Effects of ursodeoxycholic acid on conjugated bile acids and progesterone metabolites in serum and urine of patients with intrahepatic cholestasis of pregnancy

Background/Aims and Methods: The mechanism(s) behind the effects of ursodeoxycholic acid on serum steroid sulphate profiles in patients with intrahepatic cholestasis of pregnancy is not clear. Conjugated progestone metabolites and bile acids have therefore been analyzed in serum and urine of patients with intrahepatic cholestasis of pregnancy before and during treatment with ursodeoxycholic acid using chromatographic and mass spectrometric methods.Results: The concentration of glycine-/taurine-conjugated bile acids decreased from 8.9±3μmol/l (mean± SEM) before treatment to 1.8±0.6 sml/l during treatment with ursodeoxycholic acid. The total bile acid excretion in urine decreased from 56±14 to 32±5.6 μmol/g creatinine. The proportion of cholic acid in serum and urine, and of 1β-, 2β- and 6α-hydroxylated cholic acids in urine decreased markedly during ursodeoxycholic acid while the percentages of 3α, 12α-dihydroxy-3-oxo-4-cholenoic acid and chenodeoxycholic acid were unchanged. The levels in serum and excretion in urine of sulphated steroids decreased during ursodoexycholic acid, by 45–49% for disuphates and 33–35% for monosulphates. The ratios of 3α- to 3β-hydroxysteroid disulphates were lowered by ursodeoxycholic acid from 1.1 (mean) to 0.68 in serum, and from 1.2 to 0.70 in urine. The corresponding ratios for monosulphates before the during ursodeoxycholic acid were 6.9 and 4.5, respectively, in serum, and 21 and 5.2 respectively, in urine. The major monosulphates in urine, dominated by 5α-pregnane-3α, 20α-diol, were also conjugated with N-acetylglucosamine. The excretion of these double conjugates decreased from 27±8.4 to 15±5.3 μmol/g creatinine during ursodoexycholic acid. In contrast to suplhated steroids, the concentrations of glucruronides were unchanged in serum and their excretion in urine tended to increase during ursodeoxycholic acid. The metabolism of ursodeoxycholic acid was similar to that described in nonpregnant subjects. In addition to metabolites hydroxylated in the 1β-, 5β-, 6αβ and 22-positions, a 4-hydroxy-ursodeoxycholic acid was tentatively identified. This occurred predominantly as a double conjugate with glucine/taurine and glucuronic acid, as did other 4-hydroxylated bile acids of probable foetal origin.Conclusions: The results are compatible with the contention that ursodeoxycholic acid stimulates the biliary excretion of sulphated progesterone metabolites, particularly those with a 3α-hydroxy-5α(H) configuration and disulphates. The effects(s) appears to be independent of the stimulation of bile acid secretion. An effect of ursodeoxycholic acid on the reductive metabolism of progesterone cannot be excluded.     

Urinary excretion of bile acids in bile duct-ligated rats

Background. In patients with complete bile duct obstruction, the only pathway of bile acid elimination is through the urine. However, the urinary excretion of various bile acid conjugates in the presence of bile duct obstruction has not been clarified. Given this factor, the urinary excretion of various bile acids was compared in rats that were bile duct-ligated for 3 days. Methods. After urinary bladder cannulation, radiolabeled bile acids were intravenously injected, and urine samples were collected every 2 h for 6 h, and radioactivity was counted. Results. Urinary excretion (cumulative percent dose during 6 h) of taurocholate and cholate was similar (19.3% and 16.8%). Urinary excretion of tauroursodeoxycholate, lithocholate, and taurolithocholate-sulfate was less effective (12.7%, 9.8% and 2.1%, respectively). Cholate was mostly conjugated with taurine, and lithocholate was mostly conjugated with taurine and further hydroxylated. Conclusions. These results indicate that unconjugated bile acids were taken up by the liver and excreted into the blood after further biotransformation even under conditions of complete bile duct obstruction. Although bile acid sulfates are the major bile acids in the urine of patients with obstructive jaundice, monohydroxylated bile acids are considered not to be so effectively excreted into the urine, even with conjugation with taurine and sulfate, in rats. Received: October 4, 2002 / Accepted: November 8, 2002 RID="*" ID="*" Reprint requests to: H. Takikawa Acknowledgments. This work was partly supported by a Grant-in-Aid by the Japan Society for the Promotion of Science (C2-14570510).     

