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F Kuipers

Publications and source records attributed to F Kuipers.

At least 55 records · Page 3Linked to original sources
PubMed1999

Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver.

ApoE-deficient mice on low fat diet show hepatic triglyceride accumulation and a reduced very low density lipoprotein (VLDL) triglyceride production rate. To establish the role of apoE in the regulation of hepatic VLDL production, the human APOE3 gene was introduced into apoE-deficient mice by cross-breeding with APOE3 transgenics (APOE3/apoe-/- mice) or by adenoviral transduction. APOE3 was expressed in the liver and, to a lesser extent, in brain, spleen, and lung of transgenic APOE3/apoe-/- mice similar to endogenous apoe. Plasma cholesterol levels in APOE/apoe-/- mice (3.4 +/- 0.5 mM) were reduced when compared with apoe-/- mice (12.6 +/- 1.4 mM) but still elevated when compared with wild type control values (1.9 +/- 0.1 mM). Hepatic triglyceride accumulation in apoE-deficient mice was completely reversed by introduction of the APOE3 transgene. The in vivo hepatic VLDL-triglyceride production rate was reduced to 36% of control values in apoE-deficient mice but normalized in APOE3/apoe-/- mice. Hepatic secretion of apoB was not affected in either of the strains. Secretion of (3)H-labeled triglycerides synthesized from [(3)H]glycerol by cultured hepatocytes from apoE-deficient mice was four times lower than by APOE3/apoe-/- or control hepatocytes. The average size of secreted VLDL particles produced by cultured apoE-deficient hepatocytes was significantly reduced when compared with those of APOE3/apoe-/- and wild type mice. Hepatic expression of human APOE3 cDNA via adenovirus-mediated gene transfer in apoE-deficient mice resulted in a reduction of plasma cholesterol depending on plasma apoE3 levels. The in vivo VLDL-triglyceride production rate in these mice was increased up to 500% compared with LacZ-injected controls and correlated with the amount of apoE3 per particle. These findings indicate a regulatory role of apoE in hepatic VLDL-triglyceride secretion, independent from its role in lipoprotein clearance.

Animals↗
PubMed1999

Biliary phospholipid secretion is not required for intestinal absorption and plasma status of linoleic acid in mice.

Biliary phospholipids have been hypothesized to be important for essential fatty acid homeostasis. We tested this hypothesis by investigating the intestinal absorption and the status of linoleic acid in mdr2 Pgp-deficient mice which secrete phospholipid-free bile. In mice homozygous (-/-) for disruption of the mdr2 gene and wild-type (+/+) mice, dietary linoleic acid absorption was determined by 72 h balance techniques. After enteral administration, [(13)C]-linoleic acid absorption was determined by measuring [(13)C]-linoleic acid concentrations in feces and in plasma. The status of linoleic acid was determined in plasma and in liver by calculating the molar percentage of linoleic acid and the triene:tetraene ratio. Although plasma concentration of [(13)C]-linoleic acid at 2 h after enteral administration was significantly lower in (-/-) compared to (+/+) mice (P</=0.05), net intestinal absorption of dietary linoleic acid or of [(13)C]-linoleic acid was similar in (+/+) and (-/-) mice. Molar percentage of linoleic acid and the triene:tetraene ratio were not different in whole plasma or in liver of (-/-) compared to (+/+) mice. Present data indicate that biliary phospholipids are involved in the rate of appearance in plasma of enterally administered linoleic acid, but are not required for net intestinal absorption or plasma status of linoleic acid.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Bile diversion in rats leads to a decreased plasma concentration of linoleic acid which is not due to decreased net intestinal absorption of dietary linoleic acid.

