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The disulphate ester of ursodeoxycholyl-p-aminobenzoic acid (PABA-UCDA) was synthesised and compared with PABA-UDCA for its use in detection of intestinal bacteria. This compound, PABA-UDCA disulphate, had characters in common with PABA-UDCA in that it was deconjugated by cholylglycine hydrolase to release free PABA and bacteria that split glycocholic acid deconjugated PABA-UDCA disulphate. Further, in rat experiments urinary excretions of PABA were measured for six hours after oral administration of 15 mg PABA-UDCA disulphate. Ten control rats excreted (mean (SE) 188.2 (13.6) micrograms of PABA; 10 rats with an intestinal stagnant loop excreted more (530.1 (30.1) micrograms; p < 0.001): whereas 10 rats in each of three groups pretreated by oral administration of various antibiotics excreted less (polymixin B+tinidazole, 4.9 (1.6) micrograms; kanamycin, 31.0 (4.7) micrograms; clindamycin 40.9 (5.5) micrograms; p < 0.001). By contrast with PABA-UDCA, PABA-UDCA disulphate was not actively absorbed from any part of the small intestine in everted gut sac experiments, and showed poor recovery from bile after its intraileal instillation in rats. This indicated that PABA-UDCA disulphate is a single pass type substance in the gut and its oral administration test reflects the sum of the activities of bacteria in the small intestine and colon. The disulphate was easily soluble in water and this allowed its application in an in vitro test involving PABA-UDCA disulphate incubation with intraperitoneal pus (PABA-UDCA disulphate incubation test) from patients with peritonitis. This test was carried out on six patients with peritonitis, and the severity of bacterial peritonitis was expressed quantitatively. From the results obtained PABA-UDCA disulphate was considered a good material to detect intestinal bacteria.

Laminin, a glycoprotein synthesised by Ito cells, has been considered a marker of fibrogenesis. The behaviour of laminin and clinical and laboratory data in 83 patients with cirrhosis were studied to find the factors associated with increases in this glycoprotein. There were increased concentrations of laminin in 62.7% of the patients (40% of the Child's A, 64.5% of the Child's B, and 75% of the Child's C categories). Significant differences in laminin concentrations were found between the Child's grades (p = 0.009) and between patients and controls (p < 0.0001). Correlations were found between laminin concentrations and mean corpuscular volume, aspartate aminotransferase, aspartate aminotransferase: alanine aminotransferase ratio, alkaline phosphatase activity, bilirubin and glycocholic acid concentrations, and hypoalbuminaemia--that is, variables related to liver insufficiency and alcohol intake. Moreover, patients with an alcohol intake higher than 100 g/day had higher laminin concentrations than those with a lower intake (p = 0.03). Conversely, there was no significant association with portal hypertension. Multivariate analysis showed that mean corpuscular volume, bilirubin concentrations, and hypoalbuminaemia were independently associated with laminin concentrations. Poor degradation associated with liver insufficiency seems to play an important part in the increase in serum laminin concentrations in these patients.

The (14)C-glycocholate test, including the measurement of marker corrected faecal (14)C, has been assessed in the following groups of subjects: normal controls (18), patients with diarrhoea not attributable to altered bile acid metabolism (21), patients with diverticula of the small intestine (12), patients with previous resection of ileum and often proximal colon (34), and established ileostomists (10). Patients with diverticular disease had increased breath (14)CO(2) excretion, but normal faecal excretion of (14)C, and this test was more frequently abnormal than the Schilling test. Ileostomists excreted increased amounts of faecal (14)C, even when the ileum was intact and apparently normal. The pattern after resection was complex. Breath (14)C output was normal if the ileal resection was less than 25 cm in length, although some of these patients had increased faecal (14)C excretion if, in addition, at least 15 cm of proximal colon had been resected or by-passed. Longer ileal resections were associated with increased breath and/or faecal (14)C excretion, depending in part on the length of colon resected or by-passed and the 24 hour faecal volume. Fewer than half these patients had both increased breath and faecal excretion of isotope and faecal (14)C alone was occasionally normal with an ileal resection of 50 cm of more. The (14)C-glycocholate test was more frequently abnormal than the Schilling test in this group. The use of faecal marker correction had only a minor impact on the results. These data suggest that, in patients with ileal resection, faecal (14)C, like faecal weight, is determined by the extent of colonic resection as well as by the amount of ileum resected.