Intrahepatic cholestasis of pregnancy: Amelioration of pruritus by UDCA is associated with decreased progesterone disulphates in urine

Intrahepatic cholestasis of pregnancy (ICP) is characterized by pruritus, elevated bile acids, and, specifically, elevated disulphated progesterone metabolites. We aimed to study changes in these parameters during treatment with dexamethasone or ursodeoxycholic acid (UDCA) in 40 out of 130 women included in the Swedish ICP intervention trial (26 randomized to placebo or UDCA, 14 randomized to dexamethasone). Serum bile acid profiles and urinary steroid hormone metabolites were analyzed using isotope-dilution gas chromatography-mass spectrometry and electrospray-mass spectrometry. We found that all patients displayed ICP-typical serum bile acid profiles with >50% cholic acid at baseline but almost 80% UDCA upon treatment with this bile acid. In UDCA-treated patients, relative amounts of disulphated progesterone metabolites in urine decreased by 34%, 48% (P < 0.05), and 55% (P < 0.05) after 1, 2, and 3 weeks of treatment, respectively, which was significantly correlated to improvements of pruritus scores but not to serum bile acid levels. In contrast, in patients randomized to dexamethasone or placebo, no changes in steroid metabolites or pruritus scores were observed. Conclusion: UDCA treatment in ICP decreased urinary excretion of disulphated progesterone metabolites, suggesting that amelioration of pruritus is connected to stimulation of hepatobiliary excretion of progesterone disulphates.     

Portal vein bile acids in patients with severe inflammatory bowel disease

The incidence of several forms of liver disease associated with inflammatory bowel disease has been putatively ascribed to a toxic effect on the liver of portal vein bile acids abnormal in type or amount. To examine this possibility, total bile acid concentrations (sulphated and non-sulphated) were measured by gas-liquid chromatography in inferior mesentric vein serum of 19 patients undergoing colectomy for severe inflammatory bowel disease (IBD). Similar determinations were obtained on a control group of eight patients requiring colectomy for other non-inflammatory conditions. Mean values for mesenteric vein serum bile acid concentrations (μM/1) were 19·6±1·8 in controls and 16·3±2·0 in IBD. The mean sulphated bile acid fraction did not exceed 10% of total, although there was considerable variability (up to 40% of total). Lithocholic acid levels (entirely sulphated in all patients) were low. Although the IBD group showed a more than two-fold increase in mean lithocholate concentration (0·54±0·15 μM/1) over controls (0·21 ± μM/1), this difference was not statistically significant. No significant intra-group difference was noted in the non-sulphated and sulphated fractions for cholic, chenodeoxycholic, and deoxycholic acid species, respectively. No unidentified or unusual bile acids were observed. There was no correlation between bile acid measurements and liver histology. These findings fail to support the hypothesis that liver disease often found in association with severe inflammatory bowel disease represents a form of bile acid toxicity. The invariable finding of total sulphation of the lithocholic acid fraction even in the presence of severe mucosal disease was unexpected.     

Treatment of pruritus in primary biliary cirrhosis with rifampin. Results of a double-blind, crossover, randomized trial

The cause of pruritus of cholestasis is unknown. We have hypothesized that pruritus may be caused by an indirect effect of high hepatic concentrations of toxic bile acids. To test this hypothesis, we have conducted a double-blind, controlled, crossover clinical trial of rifampin, an agent that inhibits hepatic bile acid uptake and may detoxify hepatic bile acids by stimulation of mixed-function oxidases. Nine patients with primary biliary cirrhosis received 300-450 mg/day of rifampin and placebo sequentially, in random order. Each treatment was administered for 14 days, with a 14-day washout between treatments. Endpoints included patient preference, changes in a daily visual analogue scale pruritus score, and amount of cholestyramine ingested. Antipyrine elimination rates and serum bile acids were tested at the end of each treatment period. All 9 patients completed the trial and 8 of them preferred rifampin to placebo (p = 0.03). There were no adverse reactions. Visual analogue scale pruritus scores showed no significant placebo response or any effect from the order of treatment, but did show a highly significant reduction in pruritus in response to rifampin (p less than 0.002). This effect was evident within the first week of rifampin treatment. Rifampin produced a 33% reduction in antipyrine plasma half-life, but no change in fasting total serum bile acids. Cholestyramine usage did not change significantly. We conclude that rifampin is useful for short-term relief of pruritus in primary biliary cirrhosis; however, the mechanism of this effect is unknown. Longer trials are needed, as are trials in other cholestatic disorders.     

