-stimulation and operational against a concentration gradient. Uptake studies performed in membrane preparations that were highly enriched in either rER, smooth ER, or Golgi revealed that UDP-GlcNAc was taken up by the ER and by the Golgi apparatus.

Animals↗
PubMed1994

Simultaneous macroamylasemia and macrolipasemia.

The first case of the simultaneous presence of macroamylasemia and macrolipasemia in a patient with gluten enteropathy (celiac disease) is described. Both macroenzymes were formed by association of polyclonal IgA with amylase and lipase. Both macroenzymes had molecular masses > 300 kDa.

Amylases↗
PubMed1993

Topology of nucleotide-sugar:dolichyl phosphate glycosyltransferases involved in the dolichol pathway for protein glycosylation in native rat liver microsomes.

Activities of nucleotide-sugar:dolichyl phosphate glycosyltransferases (UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl 1-phosphotransferase, UDP-glucose:dolichyl phosphate glucosyltransferase and GDP-mannose:dolichyl phosphate mannosyltransferase) are not fully expressed in native microsomes and can be enhanced by pretreatment of the microsomes with detergent. To examine whether the latency of dolichyl phosphate glycosyltransferases in native microsomes reflects a lumenal orientation of the catalytic centre, we examined the effect of proteinase treatment of native microsomes on enzymic activity and investigated the relationship between enzymic activity and alteration of the permeability of the microsomal membrane barrier. The enzymic activities catalysing transfer of N-acetylglucosamine and glucose to lipid acceptors were proteinase-sensitive in native sealed microsomes. When various detergents were used to disrupt the membrane barrier, we found no relationship between activity of dolichyl phosphate glycosyltransferases and the latency of mannose-6-phosphatase, which is a marker of the permeability properties of the microsomal membrane. Permeabilization of the endoplasmic reticulum membrane by the pore-forming Staphylococcus aureus alpha-toxin did not affect glycosyltransferase activities. These results do not support the hypothesis that latency of the transferase activities is dependent on the permeability properties of the endoplasmic-reticulum membrane. Collectively our findings can best be explained by postulating that the active centres of the transferases are cytoplasmically oriented, while activation by detergent may be conformation-dependent.

Animals↗
PubMed1993

Effect of GTP on the dolichol pathway for protein glycosylation in rat liver microsomes.

Incubation of native rat liver microsomes with GTP resulted in enhanced incorporation of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc into lipid acceptors. The stimulation of GlcNAc transfer by GTP was specific for GTP; ATP exerted no effect. The GTP effect was blocked by a non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate, indicating that GTP hydrolysis was crucial. Though dolichyl pyrophosphate NN'-diacetylchitobiose [Dol-PP-(GlcNAc)2] was the main radiolabelled product formed upon incubation of GTP-treated microsomes with UDP-GlcNAc, GTP selectively stimulated UDP-GlcNAc:dolichyl phosphate (Dol-P) N-acetylglucosaminyl 1-phosphotransferase (N-acetylglucosaminyl 1-phosphotransferase). This conclusion was reached on the basis of experiments in which tunicamycin was used to selectively inhibit N-acetylglucosaminyl 1-phosphotransferase. The enhanced transformation of Dol-P to dolichyl pyrophosphate N-acetylglucosamine (Dol-PP-GlcNAc) by GTP ultimately led to enhanced protein glycosylation. GTP-induced stimulation of GlcNAc incorporation in lipid and protein by GTP was observed also in microsomes fully permeabilized with Staph. aureus alpha-toxin. These findings refute the previous proposal [Godelaine, Beaufay, Wibo and Ravoet (1983) J. Cell Biol. 97, 340-350] that increased membrane permeability constitutes the mechanism whereby GTP activates the reactions of the dolichol pathway.

Acetylglucosamine↗
PubMed1992

Are port films reliable for in vivo exit dose measurements?

The possibility of using conventional port films as an in vivo method for obtaining relative exit dose distribution in patients is evaluated. Kodak "Verification" films in "Localization" cassettes are positioned in the usual clinical conditions behind an homogeneous polystyrene phantom as well as behind a phantom containing air, wood and aluminium inhomogeneities. Taking beam divergency into account the densitometric profiles are projected back to the exit side of the phantom. They are compared to the profiles obtained with an ionization chamber used under full backscatter conditions. The agreement between the off-axis ratios determined with either method are mostly better than 2% and never exceed 5%. These phantom measurements are completed by a comparison between off-axis ratios determined on a port film for a head and neck patient and those obtained by diode dosimetry applied on the patient at the exit side of the beam. A similar agreement as between film and ion chamber on the phantoms is obtained. These encouraging results illustrate the possibilities of using conventional port films for in vivo dosimetry.

Breast Neoplasms↗
PubMed1992

Influence of shielding blocks on the output of photon beams as a function of energy and type of treatment unit.

The influence of field-defining shielding blocks on the output of a cobalt unit and of seven different accelerators (one with dual energy output) has been investigated. The quality indices range from 0.57 (cobalt-60) to 0.79. The loss in output due to shielding blocks has been calculated taking into account loss in phantom scatter only. Comparison with experimental results shows that the calculation algorithm is correct in most of the clinical conditions. However, for quality indices of 0.70 and higher, for blocks close to the central beam axis, an overestimation of the output by the algorithm has been found. The maximum deviation observed is about 5% for the highest energy and for block positions corresponding to those applied, e.g. for inverted Y-fields with narrow lumbo-aortic block spacing.

Algorithms↗
PubMed1992

Failure of expression of the phenobarbital-induced enhancement of UDP-glycosyltransferases in native, sealed endoplasmic reticulum vesicles from rat liver.