Serum cholic and chenodeoxycholic acid conjugates were measured in fasting conditions and after meals in 14 patients with bile acid malabsorption due to ileal resection. Mean serum fasting levels of both primary bile acids did not differ from the controls. After meals, serum cholic acid peaks were lower in patients with ileal resection than in control subjects (p less than 0.001), while chenodeoxycholic acid peaks were reduced in colectomised patients (p less than 0.01). In the sera from patients with ileal resection, the glycine/glycine + taurine ratio for cholic and chenodeoxycholic acid increased (p less than 0.001) from morning to evening, and glycine/glycine + taurine ratio for chenodeoxycholic acid was significantly (p less than 0.01) different from the controls in the sera collected in the evening. The results are consistent with the concept of a better intestinal conservation of chenyl, mainly of the glycine conjugated from, than of cholylconjugates, in patients with ileal resection; this is probably because of passive absorption in the intestine. The postprandial peaks of serum cholic acid conjugates may therefore be regarded as a test of ileal dysfunction, while peaks of chenodeoxycholic acid conjugates suggest colonic impairment.

We assessed ileal functional integrity in 20 consecutive patients with Crohn's disease by sequential measurement of the postprandial serum bile acid concentration. In all 14 patients with active Crohn's disease involving the terminal ileum, the mean (+/- SEM) peak response in the cholylglycine (0.4 +/- 0.04 mumol/l, n = 14) as well as in the total serum bile acid concentration (2.0 +/- 0.4 mumol/l, n = 10) was similar to that seen in a group of children who had undergone ileal resection. A significantly greater increase in the cholylglycine (1.8 +/- 0.18 mumol/l, n = 16, P less than 0.01) and in the total serum bile acid concentration (9.8 +/- 2.4 mumol/l, n = 11, P less than 0.025) was noted in normal children. In five of the six remaining patients (three with Crohn's disease shown not to involve the ileum and two of three with asymptomatic, treated Crohn's ileitis) and in seven patients with ulcerative colitis, the meal stimulated responses were normal. These preliminary results suggest that measurement of the serum bile concentration after a meal stimulus may provide a valuable index of ileal inflammation in patients with Crohn's disease.

To determine whether a defect in uptake of organic anions is present in patients with benign recurrent cholestasis, the plasma disappearance of simultaneously injected indocyanine green and cholyglycine-1-14C was measured in eight patients with asymptomatic benign recurrent cholestasis, four with the familial type, as well as 22 healthy control subjects. Evans blue was also simultaneously injected to permit correction for variation in blood volume and speed of mixing. Uptake of indocyanine green was decreased in all three patients with nonfamilial benign recurrent cholestatsis, but normal or increased in the four patients uith the familial form of the disorder. By contrast, cholyglycine-1-14C uptake was abnormal in one patient with each type of the disorder. Thus, patients with nonfamilial benign recurrent cholestasis have a defect in indocyanine green uptake, and patients with benign recurrent cholestasis are heterogeneous with respect to organic anion uptake.

The disappearance of intravenously administered cholylglycine-14C was studied in the fasting and postprandial states in seven subjects with healthy livers and 10 patients with liver disease. In neither group was there any significant difference in the pattern of 14C disappearance. In another 10 patients with liver disease there was no significant change when a loading dose of cholylglycine was given orally two hours beforehand. Clearance of bile acids seems to be unimpaired in all except severe liver disease. The apparent deterioration in endogenous bile acid removal after meals may be due simply to the increased amount of bile acids which are in circulation and available for portosystemic shunting.