Decreased bile secretion into the intestine has been associated with low plasma concentrations of essential fatty acids (EFA) in humans. We studied the mechanism behind this relationship by determining the status and absorption of the major dietary EFA, linoleic acid (LA), in control and 1-week bile-diverted rats. The absorption of LA was quantified by a balance method and by measuring plasma concentrations of [13C]LA after its intraduodenal administration. Absolute and relative concentrations of LA in plasma were decreased in bile-diverted rats (P<0.01 and P<0.001, respectively). Fecal excretion of LA was increased at least 20-fold in bile-diverted rats (0.72+/-0.11 vs. 0.03+/-0.00 mmol/day; P<0.0001). Due to increased chow ingestion by bile-diverted rats, net intestinal absorption of LA was similar between bile-diverted and control rats (1.96+/-0.14 vs. 1.91+/-0.07 mmol/day, respectively; P>0.05). After intraduodenal administration of [13C]LA, plasma concentrations were approximately 3-4-fold lower in bile-diverted rats for at least 6 h (P<0.001). Plasma concentrations of both [12C]arachidonic acid and [13C]arachidonic acid were increased in bile-diverted rats (P<0.05). We conclude that decreased plasma concentrations of LA in 1-week bile-diverted rats are not due to decreased net intestinal absorption of LA, but may be related to increased metabolism of LA.

Animals↗
PubMed1999

Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status.

The inducible nitric oxide synthase (iNOS) promoter contains nuclear factor kappaB (NF-kappaB) binding sites. NF-kappaB activation is determined, in part, by the intracellular redox status. The aim of this study was to determine the importance of the cellular glutathione status in relation to NF-kappaB activation and iNOS expression in hepatocytes in vivo and in vitro. For in vivo experiments, rats were injected with endotoxin and sacrificed 6 hours later. Glutathione was depleted by diethylmaleate. For in vitro experiments, cultured hepatocytes from untreated rats were exposed to a cytokine mixture. Glutathione levels were depleted by diethylmaleate and restored by N-acetylcysteine. iNOS expression was assessed by Western blot, reverse transcription polymerase chain reaction, nitric oxide (NO) metabolites, and immunohistochemistry. NF-kappaB binding was assessed by electrophoretic mobility shift assay. Endotoxin-induced iNOS expression in rat liver was prominent in hepatocytes, Kupffer cells, and inflammatory cells, in particular neutrophils. Glutathione depletion prevented iNOS induction in hepatocytes, but not in inflammatory cells. iNOS protein levels were in accordance with iNOS messenger RNA and NO metabolites in plasma. Glutathione depletion did not affect neutrophil infiltration. Cytokines strongly induced iNOS in cultured hepatocytes. Induction was prevented by glutathione depletion and could be restored by addition of N-acetylcysteine. NF-kappaB binding correlated with iNOS induction. In conclusion, in this study we show that iNOS induction in hepatocytes in vivo and in vitro is dependent on the intracellular glutathione status and correlates with NF-kappaB binding. Glutathione-depletion has no effect on the expression of iNOS in inflammatory cells, nor on neutrophil infiltration.

Acetylcysteine↗
PubMed1999

Regulation of hepatic transport systems involved in bile secretion during liver regeneration in rats.