Urinary excretion of bile acid glucosides and glucuronides in extrahepatic cholestasis

Recently the formation of bile acid glucosides has been described as a novel conjugation mechanism in vitro and in vivo. In 10 patients with extrahepatic cholestasis caused by carcinoma of the head of the pancreas we investigated excretion rates and profiles of urinary bile acid glucosides. Urinary bile acid glucosides and, for comparison, bile acid glucuronides were extracted and characterized according to established methods. In controls total urinary bile acid glucoside excretion was 0.22 +/- 0.03 mumol/24 hr (mean +/- S.E.M.)-in the range of bile acid glucuronide excretion (0.41 +/- 0.06 mumol/24 hr; mean +/- S.E.M.). A gas chromatography-mass spectrometry-characterized trihydroxy bile acid glucoside of still-unknown hydroxyl positions accounted for 65% of total urinary bile acid glucosides. In extrahepatic cholestasis total urinary bile acid glucoside excretion was 0.52 +/- 0.13 mumol/24 hr (mean +/- SEM), yet significantly lower than bile acid glucuronide excretion (1.53 +/- 0.13 mumol/24 hr; mean +/- SEM; p less than 0.001). In cholestasis the primary bile acid derivatives cholic and chenodeoxycholic acid glucosides amounted to 90%, whereas the trihydroxy bile acid glucoside had decreased to 5% of total bile acid glucoside excretion, indicating its alteration during enterohepatic circulation. The data establish the composition and quantity of urinary bile acid glucosides in healthy controls and cholestasis and constitute a quantitative comparison with another glycosidic conjugation reaction, bile acid glucuronidation.     

Intestinal absorption of bile acid glucuronides in rats

While the intestinal absorption of taurine, glycine, and sulfate conjugates of bile acids has been studied extensively, nothing is known about the absorption of bile acid glucuronides. In the present study, the intestinal phase of the enterohepatic circulation of two bile acid glucuronides was examined. [3-³ H]cholic acid 3-O--d-glucuronide or [3^–3 H]lithocholic acid 3-O--d-glucuronide was perfused through isolated segments of ileum or jejunum with intact blood supply in rats prepared with a biliary fistula. [¹⁴C]Taurocholic acid was perfused simultaneously with each glucuronide to compare glucuronide absorption with that of an actively transported bile acid. Intestinal absorption was determined by measuring the rate of secretion of labeled bile acid in bile. The absorption of [³H]cholic acid glucuronide by the ileum and jejunum was one fortieth and one eighth, respectively, that of [¹⁴C]taurocholic acid. Comparison of the two glucuronides show that [³H]lithocholic acid glucuronide absorption was 18 and 10 times greater than [³H]cholic acid glucuronide absorption from the jejunum and ileum, respectively. Collectively, the above observations suggest that glucuronidation of bile acids markedly reduces absorption from the small intestine.This work was supported in part by the National Institutes of Health, grant HD-14198, and the March of Dimes Foundation, grant G-305.     

Biliary excretion of sulfated bile acids and organic anions in zone 1- and zone 3-injured rats

Background and Aims: There are several reports on the biliary excretion of bile acids and organic anions in zone 1‐ and zone 3‐injured rat liver, but the results are controversial. In order to dissolve the discrepancy between previous works about the role of hepatic zonation on the hepatic handling of the substrates of multidrug resistance protein 2, the biliary excretion of sulfated bile acids, pravastatin and phenolphthalein glucuronide was studied in zone 1‐ and zone 3‐injured rats. Methods: Zone 1 and zone 3 injury were caused by allyl alcohol and bromobenzene, respectively. Bile acid sulfates, pravastatin and phenolphthalein glucuronide were administered i.v. to bile duct‐cannulated rats, and their biliary excretion was studied. Results: The biliary excretion of a tracer dose of taurolithocholate‐3‐sulfate and its excretory maximum were unchanged in zone 1 injury, but were diminished in zone 3 injury, whereas the biliary excretion of taurochenodeoxycholate‐3‐sulfate was unchanged in zone 1 and zone 3 injury. The biliary excretion of pravastatin and phenolphthalein glucuronide was markedly decreased only in zone 3 injury, whereas the excretory maximum of phenolphthalein glucuronide was decreased in both zone 1 and zone 3 injury. Conclusions: These findings indicate that zone 3 is important for the biliary excretion of substrates of multidrug resistance protein 2.     

