Quantitation of serum bile acids by isotope dilution with 13C-labelled homologs

Quantitation of individual bile acids in serum can be carried out with radioimmunoassays or with gas chromatography. The most specific measurements are obtained with combined gas chromatography/mass spectrometry employing stable isotope labelled bile acids as internal standards. So far only the use of deuterated internal standards has been described for this purpose. 24-13C-labelled bile acids have not been used since correction for the natural abundance of the 13C contribution has to be made. Furthermore, the maximal degree of labelling of 13C-labelled bile acids is about 90%. This requires additional correction for the percentage of unlabelled molecules. Using 13C-labelled bile acids as internal standards and adequate corrections for isotope interferences we have measured individual serum bile acids in healthy volunteers by inverse isotope dilution with coefficient of variation (CV) values of 5.4-6.2% determined for the total procedure of sample preparation and analytical technique. In fasting serum of 15 healthy volunteers chenodeoxycholic acid averaged 0.98 +/- SD 0.77 mumol/l, cholic acid 0.49 +/- 0.39 mumol/l and deoxycholic acid 1.07 +/- 0.68 mumol/l. Two hours postprandially these values increased to 2.42 +/- 1.46 mumol/l for chenodeoxycholic acid, 0.81 +/- 0.45 mumol/l for cholic acid and 1.66 +/- 1.02 mumol/l for deoxycholic acid. These data agree well with those described in the literature obtained with deuterated internal standards. It may be concluded that 24-13C-labelled bile acids are suitable as internal standards for quantitation of serum bile acids, if corrections for isotope interferences are made.     

Delayed postprandial plasma bile acid response in coeliac patients with slow mouth-caecum transit

Coeliac patients are known to have an expanded bile salt pool which recirculates slowly due, at least in part, to impaired gall bladder contractility. We have investigated the possibility that delayed small bowel transit of chyme and bile may also contribute to this sluggish recycling. Plasma cholylglycine, total bile acids and cholecystokinin concentrations were measured after a lactulose-labelled test meal whose mouth-caecum transit time (M-C TT) was assessed by the breath hydrogen technique. Overall there were no significant differences in plasma bile acid profiles between seven healthy controls and a group of 25 coeliac patients. However, when subjects were divided according to their M-C TT, the 10 with the slowest transit were found to have significant elevation of fasting levels when compared with the 10 with the fastest transit, fasting total bile acids being 3.4 +/- 1.3 versus 0.7 +/- 0.6 mumol/l (P less than 0.02) and fasting cholylglycine being 0.43 +/- 0.17 versus 0.06 +/- 0.04 mumol/l (P less than 0.05) respectively. Peak bile acid levels did not differ significantly between subjects with fast or slow transit. However, subjects with slow transit were found to have a delay in the return of plasma bile acid levels to fasting levels so that the 4 h postprandial levels were significantly elevated when compared with those observed in the subjects with fast transit (total bile acids 3.6 +/- 1.2 versus 0.19 +/- 0.1 mumol/l and cholylglycine 0.70 +/- 0.13 versus 0.24 +/- 0.07 mumol/l respectively, both P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of serum bile acids by glass capillary gas-liquid chromatography.

Bile acids were extracted from serum samples by chromatography on Amberlite XAD-2 and, after alkaline or enzymic hydrolysis, purified by chromatography on aluminium oxide. The quantitation was carried out by gas-liquid chromatography with an OV-101 glass capillary column using their methyl ester trimethylsilyl derivatives. The mean total amount of cholic, chenodeoxycholic and deoxycholic acids in a group of healthy fasting women was 2.14 mumol/l, in a group of fasting pregnant women at 8-12 weeks of gestation 1.13 mumol/l and at 38-41 weeks of gestation 2.10 mumol/l. In patients with cholestasis of pregnancy the total bile acid levels varied from 6 to 86 mumol/l.     