We have studied serum fasting and postprandial primary bile salt concentrations in a group of 10 consecutive hyperlipidaemic subjects. The efficiency of hepatic bile salt clearance in the same subjects was aslo studied using an injected dose of sodium glycocholate. No increase in serum fasting or postprandial concentrations of the primary bile salts were observed and hepatic bile salt clearance was only marginally abnormal in one subject. The presence of hyperlipidaemia does not invalidate the use of serum conjugated bile salt analysis for the detection of liver diseases.

The plasma clearance of a tracer dose of 14C-glycocholic acid, and fasting total serum bile acid concentrations were measured in 14 control subjects and in 38 patients with acute and chronic liver disease. In controls plasma clearance was 415 +/- 24 ml min-1 m-2 (mean +/- SEM), equivalent to a 'first-pass' extraction by the liver of 85%. Clearance was not significantly different from controls in patients with acute hepatitis or active chronic hepatitis, nor in anicteric patients with primary biliary or alcoholic cirrhosis. Thus bile acid clearance was impaired only in icteric chronic liver disease. In contrast, serum bile acid concentrations were abnormal in all but seven patients, six of whom had active chronic hepatitis in complete biochemical remission. The pattern of plasma disappearance of injected 14C-glycocholic acid was biexponential in controls and patients with liver disease, and computer analysis of the curves suggested that there was significant distribution of bile acid outside the vascular space. The preservation of bile acid clearance in anicteric chronic liver disease confirms that it is dependent more on liver blood flow than on liver cell function.

The 14C-glycocholic acid test (14C-GCA) has been assessed in 27 patients who have had resection of the ileum and colon for Crohn's disease and in 19 patients with unoperated stable Crohn's disease. The incidence of increased breath output of 14CO2 and faecal output of 14C was significantly greater in operated patients. Half the unoperated patients had normal results and, of the others, a modest increase in faecal 14C was the usual finding. There was no correlation between the 14C-GCA test, the Schilling test, and the extent and severity of the radiological signs in the unoperated patients. Metronidazole therapy was poorly tolerated and had little or no influence on symptoms but did decrease the excretion of 14CO2 in the breath when the 14C-GCA test was repeated. Cholestyramine therapy was beneficial in the majority of resected patients with diarrhoea and an increased faecal 14C excretion and, on repeat testing, there was a significant increase in the output of breath 14CO2. The beneficial effect was less marked in the unoperated patients and the breath 14CO2 output remained normal in those retested. Interruption of the enterohepatic circulation of bile acids did not seem to make an important contribution to the symptoms of patients with stable unoperated (and uncomplicated) Crohn's disease, even when the ileum was extensively involved.

The plasma disappearance of a tracer dose of cholyl-l14C-glycine has been examined in 12 control subjects and in 32 patients with hepatocellular dysfunction. Simple analysis of the data did not detect hepatic dysfunction except in severe hepatocellular disease. The greatest degree of discrimination between normal subjects and patients with mild liver disease was obtained by taking the ratio of the plasma retention at 60 minutes to that at 10 minutes; it was similar to that obtained with serum gamma-glutamyl transferase. The two hour post-prandial plasma "total" bile acid concentration gave complete separation between the control subjects and patients with liver disease.

Duodenal bile acids have been estimated in three age groups in infancy from birth to 7 months, and age-related changes have been shown. The lowest concentrations of duodenal bile acids occurred in the youngest infants, and increasing concentrations were found with increasing age. Taurine conjugated bile acids predominated in early infancy, while in older infants bile acids were mainly conjugated with glycine. The probable presence of taurolithocholic acid in the duodenal bil of 2 newborns before the establishment of a gastrointestinal microflora remains to be confirmed, but could result either from transplacental passage of secondary bile acids or from endogenous synthesis by the fetal liver.

Fasting and postprandial plasma concentrations of glycine and taurine conjugates of cholic, chenodeoxycholic, and deoxycholic acid were measured by a high pressure liquid chromatography-enzymatic assay in nine healthy subjects. The mean value of each bile acid concentration increased significantly (2.4-4.7 times) in the postprandial period. The total glycine taurine ratio of 2.5 in the fasting state increased significantly to a maximum value of 3.3 at one to 1 1/2 hours postprandially and then declined. This shift in glycine taurine ratio shows, that the relative increase in concentrations of glycine conjugates exceeds the relative increase in concentrations of taurine conjugates in the early postprandial period, and supports the view that there is significant absorption of glycine conjugated bile acids from the proximal small intestine.