Sulphated and unsulphated bile acids in serum, bile, and urine of patients with cholestasis.

Samples of serum, bile, and urine were collected simultaneously from patients with cholestasis of varying aetiology and from patients with cirrhosis; their bile acid composition was determined by gas/liquid chromatography and mass spectrometry. In cholestasis, the patterns in all three body fluids differed consistently and strikingly. In serum, cholic acid was the major bile acid and most bile acids (greater than 93%) were unsulphated, whereas, in urine, chenodeoxycholic was the major bile acid, and the majority of bile acids (greater than 60%) were sulphated. Secondary bile acids were virtually absent in bile, serum, and urine. The total amount of bile acids excreted for 24 hours correlated highly with the concentration of serum bile acids; in patients with complete obstruction, urinary excretion averaged 71-6 mg/24 h. In cirrhotic patients, serum bile acids were less raised, and chenodeoxycholic acid was the predominant acid. In healthy controls, serum bile acids were consistently richer in chenodeoxycholic acid than biliary bile acids, and no bile acids were present in urine. No unusual monohydroxy bile acids were present in patients with primary biliary cirrhosis, but, in several patients, there was a considerable amount of hyocholic acid present in the urinary bile acids. The analyses of individual bile acids in serum and urine did not appear to provide helpful information in the differential diagnosis of cholestasis. Thus, in cholestasis, conjugation of chenodeoxycholic acid with sulphate becomes a major biochemical pathway, urine becomes a major route of bile acid excretion, and abnormal bile acids are formed.     

Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the α-subunit of the human cardiac Na+ channel

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na⁺ current (I_Na,α) in human embryonic kidney (HEK293t) cells transfected with the α-subunit of the human cardiac Na⁺ channel (hH1_α). Extracellular application of 0.01 to 30 μM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I_Na,α with an IC₅₀ of 0.51 ± 0.06 μM. The EPA-induced suppression of I_Na,α was concentration- and voltage-dependent. EPA at 5 μM significantly shifted the steady-state inactivation relationship by −27.8 ± 1.2 mV (n = 6, P < 0.0001) at the V_1/2 point. In addition, EPA blocked I_Na,α with a higher “binding affinity” to hH1_α channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 μM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 μM EPA, from 2.1 ± 0.8 ms for control to 34.8 ± 2.1 ms (n = 5, P < 0.001). The effects of EPA on I_Na,α were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), α-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 μM and all-trans-retinoic acid at 10 μM had similar effects on I_Na,α as EPA. Even 5 μM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited I_Na,α. In contrast, 5 μM EPA ethyl ester did not alter I_Na,α (8 ± 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress I_Na,α with high “binding affinity” to hH1_α channels in the inactivated state and prolong the duration of recovery from inactivation.     

Uptake of bacterial lipopolysaccharide and expression of tumor necrosis factor-α-mRNA in isolated rat intrahepatic bile duct epithelial cells

AIM: To study the uptake of bacterial lipopolysaccharides (LPS) and expression of tumor necrosis factor α-mRNA (TNF-α-mRNA) with cultured rat intrahepatic bile duct epithelial cells.METHODS: By using fluorescent, immunohistochemical and in situ hybridization techniques, the uptake of Escherichia coli LPS and expression of TNF-α-mRNA with isolated rat intrahepatic bile duct epithelial cells were observed with confocal laser scanning microscopy.RESULTS: Positive reactions to LPS were found in the cytoplasm of isolated intrahepatic bile duct epithelial cells after incubation with LPS for 15 min and the FITC fluorescent intensity against LPS was significantly higher than that of the controls (121.45 μFI/μm² ± 15.62 μFI/μm² vs 32.12 μFI/μm² ± 9.64 μFI/μm², P < 0.01). After incubation with LPS for 3 h, fluorescein isocyanate (FITC) fluorescent intensities of the expression of TNF-α-mRNA with fluorescent in situ hybridization in the cytoplasm and nuclei of the cultured bile duct epithelial cells were significantly higher than those of the controls (189.15 μFI/μm² ± 21.33 μFI/μm² vs 10.00 μFI/μm² ± 8.99 μFI/μm², 64.85 μFI/μm² ± 14.99 μFI/μm² vs 21.20 μFI/μm² ± 2.04 μFI/μm², respectively (P < 0.01)). The increase of FITC fluorescent intensity of TNF-α-mRNA expression in the cytoplasm peaked at 6 h after incubation (221.38 μFI/μm² ± 22.99 μFI/μm²). At various time points after incubation with LPS, the increase of fluorescent intensities of TNF-α-mRNA in the cytoplasm were much higher than those in the nuclei (P < 0.01).CONCLUSION: LPS can act on and enter into isolated intrahepatic bile duct epithelial cells and stimulate the expression of TNF-α-mRNA.     