Assay of the major bile acids in serum by isotope dilution-mass spectrometry

A specific method for determination of cholic, chenodeoxycholic and deoxycholic acid in serum based on deuterium-labelled standards and selected ion monitoring is described. The coefficient of variation of the method as calculated from replicate determinations was in the range 1.4-3.3% for all three bile acids. Recovery experiments were in accord with the high accuracy expected for this type of assay. In analyses of sera from 23 healthy subjects, the following concentrations were obtained in the fasting state: cholic acid, 0.29 +/- 0.81 mumol/l; chenodeoxycholic acid, 0.18 +/- 0.32 mumol/l; deoxycholic acid, 0.77 +/- 0.38 mumol/l (mean +/- SD). The method may be suitable as a reference method and in experiments with a high need for accuracy.     

Analysis of conjugated and unconjugated bile acids in serum and jejunal fluid of normal subjects

A reliable method is described for the determination of conjugated and unconjugated bile acids in serum and jejunal fluid. Bile acids are extracted using reverse-phase octadecylsilane bonded silica cartridges and are separated into their unconjugated and conjugated fractions using the lipophilic anion exchanger diethylaminohydroxypropyl Sephadex LH-20 (DEAP-LH-20). The conjugated fraction can be separated into a glycine and a taurine fraction, using the same anion exchanger. The bile acids are measured using a hydroxysteroid dehydrogenase-fluorimetric assay for serum and a hydroxysteroid dehydrogenase-photometric assay for jejunal fluid. The normal fasting serum value of total 3 alpha-hydroxy bile acids amounts to 3.5 +/- 2.8 mumol/l (mean +/- SD, range 1.4-10.8, n = 22). The corresponding unconjugated bile acid fraction amounts to 39.9 +/- 11.2% (range 20.7-64.6%) of total bile acids. The concentration of conjugated bile acids became significantly elevated 30, and 60 min after a standard meal, whereas that of unconjugated bile acids remained unchanged. In jejunal fluid only conjugated bile acids are found, as well in fasting subjects as postprandial, 30 or 60 min after a standard meal.     

Changes in fecal composition, intestinal transit, bile acid metabolism and intestinal fermentation in long-term nutrition with high molecular weight formula diet

Treatment with formula diets becomes more and more popular in many patients. The influence of those diets on gut functions is as yet poorly known. We studied in ten healthy volunteers the effects of a high molecular liquid diet. Despite of a sufficient energy supply the volunteers lost significantly weight which may be related to an acceleration of small bowel transit (60 +/- 9 min vs. 31 +/- 5 min; control vs. diet period). Whole gut transit did not change significantly (52 +/- 3 h vs. 56 +/- 3 h). The fecal excretion of bile acids decreased significantly (293 +/- 35 mg/24 h vs. 151 +/- 10 mg/24 h) which was particularly due to a decrease of primary bile acids. The serum bile acid concentrations behaved in a similar way (total bile acids: 3.19 +/- 0.66 mumol/l vs. 1.71 +/- 0.21 mumol/l). Neither the determination of unconjugated serum bile acids nor hydrogen breath testing did indicate increase of bacterial growth. In conclusion, chronic nutrition with formula diets causes significant changes of gut functions.     

Unconjugated serum bile acids as a marker of small intestinal bacterial overgrowth

Non-invasive methods to detect small intestinal bacterial overgrowth often lack specificity in patients who have undergone an ileal resection or have an accelerated intestinal transit. Since elevated serum unconjugated bile acid levels have been found in patients with clinical signs of bacterial overgrowth, we studied the clinical value of unconjugated serum bile acids as a marker of small intestinal bacterial overgrowth. Patients with culture-proven bacterial overgrowth had significantly elevated fasting unconjugated serum bile acid levels (median and range: 4.5; 1.4-21.5 mumol l-1) as compared to healthy subjects (0.9; 0.3-1.7 mumol l-1, P less than 0.005), to persons with an accelerated intestinal transit (1.0; 0.3-1.9 mumol l-1, P less than 0.005) and to persons who have undergone an ileal resection (2.1; 0.7-3.6 mumol l-1, P less than 0.005). The same was true 30 and 60 min after ingestion of a Lundh meal. Serum unconjugated bile acid levels above 4 mumol l-1 were found in eight of 10 patients with culture-proven small intestinal bacterial overgrowth whereas serum levels above 4 mumol l-1 were found in none of the patients from the three control groups. These results suggest that determination of unconjugated serum bile acids is of clinical value in the evaluation of patients suspected of small intestine bacterial overgrowth.     