We investigated the expression of hepatic transport systems involved in bile secretion during liver regeneration after partial hepatectomy (PH) in rats. Initial studies showed maximal BrdU incorporation 24 hours after PH. Therefore, transporter expression and bile secretion were analyzed in detail at this time. The mRNA levels of the multidrug resistance genes mdr1a and mrp1 slightly increased, whereas mdr1b mRNA levels showed an extensive increase after PH. The mRNA levels of the conjugate transporter, mrp2, decreased slightly, whereas mrp2 protein levels did not change. Bilirubin secretion did not change, but the biliary glutathione secretion markedly decreased and the hepatic GSH content increased. The messenger RNA levels of the bile salt uptake transporters ntcp, oatp1, and oatp2 and the bile salt exporter, bsep/spgp, all decreased with ntcp showing the most prominent decrease. Protein levels of ntcp dramatically decreased whereas oatp2 only slightly decreased. Oatp1 protein expression slightly increased and bsep/spgp protein levels did not change. Decreased levels of bile salt uptake systems were associated with a 10-fold increase in the plasma bile salt concentration, yet, bile flow and bile salt secretion were increased when expressed per gram liver and unaffected when expressed on the basis of body weight. In conclusion, during the initial phase of rat liver regeneration ntcp is down-regulated whereas other transporter proteins involved in bile secretion are only slightly affected. Despite increased serum bile salt levels the remnant liver is not cholestatic: bile flow is maintained by uptake of bile salts probably via oatp isoforms and their secretion via bsep/spgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Acyl-coenzyme A:cholesterol acyltransferase inhibitor, avasimibe, stimulates bile acid synthesis and cholesterol 7alpha-hydroxylase in cultured rat hepatocytes and in vivo in the rat.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors are currently in clinical development as potential lipid-lowering and antiatherosclerotic agents. We investigated the effect of avasimibe (Cl- 1011), a novel ACAT inhibitor, on bile acid synthesis and cholesterol 7alpha-hydroxylase in cultured rat hepatocytes and rats fed different diets. Avasimibe dose-dependently decreased ACAT activity in rat hepatocytes in the presence and absence of beta-migrating very low-density lipoproteins (betaVLDL) (by 93% and 75% at 10 micromol/L) and reduced intracellular storage of cholesteryl esters. Avasimibe (3 micromol/L) increased bile acid synthesis (2.9-fold) after preincubation with betaVLDL and cholesterol 7alpha-hydroxylase activity (1.7- and 2.6-fold, with or without betaVLDL), the latter paralleled by a similar induction of its messenger RNA (mRNA). Hepatocytes treated with avasimibe showed a shift from storage and secretion of cholesteryl esters to conversion of cholesterol into bile acids. In rats fed diets containing different amounts of cholesterol and cholate, avasimibe reduced plasma cholesterol (by 52% to 71%) and triglyceride levels (by 28% to 62%). Avasimibe did not further increase cholesterol 7alpha-hydroxylase activity and mRNA in cholesterol-fed rats, but prevented down-regulation by cholate. Avasimibe did not affect sterol 27-hydroxylase and oxysterol 7alpha-hydroxylase, 2 enzymes in the alternative pathway in bile acid synthesis. No increase in the ratio of biliary excreted cholesterol to bile acids was found, indicating that ACAT inhibition does not result in a more lithogenic bile. Avasimibe increases bile acid synthesis in cultured hepatocytes by enhancing the supply of free cholesterol both as substrate and inducer of cholesterol 7alpha-hydroxylase. These effects may partially explain the potent cholesterol-lowering effects of avasimibe in the rat.

Acetamides↗
PubMed1999

The unique acyl chain specificity of biliary phosphatidylcholines in mice is independent of their biosynthetic origin in the liver.

The liver synthesizes phosphatidylcholine (PC) de novo from choline via the CDP-choline pathway, and from phosphatidylethanolamine (PE) via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Significant amounts of PC, which are highly specific in their acyl chain composition, are secreted into bile by the liver. To determine whether either of the 2 PC biosynthetic routes is sufficient to provide physiological PC concentrations in bile, or is responsible for the unique acyl chain composition of bile PC, we analyzed gallbladder bile composition in mice that synthesized PC either via the PEMT pathway (induced by feeding a choline-deficient diet) or the CDP-choline pathway (based on genetic PEMT-deficiency). The PC concentration in gallbladder bile of mice that synthesize PC mainly via the CDP-choline pathway was comparable with control mice that synthesize PC via both pathways, whereas it was reduced by approximately 40% in mice that synthesize PC via the PEMT pathway. The acyl chain composition of bile PC was similar irrespective of the active PC biosynthetic pathway in the liver. These data demonstrate that the CDP-choline pathway alone, but not the PEMT pathway alone, can account for physiological concentrations of PC in gallbladder bile. Moreover, the specificity of biliary PC fatty acyl composition is determined independently from the synthetic origin of PC.

Animals↗
PubMed1999

Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum gamma-glutamyltransferase activity. Patients with normal gamma-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24. Also, patients with bile acid synthesis defects have low gamma-glutamyltransferase activity. We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations. METHODS: BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients. Bile salt kinetics were studied in 1 patient. RESULTS: Sixteen of 28 liver samples showed no canalicular BSEP staining. Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples. Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients. No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal. Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 +/- 0.2 mmol/L (n = 9; <1% of normal) and in BSEP-positive PFIC patients 18.1 +/- 9.9 mmol/L (n = 3; 40% of normal). The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients. CONCLUSIONS: The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression. Biliary secretion of bile salts is greatly reduced in BSEP-negative patients.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Localization of the Wilson's disease protein in human liver.