In a group of dogs in whom fulminant liver failure had been induced, perfusion of blood through activated charcoal resulted in a significantly longer survival than that of a similar group of dogs whose blood was not so treated. An otherwise progressive rise in blood ammonia concentration was halted in the treatment group. In another group of dogs with fulminant liver failure perfusion of blood through the resin Amberlite XAD-2 was associated with a fall in the serum bilirubin concentration and complete clearance from the blood of (14)C-labelled sodium glycocholate. Survival in this group of animals was not significantly prolonged. This was due at least in part to the occurrence of haemorrhage due to thrombocytopenia. Platelets adhere to the resin but do not adhere to the same degree to charcoal coated with a thin layer of polymer.

Bile acid studies were performed in patients with Crohn's disease, radiologically confined to the colon. The bile acid pool size of 10 patients with isolated Crohn's colitis was significantly lower than that of 10 normal control subjects (P less than 0.001) and of 10 ulcerative colitis patients (P less than 0.005). Measurements of 14C-excretion in breath and in 24 hours stool collections after the administration of 5 muCi 14C-glycocholate showed a normal 14C-excretion in breath and usually a markedly increased loss of 14C in the stool (greater than 7% of the dose). The simultaneous administration of 5 muCi 3H-polyethylene glycol MW 4000 (3H-PEG) as a marker indicated that the 14C/3H ratio in the patients with Crohn's colitis was significantly greater than in a control series of patients with diarrhoea not due to bile acid malabsorption. Studies on the composition of duodenal bile showed a significantly decreased concentration of deoxycholic acid in duodenal bile. These observations suggest bile acid malabsorption in patients with Crohn's disease apparently confined to the colon.

Isotopic bile salt studies have been performed in 13 cholecystectomy patients and 10 matched controls using labelled taurocholate and deoxycholic acid. Cholecystectomy subjects have reduced pools of both primary bile salts, cholate and chenodeoxycholate, while the deoxycholate pool remains normal in size. As a result of these changes, the total bile salt pool is reduced to almost half its normal size and deoxycholate becomes the predominant bile salt. The half-life of taurocholate is reduced but, because its pool size is diminished, the daily synthesis of taurocholate remains normal. There is accelerated transfer of (14)C from taurocholate-24-(14)C to its metabolites in bile, especially deoxycholate conjugates. In four subjects studied pre- and postoperatively similar changes occurred in all the above parameters. All these data can be explained by the fact that the bile salt pool circulates during fasting as well as during digestion. The consequences of this are (1) increased exposure of bile salts to intestinal bacteria and hence increased bacterial degradation; (2) continuous passage of the bile salt pool through the liver, and therefore continuous and presumably enhanced feedback inhibition of hepatic bile salt synthesis. The reservoir function of the gallbladder influences the size, kinetics, metabolism, and composition of the bile salt pool. We suggest that no study of bile salt metabolism is complete without some assessment of gallbladder status.

Twenty-four insulin-dependent diabetics, including seven with diabetic diarrhoea, were studied by means of the 14C-glycocholate (14C-GCA) test and various tests for autonomic dysfunction. The breath component of het test was abnormal in four of the seven patients with diarrhoea and one of the other diabetics. Three patients with diarrhoea and a positive breath test result responded to antibiotics, whereas two with diarrhoea and a negative test result did not. High faecal 14C, suggesting bile acid malabsorption, was found in only one patient with diarrhoea and he had previously failed to respond to cholestyramine. These results suggest that bacterial overgrowth in the small intestine does occur in some but not all patients with diabetic diarrhoea and that the 14C-GCA test can predict the response to antibiotics. All the patients with diabetic diarrhoea had good evidence of autonomic dysfunction.