Carnitine deficiency and hyperammonemia in children receiving valproic acid with and without other anticonvulsant drugs

Plasma ammonia and total and free carnitine were measured in 84 children requiring anticonvulsant drugs: 32 patients (group A) on valproic acid alone, 28 children (group B) on polytherapy including valproic acid, and 24 patients (group C) on polytherapy without valproic acid. The other anticonvulsant drugs used in groups B and C were carbamazepine and phenobarbital. Plasma ammonia concentrations were elevated in both group A and B compared with controls. Group B patients showed significantly higher hyperammonemia than group A (59.9 +/- 16.3 micrograms/dl vs. 36.7 +/- 12.4 micrograms/dl; P < 0.05). Group C patients had plasma ammonia levels similar to those of controls (31.1 +/- 14.7 micrograms/dl vs. 29.7 +/- 12.1 micrograms/dl; NS). In both group A and group B patients, plasma ammonia levels were correlated with the valproic acid dosage (r = 0.32, P < 0.01) and with serum concentrations of valproic acid (r = 0.41, P < 0.001). Moreover, a significant correlation between plasma ammonia and duration of valproic acid therapy was found in the patients as a whole (r = 0.31, P < 0.01). Plasma total and free carnitine concentrations were significantly reduced in groups A and B (total carnitine 36.9 +/- 6.9 mumol/l vs. 32.9 +/- 9.7 mumol/l; free carnitine 28.9 +/- 5.1 mumol/l vs. 25.7 +/- 4.3 mumol/l, respectively) compared with group C patients who did not receive valproic acid and in whom values were similar to controls (total carnitine 46.1 +/- 9.0 mumol/l vs. 47.7 +/- 10.1 mumol/l; free carnitine 40.1 +/- 7.1 mumol/l vs. 42.9 +/- 8.0 mumol/l, respectively). Twenty-eight patients (18 of group A and 10 of group B) were re-evaluated and showed a complete normalization of plasma ammonia, and total and free carnitine levels which were similar to controls. Our data suggest that hyperammonemia is an important problem in patients receiving valproic acid, particularly in association with other anticonvulsant drugs. This increase of plasma ammonia and the concomitant reduction of carnitine seem to be transient and completely reversible.     

Hepatic storage and biliary transport maximum of taurocholate and taurochenodeoxycholate in the dog.

The purpose of this work was to validate for taurocholate and taurochenodeoxycholate the multiple infusion method of Wheeler et al. previously used for the study of hepatic sulphobromophthalein transport and to obtain numerical estimates of the relative storage capacity and secretory transport maximum of both bile acids. Experiments were performed in anaesthetized dogs after depletion of the endogenous bile acid pool. Bile was collected continuously to prevent recirculation of bile acids and to measure their secretion rates. Taurocholate or taurochenodeoxycholate were infused intravenously at 3 different rates and blood samples were collected every ten min to measure serum bile acid concentrations. Extrahepatic distribution spaces of taurocholate and taurochenodeoxycholate were measured by an isotope dilution method. Serum bile acid concentrations varied linearly with time during the last 30 min of each infusion period. A linear relationship was found between the calculated hepatic removal rate and the rate of change of serum bile acid concentration. The mean values of relative storage capacity were (in mumol.mumol-1.l-1.kg body weight-1) 0.16 +/- SD 0.07 for taurocholate and 0.08 +/- SD 0.03 for taurochenodeoxycholate. The mean values for secretory transport maximum were (in mumol.min-1.kg body weight-1) 4.81 +/- SD 1.24 for taurocholate and 3.56 +/- SD 0.60 for taurochenodeoxycholate. The values of secretory transport maximum with the multiple infusion method were only slightly higher than those obtained by direct measurement of biliary secretion. The values of relative storage capacity obtained during infusions resulting in decreasing plasma concentrations were usually lower than those obtained when the plasma concentration increased. This suggests that the limitations of the method previously noted for sulphobromophthalein may apply to bile acids.     

