Serum unconjugated bile acids as a test for intestinal bacterial overgrowth in dogs.

Small intestinal bacterial overgrowth (SIBO) has a high incidence in dogs and, as in humans, is difficult to diagnose. The aim of this study was to determine the diagnostic significance of serum unconjugated bile acid concentrations in dogs with bacterial overgrowth. Fasting sera were obtained from 23 dogs: 10 with culture-proven SIBO, 8 with indirectly diagnosed SIBO (normal pancreatic function but small intestinal disease associated with subnormal serum cobalamin and supranormal folate concentrations), and 5 healthy controls. Unconjugated bile acids were determined using gas chromatography-mass spectrometry after isolation by liquid-solid extraction and anion-exchange chromatography. Mean serum unconjugated bile acid concentrations were significantly elevated in dogs with SIBO (mean +/- SD: 0.91 +/- 1.03 micromol/liter), and in dogs with indirectly diagnosed SIBO (2.11 +/- 2.20 micromol/liter) compared to clinically healthy dogs (0.015 +/- 0.015 micromol/liter, P < 0.005). Cholic acid was the predominant unconjugated bile acid in the serum of dogs with SIBO. In conclusion serum unconjugated bile acid concentrations of healthy dogs are significantly lower than reported values for humans, and this fraction represents a relatively small proportion (0-2.3%; mean 0.8%) of the total bile acids in dogs. Unconjugated bile acids increased 10- to 20-fold in dogs with SIBO indicating the clinical utility of serum unconjugated bile acids for diagnosis of intestinal bacterial overgrowth in dogs.

Differences in the metabolism and disposition of ursodeoxycholic acid and of its taurine-conjugated species in patients with primary biliary cirrhosis.

The clinical effectiveness of ursodeoxycholate in the treatment of liver disease may be limited by its poor absorption and extensive biotransformation. Because in vitro and in vivo studies suggest that the more hydrophilic bile acid tauroursodeoxycholate has greater beneficial effects than ursodeoxycholate, we have compared for the first time the absorption, metabolism, and clinical responses to these bile acids in patients with primary biliary cirrhosis (PBC). Twelve female patients with PBC were sequentially administered tauroursodeoxycholate and ursodeoxycholate (750 mg/d for 2 months) in a randomized, cross-over study. Bile acids were measured in serum, duodenal bile, urine, and feces by gas chromatography-mass spectrometry (GC-MS). Biliary ursodeoxycholate enrichment was higher during tauroursodeoxycholate administration (32.6% vs. 29.2% during ursodeoxycholate; P <.05). Lithocholic acid concentration was consistently higher in all biological fluids during ursodeoxycholate administration. Fecal bile acid excretion was the major route of elimination of both bile acids; ursodeoxycholate accounted for 8% and 23% of the total fecal bile acids during tauroursodeoxycholate and ursodeoxycholate administration, respectively (P <.05). Tauroursodeoxycholate was better absorbed than ursodeoxycholate, and, although it was partially deconjugated and reconjugated with glycine, it underwent reduced biotransformation to more hydrophobic metabolites. This comparative study suggests that tauroursodeoxycholate has significant advantages over ursodeoxycholate that may be of benefit for long-term therapy in PBC.

Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease.

We describe a metabolic defect in bile acid synthesis involving a deficiency in 7alpha-hydroxylation due to a mutation in the gene for the microsomal oxysterol 7alpha-hydroxylase enzyme, active in the acidic pathway for bile acid synthesis. The defect, identified in a 10-wk-old boy presenting with severe cholestasis, cirrhosis, and liver synthetic failure, was established by fast atom bombardment ionization-mass spectrometry, which revealed elevated urinary bile acid excretion, a mass spectrum with intense ions at m/z 453 and m/z 510 corresponding to sulfate and glycosulfate conjugates of unsaturated monohydroxy-cholenoic acids, and an absence of primary bile acids. Gas chromatography-mass spectrometric analysis confirmed the major products of hepatic synthesis to be 3beta-hydroxy-5-cholenoic and 3beta-hydroxy-5-cholestenoic acids, which accounted for 96% of the total serum bile acids. Levels of 27-hydroxycholesterol were > 4,500 times normal. The biochemical findings were consistent with a deficiency in 7alpha-hydroxylation, leading to the accumulation of hepatotoxic unsaturated monohydroxy bile acids. Hepatic microsomal oxysterol 7alpha-hydroxylase activity was undetectable in the patient. Gene analysis revealed a cytosine to thymidine transition mutation in exon 5 that converts an arginine codon at position 388 to a stop codon. The truncated protein was inactive when expressed in 293 cells. These findings indicate the quantitative importance of the acidic pathway in early life in humans and define a further inborn error in bile acid synthesis as a metabolic cause of severe cholestatic liver disease.