BACKGROUND & AIMS: Wilson's disease is an autosomal-recessive disorder of copper metabolism that results from the absence or dysfunction of a copper-transporting P-type adenosine triphosphatase that leads to impaired biliary copper excretion and disturbed holoceruloplasmin synthesis. To gain further insight into the role of the Wilson's disease protein in hepatic copper handling, its localization in human liver was investigated. METHODS: By use of a specific antibody, localization of the Wilson's disease protein was studied in liver membrane fractions and liver sections by immunoblotting, immunohistochemistry, and double-label confocal scanning laser microscopy. RESULTS: The 165-kilodalton protein, found by immunoblotting, was most abundant mainly in isolated plasma membrane fractions enriched in canalicular domains. Immunohistochemistry revealed intracellular punctuate staining of hepatocytes in certain regions of the liver, whereas a canalicular membrane staining pattern was observed in other regions. Double-labeling studies showed that in the latter regions the transporter is present mainly in vesicular structures just underneath the canalicular membrane that are positive for markers of the trans-Golgi network. A weak staining of the canalicular membrane, identified by staining for P-glycoprotein, was observed. CONCLUSIONS: These results show that in human liver the Wilson's disease protein is predominantly present in trans-Golgi vesicles in the pericanalicular area, whereas relatively small amounts of the protein appear to localize to the canalicular membrane, consistent with a dual function of the protein in holoceruloplasmin synthesis and biliary copper excretion.

Adenosine Triphosphatases↗
PubMed1999

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) induce hepatic expression of the phospholipid translocase mdr2 in rats.

BACKGROUND & AIMS: Biliary cholesterol secretion is coupled to that of phospholipids in a process controlled by mdr2 P-glycoprotein activity and bile salt secretion. Statins, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been shown to affect hepatobiliary lipid secretion in rats. The aim of this study was to relate the effects of statins on bile formation to the expression of mdr2 and other hepatic adenosine triphosphate-dependent transport proteins involved in bile formation in rats. METHODS: Rats received simvastatin- or pravastatin-containing chow continuously for 5 days. In one group of rats, simvastatin treatment was withdrawn 9-12 hours before the end of the experiment to induce biliary cholesterol hypersecretion (rebound). Bile and liver tissue were collected for lipid analysis, and hepatic messenger RNA (mRNA) and protein levels were studied by reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULTS: Simvastatin feeding did not alter biliary bile salt secretion. Secretion of phospholipids and cholesterol was stimulated by 74% and 90%, respectively, in the simvastatin-continuous group and by 72% and 235%, respectively, in the rebound group compared with controls. mdr2 mRNA levels increased only in the continuous group. mdr2 protein levels increased in both simvastatin-fed groups. Induction was most pronounced in periportal hepatocytes. mdr1b mRNA levels were moderately increased in both simvastatin-fed groups. Levels of other hepatic transport proteins did not change. Similar results were obtained in pravastatin-fed rats. CONCLUSIONS: Statins increase expression of mdr2 and mdr1b in rats, revealing a novel effect of these commonly used drugs.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Biliary fibrosis associated with altered bile composition in a mouse model of erythropoietic protoporphyria.