Radioimmunoassay of bile acids in tissue, bile, and urine

Two commercially available (Abbott Labs.) radioimmunoassays for determination of conjugated cholic acid and sulfoglycolithocholic acid in serum have been modified for bile acid measurements in alcoholic tissue extracts, bile, and urine. The specificity of both radioimmunoassays has been determined with regard to 27 free and conjugated bile acids. After filtration, bile acids can be measured in urine and bile without prior extraction. Tissue is homogenized and the bile acids are extracted into methanol. Urinary excretion by 64 healthy humans was 2.09 (SD 1.09) mumol of conjugated cholic acid and 8.44 (SD 8.03) mumol of sulfated glycolithocholic acid per 24 h. In liver from 10 patients with various noncholestatic liver disease, the mean concentration of conjugated cholic acid was 32.4 (SD 15.9) nmol/g wet weight. In the liver of 27 male Wistar rats, the concentration of conjugated cholic acid was 41.3 (SD 11.7) nmol/g of tissue, of sulfoglycolithocholic acid 5.1 (SD 2.3) nmol/g of tissue.     

Blood plasma fluoride in haemodialysed patients

Blood plasma fluoride was determined in 15 chronic haemodialysed patients (60.2 +/- 7.2 yr old) before and after a 4-h dialysis using dialysates with very low fluoride level, and in two control groups, the first of 20 healthy younger subjects (45.9 +/- 3.4 yr old), the second of 8 healthy older subjects (69.1 +/- 6.8 y old). Before haemodialysis the fluoride concentration (1.31 +/- 0.31 mumol/l; 24.8 +/- 5.9 micrograms/l), was higher than in both control groups (0.35 +/- 0.16 mumol/l; 6.6 +/- 3.1 micrograms/l and 0.44 +/- 0.16 mumol/l 8.4 +/- 3.0 micrograms/l, respectively). During dialysis, the mean fluoride concentration fell to 0.94 +/- 0.26 mumol/l, remaining however, significantly higher than in control subjects. The use of fluoride-free dialysates seems to partially compensate the effect of renal impairment since plasma fluoride is only moderately increased in these patients.     

Bile acids and their sulphated and glucuronidated derivatives in bile, plasma, and urine of children with intrahepatic cholestasis: effects of phenobarbital treatment

Abstract. Bile acids and their sulphated and glucuronidated derivatives were studied in three children with persistent intrahepatic cholestasis, two children with intrahepatic biliary hypoplasia, and four healthy children. In children with cholestasis, biliary bile acids consisted of 11(±0–3) % 3 β-hydroxy-delta-5-cholenoic acid, 2-1(± 0–6) % lithocholic acid, 2-2(± 11) % deoxy-cholic acid, 5–8(±2-2) % ursodeoxycholic acid, 39-1(± 1 -4) % chenodeoxycholic acid, 0–5(± 0 2) % hyo-cholic acid, and 49-3(± 3 0) % cholic acid. Of these bile acids 121 (±l 9) % were sulphated and 4–5 (±0 6) % were glucuronidated. In healthy children, biliary bile acids consisted of 0–7 (±0–4) % lithocholic acid, 3–4 (±0 8.) % deoxycholic acid, 0–1 (±0 1) % ursodeoxycholic acid, 32-7 (±6 9) % chenodeoxycholic acid, and 631 (±7 1) % cholic acid. Of these bile acids, 0–6±0 1 % were sulphated and 0–2 ±0 1% were glucuronidated (mean ± SEM). In the urine of healthy children, 3-3(±0 6) mg/24 h bile acids (1–5±0 3 mg sulphates and 0–1 ±0 1 mg glucuronides) were excreted, in the urine of children with cholestasis 61-4 (± 10 2) mg/24 h (30 2 ±7 1 mg sulphates and 5 6 ±1 2 mg glucuronides) were excreted. Thus in children with cholestasis the amounts of sulphated and glucuronidated bile acids are greater than in healthy controls. Substantial amounts of sulphated and glucuronidated bile acids are excreted in bile and urine of these patients. Phenobarbitone treatment in the five children with cholestasis led to a reduction of serum bile acids from 90 4 (± 13 2) μg/ml to 39 3(±3 6) μ//ml, a relative increase of bile acid glucuronides in bile from 45 (±0 6)% to 8 l(±0 6)%, a slight alteration of the bile acid sulphates in bile from 121(±l 9) % to 111 (± 1 2)% and no alteration of the bile acid spectrum. Urinary excretion of bile acids decreased from 61 4 (± 10 2) mg/24 h to 34 7(±3 0) mg/24 h. Phenobarbitone treatment of children with cholestasis thus induced glucuronidation of bile acids but had no significant effect on sulphation or on formation of individual bile acids.     