Genetic and morphological findings in progressive familial intrahepatic cholestasis (Byler disease [PFIC-1] and Byler syndrome): evidence for heterogeneity.

Byler disease (ByD) is an autosomal recessive disorder in which cholestasis of onset in infancy leads to hepatic fibrosis and death. Children who have a clinically similar disorder, but are not members of the Amish kindred in which ByD was described, are said to have Byler syndrome (ByS). Controversy exists as to whether ByD and ByS (subtypes of progressive familial intrahepatic cholestasis [PFIC]) represent one clinicopathological entity. The gene for ByD has been mapped to a 19-cM region of 18q21-q22. PFIC caused by a lesion in this region, including ByD, can be designated PFIC-1. Examination of haplotypes in siblings with ByS in two unrelated non-Amish families showed that the gene(s) responsible for their disorder(s) did not lie in the PFIC-1 candidate region. On light microscopy and transmission electron microscopy (TEM), liver tissue differed between Amish children with PFIC-1, who had coarsely granular bile and at presentation had bland intracanalicular cholestasis, and the children with ByS in the two non-Amish families, who had amorphous or finely filamentous bile and at presentation had neonatal hepatitis. Bile acid composition of bile also differed: In the Amish children with PFIC-1 and in one ByS family, the proportional concentration of chenodeoxycholic acid (CDCA) in bile was low compared with normal bile; in the other ByS family, it was only slightly reduced. Genetic analysis and light microscopy and TEM of liver may help distinguish PFIC-1 from other forms of ByS.

Effect of psyllium in hypercholesterolemia at two monounsaturated fatty acid intakes.

We performed two studies to determine whether the lipid-lowering effect of viscous soluble fiber was modified by monounsaturated fatty acid (MUFA). First, psyllium (1.4 g/MJ) was compared with wheat bran (control) in 1-mo metabolic diets by using a randomized crossover design (n = 32 hyperlipidemic subjects). The background diet contained approximately 6% of energy as MUFA (20% of total fat). The second study (n = 27 hyperlipidemic subjects) was similar to the first but the background diet contained approximately 12% MUFA (29% of total fat) because of the addition of canola oil. At both fat intakes, psyllium resulted in significant reductions in total, low-density-lipoprotein (LDL), and high-density-lipoprotein (HDL) cholesterol compared with the wheat bran control. For the psyllium diet at 6% compared with 12% MUFA, the decreases in LDL cholesterol were 12.3 +/- 1.5% (P < 0.001) and 15.3 +/- 2.4% (P < 0.001), respectively. With the higher-MUFA diet triacylglycerol fell significantly over the control phase (16.6 +/- 5.5%, P = 0.006) and the ratio of LDL to HDL cholesterol fell significantly over the psyllium phase (7.3 +/- 2.8%, P = 0.015). Psyllium and MUFA intakes were negatively related to the percentage change in the ratio of LDL to HDL cholesterol (r = -0.34, P = 0.019 and r = -0.44, P = 0.002, respectively). Chenodeoxycholate synthesis rate increased (30 +/- 13%, P = 0.038) with the psyllium diet in the 12 subjects in whom this was assessed. We conclude that psyllium lowered LDL- and HDL-cholesterol concentrations similarly at both MUFA intakes. However, there may be some advantage in combining soluble fiber and MUFA to reduce the ratio of LDL to HDL cholesterol.

Additive hypocholesterolemic effect of psyllium and cholestyramine in the hamster: influence on fecal sterol and bile acid profiles.