BACKGROUND & AIMS: Reduced activity of ferrochelatase in erythropoietic protoporphyria (EPP) results in protoporphyrin (PP) accumulation in erythrocytes and liver. Liver disease may occur in patients with EPP, some of whom develop progressive liver failure that necessitates transplantation. We investigated the mechanisms underlying EPP-associated liver disease in a mouse model of EPP. METHODS: Liver histology, indicators of lipid peroxidation, plasma parameters of liver function, and bile composition were studied in mice homozygous (fch/fch) for a point mutation in the ferrochelatase gene and in heterozygous (fch/+) and wild-type (+/+) mice. RESULTS: Microscopic examination showed bile duct proliferation and biliary fibrosis with portoportal bridging in fch/fch mice. PP content was 130-fold increased, and thiobarbituric acid-reactive substances (+30%) and conjugated dienes (+75%) were slightly higher in fch/fch than in fch/+ and +/+ livers. Levels of hepatic thiols (-12%) and iron (-52%) were reduced in fch/fch livers. Liver enzymes and plasma bilirubin were markedly increased in the homozygotes. Plasma bile salt levels were 80 times higher in fch/fch than in fch/+ and +/+ mice, probably related to the absence of the Na(+)-taurocholate cotransporting protein (Ntcp) in fch/fch liver. Paradoxically, bile flow was not impaired and biliary bile salt secretion was 4 times higher in fch/fch mice than in controls. Up-regulation of the intestinal Na(+)-dependent bile salt transport system in fch/fch mice may enhance efficiency of bile salt reabsorption. The bile salt/lipid ratio and PP content of fch/fch bile were increased 2-fold and 85-fold, respectively, compared with +/+, whereas biliary glutathione was reduced by 90%. Similar effects on bile formation were caused by griseofulvin-induced inhibition of ferrochelatase activity in control mice. CONCLUSIONS: Bile formation is strongly affected in mice with impaired ferrochelatase activity. Rather than peroxidative processes, formation of cytotoxic bile with high concentrations of bile salts and PP may cause biliary fibrosis in fch/fch mice by damaging bile duct epithelium.

Animals↗
PubMed1999

Validation in an animal model of the carbon 13-labeled mixed triglyceride breath test for the detection of intestinal fat malabsorption.

OBJECTIVE: To determine, in a rat model of fat malabsorption, the potency of the carbon 13-labeled mixed triglyceride ((13)C-MTG) breath test as a noninvasive, patient-friendly replacement for classic fat balance studies. STUDY DESIGN: Comparison of the percentage of fat absorption, detected by fat balance, with the (13)CO(2) recovery of the (13)C-MTG breath test in rats fed high-fat chow and varying amounts of the lipase inhibitor, orlistat (0, 50, 200, and 800 mg per kilogram of chow), for 5 days. RESULTS: On orlistat administration, total fat absorption decreased from 80.2% +/- 2.2% to 32.8% +/- 3.7% (mean +/- SEM, 0 mg and 800 mg of orlistat per kilogram of chow, respectively; P <.001). Correspondingly, breath (13)CO(2) recovery from (13)C-MTG at 6 hours decreased from 84.5% +/- 7.8% to 42.0% +/- 1.5% of the dose (0 mg and 800 mg of orlistat per kilogram of chow, respectively; P <.001). The 6-hour recovery of breath (13)CO(2) appeared to be highly correlated with the percentage of fat absorption (r = 0.88, P <.001). In rats with fat absorption higher than 70%, however, the coefficient of variation of the (13)C-MTG breath test was 3-fold larger than that of the fat balance. CONCLUSIONS: The (13)C-MTG breath test could potentially replace the fat balance method for comparing fat absorption efficacy between groups. Yet, a considerable interindividual variation of the (13)C-MTG breath test under conditions of relatively mild fat malabsorption does not support its application for diagnostic purposes in individuals.

Animals↗
PubMed1999

Decreased Na+-dependent taurocholate uptake and low expression of the sinusoidal Na+-taurocholate cotransporting protein (Ntcp) in livers of mdr2 P-glycoprotein-deficient mice.