Conflicting data have been published regarding the effects of phenobarbital treatment on bilirubin UDP-glucuronyltransferase activity in native liver microsomes. Recent evidence suggests that the bilirubin UDP-glycosyltransferase system faces the interior of microsomal vesicles, and that expression of its activities in sealed microsomes may be rate-limited by transport of UDP sugars across the membrane. These observations raise the possibility that the reported variability in the effects of phenobarbital may reflect differences in integrity of the membrane in microsomal preparations. We examined the effect of phenobarbital on bilirubin UDP-glucosyltransferase and the UDP-glucuronyltransferase activities towards bilirubin, 4-nitrophenol, and 1-naphthol using native rat liver microsomes with verified vesicle integrity. Phenobarbital-induced microsomes in which the membrane permeability barrier was eliminated by pretreatment with detergent displayed markedly higher UDP-glycosyltransferase activities towards all tested substrates compared with activities in similarly disrupted microsomes from untreated rats. In contrast, none of the transferase activities tested were significantly enhanced by phenobarbital treatment when the enzymic activities were assayed in sealed microsomes. Addition to the enzyme assay mixture of UDPGlcNAc, a presumed physiological activator of the UDP-glucuronyltransferases, failed to expose the enhanced UDP-glucuronyltransferase concentration in phenobarbital-induced sealed microsomes. Our findings are consistent with the idea that transport of UDP sugar across the membrane may be rate-limiting for expression of UDP-glycosyltransferase activities in sealed microsomes. Quantitative assessment of membrane integrity is an essential prerequisite in experiments designed to study the regulation of the microsomal UDP-glycosyltransferase system.

Animals↗
PubMed1991

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.

Bilirubin↗
PubMed1991

Is it necessary to repeat quality control procedures for head and neck patients?

Using serial verification films for detection of localization errors and in vivo measurements of the delivered dose, a comparison was made of the information obtained from a single check on the first treatment session or from repeated checks in subsequent irradiations, leading to an assessment of the predictive value of a single check. A total number of 215 films and 261 entrance dose measurements have been performed on 34 fields for 10 head and neck patients. The patients are immobilized with individual plastic masks fixed on the couch and treated on a 6 MV linac, supplied with an automatic verification system excluding the couch parameters. The global results show Gaussian frequency distributions with standard deviations of 4 mm for port film measurements and 3.4% for the dose measurements. Large errors (greater than 5 mm displacement and greater than 4% deviation from the expected dose) have been detected in 16% in the cranio-caudal direction and 24% in the antero-posterior direction with port films and in 15% of the in vivo measurements. In order to identify the nature of the errors, which can be random or systematic, the first measurement is taken as the reference value and shows that consecutive measurements on the same field were reproducible with standard deviations of respectively 2.5 mm and 1.8%. This means that a large part of the spread of the global results can be explained by systematic errors in the treatment preparation chain. With the first check, 6 out of 10 systematic localization errors and 7 out of 7 systematic errors leading to erroneous dose delivery have been detected. Therefore, most of the systematic errors, which affect the overall quality of the treatment, can be identified with the first check. The four systematic localization errors, missed with the first film, were of rather limited size: only one of them showed a mean displacement larger than 7 mm. Because the first measurement is an acceptable indication of the overall quality of the treatment delivery, the authors propose a code of practice for checking the treatment quality at the patient level.

Dose-Response Relationship, Radiation↗
PubMed1990

Unconjugated and conjugated bilirubin pigments during perinatal development. II. Studies on serum of healthy newborns and of neonates with erythroblastosis fetalis.

A recently developed reverse-phase high-performance liquid chromatography procedure was applied to the analysis of bilirubins present in serum of neonates. In normal newborns, unconjugated (UCB) and conjugated bilirubins (CB) rose in parallel from day 2 to day 4: the average conjugated fraction remained between 1.2 and 1.6% of total pigment, significantly below the 3.6% documented in adults. However, the absolute concentration of conjugates was 2- to 6-fold higher than the serum levels in adults. Bilirubin conjugates were detected in all samples, but only 20% of these were bilirubin diconjugates, in contrast to a mean of 54% in the adult population. The increased absolute concentrations of UCB and CB together with the decreased fractional amount of conjugates and with the decreased diconjugates seem to be the result of an increased bilirubin production combined with a relative defective biliary secretion. Serum bile acids were high but did not correlate with the levels of bilirubin conjugates. The pattern of serum bilirubins was not different in 4 newborns with hemolytic syndromes. Cholestasis occurred in another child with hemolysis; this was evident from the high concentration and the high fractional amount of conjugates and from the high serum bile acids level.

Bile Acids and Salts↗
PubMed1989

Chromatographic analysis and structure determination of biliverdins and bilirubins.

Recent applications of thin-layer chromatographic (TLC) and high-performance liquid chromatographic (HPLC) procedures has revealed an unexpected wide variety of naturally occurring unconjugated and conjugated bilirubins. Biliverdins seems to occur only in unconjugated forms, mainly as the IX alpha isomer. Several synthetic biliverdins and bilirubins present interesting models for biochemical and metabolic studies. Owing to recent recognition of the astounding heterogeneity of natural bilirubins and to the various artifactual changes that bile pigments can undergo, considerable confusion has existed, and still exists, with regard to the nomenclature of the bile pigments and their derivatives. To set a background for further discussion, the present review starts with a brief discussion of nomenclature and of the various characteristic forms of lability of the bile pigments. TLC and HPLC procedures for preparation and analysis of unconjugated biliverdins and bilirubins and their methyl ester and sugar ester conjugates, as well as procedures for analysis of bilirubin-protein conjugates, are then discussed. Since, in view of the lability and pronounced heterogeneity of bile pigments, it is important to assess the composition and nature of chromatographically isolated pigments, the review is concluded by a brief evaluation of various structural tests.

Animals↗
PubMed1989

Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver.

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

Animals↗