Recent findings suggest that the effects of cholestyramine and psyllium in combination could be additive for cholesterol-lowering. We therefore examined the effect of both agents, alone and in combination, on lipoprotein cholesterol and neutral and acidic steroid excretion in the hamster. Animals (n = 8/group) were fed for 21 days, either a basal chow diet supplemented with 10% palm oil and 0.2% cholesterol, or one of four treatments consisting of the basal diet plus: 5.5% cellulose; 5% psyllium with 0.5% cellulose; 0.5% cholestyramine with 5% cellulose; or 5% psyllium with 0.5% cholestyramine. Psyllium and cholestyramine both had significant hypocholesterolemic effects, but in combination produced additive reductions in lipoprotein and hepatic cholesterol. Psyllium, cholestyramine, and the combination increased total bile acid excretion by 26%, 57%, and 79%, respectively. Psyllium affected only unconjugated bile acid excretion while cholestyramine also increased the excretion of conjugated and primary bile acids. Neither agent, nor the combination, affected fecal neutral sterol excretion. We conclude that, while both agents lower cholesterol by a mechanism of increased bile acid excretion, these studies indicate that psyllium does not bind bile acids in vivo and lend further support for the concomitant use of these agents for cholesterol-lowering.

delta 22-Ursodeoxycholic acid, a unique metabolite of administered ursodeoxycholic acid in rats, indicating partial beta-oxidation as a major pathway for bile acid metabolism.

We describe for the first time the identification of 3 alpha, 7 beta-dihydroxy-5 beta-chol-22-en-24-oic acid (delta 22-UDCA) in the plasma, bile, intestinal contents, and liver tissue of Sprague-Dawley rats after intravenous and oral administration of ursodeoxycholic acid (UDCA). Infusion of [2,2,4,4-2H4]UDCA confirmed delta 22-UDCA to be a specific metabolite of UDCA. Definitive confirmation of this unique and major metabolite was established by liquid secondary ionization mass spectrometry and gas chromatography-mass spectrometry by comparison of the retention index and mass spectrum with an authentic standard of delta 22-UDCA. When rats were fed a diet containing 1.0% UDCA, high concentrations of delta 22-UDCA were found in the plasma (40.3 +/- 11.8 mumol/L) and liver tissue (300.9 +/- 64.2 nmol/g of tissue), and these represented 36% and 57%, respectively, of the UDCA concentration. In animals fed 0.4% and 1.0% UDCA, the mass of delta 22-UDCA in the jejunum was high (7.5 +/- 0.9 and 6.6 +/- 0.6 mg, respectively), accounting for 50-60% of the total UDCA, but diminished markedly along the intestine, accounting for < 3% of the total UDCA in the colon. Although delta 22-UDCA was not found in biological samples from control rats, delta 22-beta-muricholic and delta 22-omega-muricholic acids were normal constituents of plasma and intestinal contents and were major muricholate isomers in liver tissue and bile. Synthesis of delta 22-bile acids appears to be highly specific toward bile acids possessing a functional 7 beta-hydroxyl group. We presume that, in common with pathways for endogenous bile acid synthesis, partial side-chain oxidation of UDCA occurs in the peroxisome with formation of alpha/beta unsaturation; since UDCA has only a 5-carbon side chain, release of propionic or acetic acid is not possible, beta-oxidation proceeds no further, and delta 22-UDCA is formed. While the mechanism of formation and physiological significance of delta 22-bile acids remain to be established, our data indicate that partial beta-oxidation is a quantitatively important pathway for endogenous bile acid synthesis and for UDCA metabolism in this species.

A new cause of progressive intrahepatic cholestasis: 3 beta-hydroxy-C27-steroid dehydrogenase/isomerase deficiency.