BACKGROUND/AIMS: Ntcp-mediated uptake of bile salts at the basolateral membrane of hepatocytes is required for maintenance of their enterohepatic circulation. Expression of Ntcp is reduced in various experimental models of cholestasis associated with increased plasma bile salt concentrations. Mdr2 P-glycoprotein-deficient mice lack biliary phospholipids and cholesterol but show unchanged biliary bile salt secretion and increased bile flow. These mice are evidently not cholestatic, but plasma bile salt concentrations are markedly increased. The aim of this study was to investigate the role of Ntcp in the elevated bile salt levels in mdr2 P-glycoprotein-deficient (-/-) mice. METHODS: Plasma membranes were isolated from male wild-type (+/+) and mdr2 (-/-) mice for measurement of Na+-dependent taurocholate transport and assessment of Ntcp protein levels by Western blotting. Northern blot analysis and competitive reverse transcription-polymerase chain reaction were used to determine hepatic Ntcp mRNA levels. RESULTS: Kinetic analysis showed a 2-fold decrease in the Vmax of Na+-dependent taurocholate transport, with an unaffected Km in (-/-) mice compared with (+/+) controls. Ntcp protein levels were 4-6-fold reduced in plasma membranes of (-/-) mice relative to sex-matched controls. Surprisingly, hepatic Ntcp mRNA levels were not significantly affected in the (-/-) mice. CONCLUSIONS: Elevated plasma bile salt levels in mdr2 P-glycoprotein-deficient mice in the absence of overt cholestasis are associated with reduced Ntcp expression and transport activity. This is due to posttranscriptional down-regulation of Ntcp.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Different pathways of canalicular secretion of sulfated and non-sulfated fluorescent bile acids: a study in isolated hepatocyte couplets and TR- rats.

BACKGROUND/AIMS: Fluorescent bile acids have proved useful for characterizing bile salt transport mechanisms. The aim of this study was to further validate the use of lysyl-fluorescein conjugated bile acid analogues as surrogate bile acids. METHODS: We analyzed biliary excretion kinetics of cholyl lysyl fluorescein (CLF), lithocholyl lysyl fluorescein (LLF) and sulfo-lithocholyl lysyl fluorescein (sLLF), both in the isolated rat hepatocyte couplet model and in TR- rats with a selective canalicular transport defect of non-bile acid organic anions. RESULTS: CLF and LLF, which like their natural nonsulfated bile acid congeners are expected to be handled by the canalicular bile salt export pump, were transferred into the bile canaliculus much faster than sLLF, a putative substrate for the canalicular multispecific organic anion transporter in both the in vivo and the in vitro models employed. The contention that different transport systems are involved in sulfated and non-sulfated lysyl fluorescein conjugated bile acids biliary excretion was supported further by studies using TR- rats, in which the cumulative biliary excretion of sLLF was reduced to 6% as compared with that of normal Wistar rats, in good agreement with values for its naturally-occurring radiolabeled parent compound sulfoglycolithocholate. In contrast, CLF and LLF were reduced to 66% and 52%, similar values to these for their congeners, [14C] glycocholate and [14C] lithocholate. CONCLUSION: The close similarity in behavior of lysyl fluorescein conjugated bile acids to that of their naturally-occurring parent compounds in these different models gives support for both sulfated and nonsulfated lysyl fluorescein conjugated bile acids as substitute molecules for studies of bile acid transport.

Animals↗
PubMed1999

Variability of human hepatic UDP-glucuronosyltransferase activity.

The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases using photoaffinity labeling, immunoblotting and enzymatic assays. There was wide inter-individual variation in photoincorporation of the photoaffinity analogs, [32P]5-azido-UDP-glucuronic acid and [32P]5-azido-UDP-glucose and enzymatic glucuronidation of substrates specific to the two subfamilies of UDP-glucuronosyltransferases. However, the largest differences were between subjects with liver disease. Glucuronidation activities toward one substrate from each of the UDP-glucuronosyltransferases subfamilies, 1A and 2B, for control and liver disease, respectively, were 1.7-4.5 vs 0.4-4.7 nmol/mg x min for hyodeoxycholic acid (2B substrate) and 9.2-27.9 vs 8.1-75 nmol/mg x min for pchloro-m-xylenol (1A substrate). Microsomes from a patient with chronic tyrosinemia (HL32) photoincorporated [32P]5-azido-UDP-glucuronic acid at a level 1.5 times higher than the other samples, was intensely photolabeled by [32P]5-azido-UDP-glucose and had significantly higher enzymatic activity toward p-chloro-m-xylenol. Immunoblot analysis using anti-UDP-glucuronosyltransferase antibodies demonstrated wide inter-individual variations in UDP-glucuronosyltransferase protein with increased UDP-glucuronosyltransferase protein in HL32 microsomes, corresponding to one of the bands photolabeled by both probes. Detailed investigation of substrate specificity, using substrates representative of both the 1A (bilirubin, 4-nitrophenol) and 2B (androsterone, testosterone) families was carried out with HL32, HL38 (age and sex matched control) and HL18 (older control). Strikingly increased (5-8-fold) glucuronidation activity was seen in comparison to HL18 only with the phenolic substrates. The results indicate that one or more phenol-specific UDP-glucuronosyltransferase 1A isoforms are expressed at above normal levels in this tyrosinemic subject.