Bile acids in plasma of patients without kidneys

Bile acids were measured in plasma of three non-fasting bilaterally nephrectomized patients in chronic haemodialysis. After separation of the bile acid mixture according to mode of conjugation and cleavage of amide and sulphate ester bonds, the bile acids were analysed as their methylester trimethylsilyl ethers on gas-liquid chromatography-mass spectrometry (GC-MS). The total bile acid concentration was 4.10-13.70 mumol X l(-1) (N: less than 14 mumol X l(-1)) mostly glycine conjugates. Unconjugated bile acids were found in significant amounts in one patient and in trace amounts in another patient. Chenodeoxycholic acid was the predominant individual bile acid constituting 1.89-6.79 mumol X l(-1) (N: less than 10 mumol X l(-1)). The oxidoreduction product of this bile acid: ursodeoxycholic acid, was found in all patients, whereas the 7 alpha-dehydroxylation product: lithocholic acid, was absent. 1 beta-hydroxy-deoxycholic acid was a major constituent in two of the cases. Hyocholic acid, the 6 alpha-hydroxylation product of chenodeoxycholic acid was detected in two cases. Tetrahydroxy bile acids and sulphated bile acids were not found in significant amounts.     

Simultaneous measurement of free and esterified fatty acids by gas chromatography from normal and type IV hyperlipoproteinaemic sera

A transmethylated reaction of esterified fatty acids with sodium- methoxide in a mixture of serum, petroleum ether and methanol is presented. In the conditions used the free fatty acids in the sample were not esterified. 1-2 microliters of the organic phase was injected into an OV-351 fused silica capillary column of gas liquid chromatography (GLC) fitted with flameionizing detector (FID) temperature program, and calculating integrator. By this method both free and esterified fatty acids were measured in a single run. The development of the present method for the better quantitation of free fatty acids is in progress as using gas chromatography/mass spectrometry for identification of the peaks. In thirteen healthy subjects, the serum free fatty acid content was 538 +/- 176 mumol/l and free glycerol 107 +/- 39 mumol/l, while in seven type IV hyperlipoproteinaemic sera the corresponding values were 1049 +/- 529 and 86 +/- 38 mumol/l. The most prominent differences between healthy and type IV hyperlipoproteinaemic subjects for esterified fatty acids were found in palmitic, oleic and arachidonic acids. The correlations between free and bound fatty acids has been discussed.     

Increased concentrations of endogenous 13-cis- and all-trans-retinoic acids in diffuse idiopathic skeletal hyperostosis, as demonstrated by HPLC.

Endogenous 13-cis- and all-trans-retinoic acids have been quantitated in human serum using a solvent extraction procedure followed by isocratic reversed phase high performance liquid chromatography and UV detection. In healthy adults, after an overnight fasting period, the concentrations of 13-cis- and all-trans-retinoic acids yielded 5.3 +/- 2.43 nmol/l and 11.8 +/- 3.3 nmol/l, respectively (mean +/- SD). The method has been successfully applied to the analysis of both isomers in serum from patients with idiopathic skeletal hyperostosis in whom, the 13-cis- as well as all-trans-retinoic acid levels were raised as compared to the control group.     

Assessment of taurine deficiency in cystic fibrosis

Bile acid taurine deficiency is common in cystic fibrosis (CF) and is thought to be associated with impaired fat absorption. The relationship between the glycine: taurine bile acid conjugation ratio (bile acid G/T ratio) and taurine concentrations in plasma, urine and leucocytes was examined in 27 CF children aged 4-15 yr. The bile acid G/T ratio was elevated in serum in 14 of the 27 and in duodenal juice in 5 of 6 children. Subgroups of CF children with elevated and normal bile acid G/T ratio and controls (n = 8) all had similar plasma, urine and leucocyte taurine concentrations. The results suggest that either taurine measurement in plasma, urine and leucocytes does not accurately reflect stores elsewhere in CF, or that taurine deficiency is confined to bile acids.     