Comparative regulation of hepatic sterol 27-hydroxylase and cholesterol 7α-hydroxylase activities in the rat, guinea pig, and rabbit: Effects of cholesterol and bile acids

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7α-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 5β-cholestane-3α,7α-diol, and 5β-cholestane-3α,7α,12α-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial sterol 27-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7α-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7α-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7α-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.     

Effects of ursodeoxycholate, its glucuronide and disulfate and beta-muricholate on biliary bicarbonate concentration and biliary lipid excretion.

We previously reported that high-dose ursodeoxycholate (UDC) infusion in rats resulted in extensive glucuronidation of UDC, and speculated that the glucuronidation causes bicarbonate-rich hypercholeresis induced by UDC (Takikawa, H., Sano, N., Narita, T. and Yamanaka, M. Hepatology 1990; 11: 743-749). To test this hypothesis, UDC, UDC-3-O-glucuronide, UDC-3,7-disulfate and beta-muricholate were separately and intravenously infused into rats (1 mumol/min per 100 g), and biliary bicarbonate concentration was measured. The effects of these bile acids on biliary lipid secretion were also studied. All four bile acids increased bile flow and biliary bile acid excretion. UDC and beta-muricholate significantly increased biliary bicarbonate concentration, whereas UDC glucuronide and disulfate did not. Independence of UDC glucuronide excretion and biliary bicarbonate concentration was also confirmed in EHBR, a hyperbilirubinemic mutant Sprague-Dawley rat. In this case biliary bicarbonate concentration also increased in spite of the absence of UDC glucuronide in the bile after UDC infusion. Biliary phospholipid secretion was increased with UDC, unchanged with beta-muricholate, and decreased with UDC glucuronide and disulfate. Biliary cholesterol secretion was increased with UDC, unchanged with beta-muricholate and UDC glucuronide, and decreased with UDC disulfate. These data indicate that glucuronidation is not the cause of bicarbonate-rich hypercholeresis induced by UDC but that glucuronidation and sulfation change the effect of UDC on biliary lipid secretion.     

Clinical and genetic features of congenital bile acid synthesis defect with a novel mutation in AKR1D1 gene sequencing: Case reports

Rationale:Congenital bile acid synthesis defect (BASD) is a rare disease caused by mutations in the aldo-keto reductase 1D1 gene, which encodes the primary Δ4-3-oxosteroid 5β-reductase enzyme. Early disease diagnosis is critical for early treatment with bile acid replacement therapy, with an excellent chance for recovery. In contrast, protracted diagnosis and treatment may lead to poor outcomes, including decompensated hepatic cirrhosis, liver transplant, and even death.Patient concerns:Three clinical congenital bile acid synthesis defect cases in the Vietnamese population are herein reported. These pediatric patients presented with symptoms of prolonged postpartum jaundice and abnormal loose stool (mucus, lipids, and white). The clinical examinations showed hepatosplenomegaly. Urinalysis showed a very low fraction of primary bile acids and atypical 3-oxo-Δ4- bile acids in all three patients.Diagnoses:The patients were diagnosed with primary Δ4-3-oxosteroid 5β-reductase deficiency. Next-generation gene sequencing revealed two homozygous mutations in the aldo-keto reductase family 1 member D1 gene. The first is a documented variant, c.797G>A (p.Arg266Gln), and the second is a novel mutation at c.155T>C (p.Ile52Thr).Interventions:Immediately after diagnosis, patients were treated with oral chenodeoxycholate 5 mg/kg/d.Outcomes:The patients’ symptoms, signs, and primary bile acids levels improved significantly.Lessons:Clinicians should consider genetic disorders related to cholestasis for effective and life-saving treatment. A prompt genetic analysis by next-generation gene sequencing enables patients to access bile acid replacement therapy earlier, significantly improving short- and long-term outcomes.     