Adolescent↗
PubMed1999

Differential effects of 17alpha-ethinylestradiol on the neutral and acidic pathways of bile salt synthesis in the rat.

Effects of 17alpha-ethinylestradiol (EE) on the neutral and acidic biosynthetic pathways of bile salt (BS) synthesis were evaluated in rats with an intact enterohepatic circulation and in rats with long-term bile diversion to induce BS synthesis. For this purpose, bile salt pool composition, synthesis of individual BS in vivo, hepatic activities, and expression levels of cholesterol 7alpha-hydroxylase (CYP7A), and sterol 27-hydroxylase (CYP27), as well as of other enzymes involved in BS synthesis, were analyzed in rats treated with EE (5 mg/kg, 3 days) or its vehicle. BS pool size was decreased by 27% but total BS synthesis was not affected by EE in intact rats. Synthesis of cholate was reduced by 68% in EE-treated rats, while that of chenodeoxycholate was increased by 60%. The recently identified Delta22-isomer of beta-muricholate contributed for 5.4% and 18.3 % (P < 0.01) to the pool in control and EE-treated rats, respectively, but could not be detected in bile after exhaustion of the pool. A clear reduction of BS synthesis was found in bile-diverted rats treated with EE, yet biliary BS composition was only minimally affected. Activity of CYP7A was decreased by EE in both intact and bile-diverted rats, whereas the activity of the CYP27 was not affected. Hepatic mRNA levels of CYP7A were significantly reduced by EE in bile-diverted rats only; CYP27 mRNA levels were not affected by EE. In addition, mRNA levels of sterol 12alpha-hydroxylase and lithocholate 6beta-hydroxylase were increased by bile diversion and suppressed by EE. This study shows that 17alpha-ethinylestradiol (EE)-induced intrahepatic cholestasis in rats is associated with selective inhibition of the neutral pathway of bile salt (BS) synthesis. Simultaneous impairment of other enzymes in the BS biosynthetic pathways may contribute to overall effects of EE on BS synthesis.

Animals↗
PubMed1998

In vivo determination of very-low-density lipoprotein-apolipoprotein B100 secretion rates in humans with a low dose of l-[1-13C]valine and isotope ratio mass spectrometry.

The aim of the present study was to determine the rate of very-low-density lipoprotein (VLDL)-apolipoprotein (apo) B100 secretion in humans with a minimized amount of l-[1-13C]valine infusion in combination with the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. To compare this method with the conventional gas chromatography/mass spectrometry (GC/MS) technique, two different dosages of l-[1-13C]valine and both analytical techniques were compared in a single study. A priming dose of l-[1-13C]valine (2 micromol/kg) followed by a constant infusion (2 micromol/kg/h) was given for 3 h, directly followed by a second priming dose (15 micromol/kg) and a constant infusion (15 micromol/kg/h) for 4 h. The fractional secretion rate obtained by GC/C/IRMS measurements from the first 3 h of infusion (mean +/- SD: 0.22 +/- 0.09 pools/h) was similar to that obtained by GC/MS during the last 4 h of infusion (0. 23 +/- 0.07 pools/h; P = 0.56). In conclusion, superior analytical accuracy and sensitivity of GC/C/IRMS enable measurements of VLDL-apo B100 secretion with much lower doses of l-[1-13C]valine and allow for reduction of experimental costs.

Apolipoprotein B-100↗