There have been a few reports of infants with severe neonatal cholestasis related to a defect in primary bile acid synthesis. To assess the importance of such deficiency among children with progressive intrahepatic cholestasis (Byler disease), screening for inborn errors in bile acid synthesis was performed by fast atom bombardment ionization-mass spectrometry of urine samples from 30 affected children. Bile acid analysis revealed a specific fast atom bombardment ionization-mass spectrometry profile for 3 beta-hydroxy-C27 steroid dehydrogenase/isomerase deficiency in five children who had jaundice, hepatosplenomegaly, and fatty stools beginning at ages ranging from 4 to 46 months. None of them had pruritus. Liver function tests showed persistently normal serum gamma-glutamyltransferase activity, low serum cholesterol and vitamin E levels, normal serum bile acid concentrations despite raised serum bilirubin levels, and decreased prothrombin time and clotting factor V. In four of the cases a similar disease was observed in siblings. Liver function returned to normal after oral ursodeoxycholic acid therapy. We conclude that 3 beta-hydroxy-C27-steroid dehydrogenase/isomerase deficiency should be considered when idiopathic cholestatic liver disease with clinical features akin to Byler disease is characterized by the association of normal serum gamma-glutamyltransferase activity, normal serum bile acid concentration, absence of pruritus, and a return to normal liver function during ursodeoxycholic acid therapy. Early identification of these children is essential because they benefit from bile acid therapy and might thus avoid the need for liver transplantation.

Tryptophan intake influences infants' sleep latency.

Formula-fed infants have depressed plasma tryptophan concentration relative to human milk-fed infants. Because tryptophan alters sleep-waking patterns in adults, a study was designed to determine whether additional dietary tryptophan could elevate plasma tryptophan concentrations of formula-fed infants to concentrations characteristic of human milk feeding and whether differences in plasma tryptophan or the ratio of plama tryptophan to the sum of the other plasma large neutral amino acids (tryptophan:LNAA) were of behavioral significance. Infants were fed a formula (13 g protein/L; whey:casein, 34:66) containing either 0, 294, 588 or 882 mumol/L of added tryptophan. Infants fed human milk or commercial formula (15 g protein/L; whey:casein, 18:82) were included for comparison. In formula-fed groups, plasma tryptophan was directly related to tryptophan intake (r = 0.46, P less than 0.0005). Infants fed commercial formula or the formula without added tryptophan had lower (P less than 0.001) plasma tryptophan compared with infants fed human milk. Only the infants fed the highest tryptophan formula had significantly higher plasma tryptophan:LNAA ratios than the other experimental groups, and these ratios were similar to those of infants fed human milk. The plasma tryptophan:LNAA ratios, not plasma tryptophan concentrations, were predictive of differences in the infants' sleep latency; infants fed formula containing the highest tryptophan had sleep latencies of 18.7 min, significantly shorter (P less than 0.05) than those of infants fed formulas containing less added tryptophan (27.7 min). Feeding infants formulas differing in tryptophan concentration produced differences in sleep latency, which could influence neurobehavioral developments.(ABSTRACT TRUNCATED AT 250 WORDS)

Ileal resection/dysfunction in childhood predisposes to lithogenic bile only after puberty.

Children with ileal resection/dysfunction since infancy have bile that is not supersaturated with cholesterol. Five sexually mature subjects (age 16-19 years) who had been previously investigated in childhood (age 4-9 years) were studied. Gallstones were found in one. Bile rich duodenal aspirates were analyzed for lipid content (molar fraction) and the cholesterol saturation index was calculated. In the postpubertal subjects, a significantly higher proportion of biliary cholesterol (22.0% +/- 4.8% vs. 3.2% +/- 0.6% and 5.7% +/- 0.5%, P less than 0.005) and significantly lower bile acids (58.1% +/- 3.9% vs. 79.7% +/- 2.3% and 78.2% +/- 1.9%, P less than 0.005) were found compared with the initial (prepubertal) samples or in samples from 20 healthy young adults. The cholesterol saturation index was significantly higher (3.1 +/- 0.7 vs. 0.6 +/- 0.1 and 1.1 +/- 0.1, P less than 0.005) whereas phospholipid content did not change (19.9% +/- 1.6% vs. 17.1% +/- 1.8% and 16.6% +/- 1.6%) as compared with themselves before puberty and as healthy young adults, respectively. It was concluded that children with ileal resection/dysfunction do not appear at risk for cholesterol cholelithiasis before puberty; however, the development of biliary cholesterol supersaturation after puberty may predispose them to gallstone formation in adulthood.