Biliary excretion of bile acids and organic anions in zone 1‐ and zone 3‐injured rats

Abstract: Aims: Hepatocytes in zone 1 of the hepatic lobule play a role in the uptake and biliary excretion of bile acids and organic anions under physiological conditions, and those in zone 3 may play a role only with a high‐dose load. To further elucidate the role of hepatic zonation on the handling of bile acids and organic anions, biliary excretion of these compounds was studied in zone 1‐ and zone 3‐injured rats. Methods: Biliary excretion of bile acids and organic anions was studied in zone 1‐ and zone 3‐injured rats, caused by allyl alcohol and bromobenzene, respectively. Results: Biliary excretion of a tracer dose of taurocholate was unchanged in the injury in both zones, but that of leukotriene C₄ was decreased in zone 1 injury. The excretory maximum of taurocholate was decreased with zone 1 and the zone 3 injuries. Biliary excretion of deoxycholate metabolites was decreased in zone 3 injury, although the profile of metabolites in the bile was unchanged. Sulfobromophthalein excretion was decreased in zone 1 injury, but unchanged in zone 3 injury. Conclusions: These findings indicate that zone 1 is very important for biliary excretion of both organic anions and bile acids. In contrast, zone 3 is considered not to have a role in biliary excretion of organic anions, but to play a role in the excretion of bile acids.     

Effect of ursodeoxycholic acid on bile acid metabolism in primary biliary cirrhosis

We have compared the effect of ursodeoxycholic acid with placebo on the clinical state, blood liver chemistries and serum and urinary bile acids in four patients with primary biliary cirrhosis. All parameters were evaluated monthly, and bile acid composition was measured by capillary gas-liquid chromatography. At the time of admission, all patients showed intense pruritus, and their serum alkaline phosphatase, AST and ALT levels were elevated 4.3, 2.7 and 2.3 times over control values. Serum bile acids were elevated almost 38-fold with 2.5 times more cholic acid than chenodeoxycholic acid. Urinary bile acid output was elevated 28 times the control values, and 36% were 1 beta-hydroxycholic acid, 1 beta-hydroxydeoxycholic acid and hyocholic acid (3 alpha,6 alpha, 7 alpha-trihydroxy-5 beta-cholanoic acid). Three months of placebo administration did not significantly affect the clinical or biochemical presentations, and the serum and urinary bile acid composition did not change. In contrast, ursodeoxycholic acid feeding (12 to 15 mg per kg per day) for 6 months abolished pruritus in two and lessened itching in two subjects and reduced serum alkaline phosphatase, AST and ALT levels by 21, 35 and 47%, respectively. The mean values for the total serum bile acid concentrations in these patients declined 26% from the pretreatment value, but the proportion of ursodeoxycholic acid increased from 3 to 40% of the total bile acids; thus, total fasting serum endogenous bile acid levels decreased almost 50%. Similar changes were noted in the urinary bile acids, in which ursodeoxycholic acid became the major bile acid, and approximately 18% were hydroxylated at C-1, C-6 and C-21.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of caveolin-3 and cholecystokinin A receptor with cholesterol gallstone disease in mice

AIM: To investigate the role of caveolin-3 (CAV3) and cholecystokinin A receptor (CCKAR) in cholesterol gallstone disease (CGD).METHODS: To establish a mouse model of CGD, male C57BL/6 mice were fed with a lithogenic diet containing 1.0% cholic acid, 1.25% cholesterol and 15% fat; a similar control group was given a normal diet. The fresh liver weights and liver-to-body weight ratio were compared between the two groups after one month. Serum lipid profile and bile composition were determined with an autoanalyzer. The Cav3 and Cckar mRNA and CAV3 and CCKAR protein levels in the liver and gallbladder were determined via real-time polymerase chain reaction and Western blot, respectively.RESULTS: Establishment of the mouse CGD model was verified by the presence of cholesterol gallstones in mice fed the lithogenic diet. Compared with mice maintained on a normal diet, those fed the lithogenic diet had significantly higher mean liver-to-body weight ratio (0.067 ± 0.007 vs 0.039 ± 0.007, P < 0.01), serum total cholesterol (4.22 ± 0.46 mmol/L vs 2.21 ± 0.11 mmol/L, P < 0.001), bile total cholesterol (1.33 ± 0.33 mmol/L vs 0.21 ± 0.11 mmol/L, P < 0.001), and bile phospholipid concentrations (3.55 ± 1.40 mmol/L vs 1.55 ± 0.63 mmol/L, P = 0.04), but lower total bile acid concentrations (726.48 ± 51.83 μmol/L vs 839.83 ± 23.74 μmol/L, P = 0.007). The lithogenic diet was also associated with significantly lower CAV3 in the liver and lower CAV3 and CCKAR in the gallbladder compared with the control mice (all P < 0.05).CONCLUSION: CAV3 and CCKAR may be involved in cholesterol gallstone disease.     

Effects of 1α-hydroxy-vitamin D3 and 1,25-dihydroxy-vitamin D3 on calcium and phosphorus metabolism in hypoparathyroidism

The effects of 1α-OH D₃ or 1,25-(OH)₂D₃ on calcium and phosphorus metabolism have been evaluated in five hypoparathyroid patients to establish the direct effects of these compounds in adult humans, uncomplicated by compensatory changes in parathyroid hormone secretion. Doses of 1–2.5 μg/day in four patients (5 μg/day in a fifth patient on diphenyl-hydantoin and phenobarbital) caused a marked increase in serum calcium concentration and urinary calcium excretion, without significant changes in renal calcium clearance or urinary hydroxyproline excretion. These results suggest that the correction of hypocalcemia involved primarily a stimulation of intestinal calcium absorption rather than a stimulation of skeletal calcium resorption. Simultaneously, there were increases in urinary phosphorus excretion and variable changes in serum inorganic phosphate concentration.These effects were produced by doses of 1α-OH D₃ and 1,25-(OH)₂D₃ which approach the dose needed to prevent rickets, in contrast to the very large doses of vitamin D or 25-OH D₃ required for comparable effects in hypoparathyroid patients. The increased relative effectiveness of these one-hydroxylated forms of vitamin D reveals a deficiency of vitamin D one-hydroxylation in hypoparathyroidism. The rapidity of action of 1α-OH D₃ and 1,25-(OH)₂D₃ was also striking. Apart from its physiologic implications, the potency of the one-hydroxylated forms of vitamin D offers significant therapeutic advantages in some patients whose hypoparathyroidism is difficult to control with vitamin D itself.     

Physiological factors influencing serum bile acid levels

This study defines the effects of fasting (prolongation of an overnight fast for a further four hours), feeding (the response to eating the three main `solid' meals of the day), and cholecystokinin-induced gallbladder contraction (75-100 units of CCK given as a bolus intravenous injection) on serum individual bile acids in five to eight healthy control subjects. The serum conjugates of the two primary bile acids, cholic and chenodeoxycholic, were measured using sensitive specific radio-immunoassays. During fasting, there was no significant change in the levels of the serum individual bile acids (conjugates of cholate, 1·28 ± 0·19; conjugates of chenodeoxycholate, 1·17 ± 0·17 μmol/l). After breakfast, the serum conjugates of cholate and chenodeoxycholate increased significantly but thereafter the mean values remained high with less consistent responses to lunch and dinner, some subjects showing a peak and trough response to all three meals, while others showed a plateau response throughout the day. After breakfast, the serum chenodeoxycholate conjugates increased more rapidly (peak at 60 minutes when the concentration reached 2·07 ± 0·30 μmol/l) and to a greater extent than the conjugates of cholate (peak at 90 minutes; 1·50 ± 0·24 μmol/l). A similar pattern of results was seen after intravenous CCK, suggesting either preferential jejunal absorption of chenodeoxycholate conjugates and/or preferential hepatic clearance of cholate conjugates. These results provide essential background data for future studies of serum individual bile acids in intestinal and hepatic disease.     

Efflux of lithocholate and its sulfate and glucuronide from isolated rat hepatocytes is normal in EHBR and is not affected by cellular ATP depletion

The efflux of lithocholate and its sulfate and glucuronide and taurocholate was studied in isolated rat hepatocytes. The velocity of the initial efflux rate from hepatocytes was in the order of taurocholate > lithocholate > lithocholate-3-O-glucuronide > lithocholate-3-sulfate. The efflux rate of these bile acids did not change in hepatocytes isolated from EHBR (Eisai hyperbilirubinemic mutant rat). Cellular ATP-depletion by fructose or rotenone did not affect the efflux rate of these bile acids. These findings seem to indicate that the efflux of these bile acids from isolated rat hepatocytes mainly reflects the sinusoidal efflux rather than the canalicular excretion, and that the efflux rate of bile acids is reduced by glucuronidation and sulfation.