Sulphur containing amino acids in chronic renal failure with particular reference to homocystine and cysteine-homocysteine mixed disulphide

We measured plasma sulphur amino acids in twenty-two patients with chronic renal failure and compared the findings with those obtained in twenty-two normal subjects. In fasting blood (08.00 hours) cysteine-homocysteine mixed disulphide was significantly increased in the renal patients, mean values (+/- SD) being 8.2 +/- 3.4 and 3.1 +/- 1.0 mumol/l respectively (P less than 0.001). The increase was positively correlated with reduced renal function, as assessed by serum creatinine (r = 0.62; P less than 0.01). Homocystine was detected in nineteen patients, the mean concentration (+/- SD) being 1.7 +/- 0.6 mumol/l; it was not found in any normal subject. Methionine levels were not different but there were significant increases in cystine (P less than 0.001) and taurine (P less than 0.05) in the patients. Similar values for these amino acids were found in a second blood sample drawn at 16.00 hours. Changes in the other neutral and acidic amino acids measured were in agreement with those reported in chronic azotaemia. We concluded that plasma levels of all the principal sulphur amino acids except methionine are elevated in chronic renal failure emphasizing the importance of the kidney in sulphur excretion. Prolonged accumulation of homocysteine and cysteine-homocysteine mixed disulphide may be relevant to the development of accelerated vascular disease in patients with chronic renal failure by producing endothelial damage.     

Non-esterified fatty acids may regulate human leucocyte sodium pump activity

Human leucocyte sodium pump activity was studied in normal fasting subjects by measuring the ouabain-sensitive 22Na+ efflux rate constants. This 22Na+ efflux rate constant was inversely related to the fasting plasma non-esterified fatty acid level (rs = -0.73, P less than 0.0001). An oral glucose load (40 g/m2 surface area) led to an increase in the leucocyte ouabain-sensitive 22Na+ efflux rate constant after 2 h (1.97 +/- 0.25 to 2.44 +/- 0.19 h-1, P less than 0.0001, n = 11). There was a concomitant fall in the plasma non-esterified fatty acid level. Incubation of leucocytes in vitro with 100 mumol/l linoleic acid inhibited the leucocyte ouabain-sensitive 22Na+ efflux rate constant (1.52 +/- 0.27 vs 0.84 +/- 0.24 h-1, P less than 0.001, n = 8). The leucocyte Na+,K+-dependent adenosine triphosphatase (Na+,K+-ATPase) activity was inhibited in vitro by long chain non-esterified fatty acids, especially when unsaturated. Non-esterified fatty acids may account for some of the Na+,K+-ATPase inhibitory activity of plasma.     

Determination of free tryptophan in plasma and its clinical applications

Free tryptophan in plasma was separated by centrifugation through an Amicon ultrafiltration membrane cone. The value obtained without control of pH was found to be lower than that obtained with control of pH by an improved method ( Hijikata et al. (1981) Anal. Biochem. 118, 10-16). For determination of the total tryptophan concentration in the plasma, high performance liquid chromatography (HPLC) was better than the method of Denckla Dewey as modified by Bloxam & Warren ( (1974) Anal. Biochem. 60, 621-625), as judged on the basis of sensitivity, recovery rate and coefficient of variance. The total tryptophan concentration in the plasma determined by HPLC was lower than that determined by the Bloxam & Warren method. The total tryptophan concentration (t-Trp), free tryptophan concentration (f-Trp) and f-Trp/t-Trp ratio were 55.8 +/- 10.2 mumol/l, 11.6 +/- 1.5 mumol/l and 0.211 +/- 0.03 (mean +/- 1 SD) respectively, in healthy subjects (controls). No significant difference was observed between the values of controls and those of patients with liver cirrhosis, hepatocellular carcinoma, liver cirrhosis with hepatocellular carcinoma and hepatic encephalopathy of liver cirrhosis without bleeding. But in liver cirrhosis with bleeding, free tryptophan concentration (f-Trp, 48.0 +/- 23.3 mumol/l, p less than 0.001) and f-Trp/t-Trp ratio (0.645 +/- 0.289, p less than 0.001) were significantly higher than those of controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous-flow determination of bile acids in serum, and its clinical application

We describe a highly sensitive and accurate automated continuous-flow method for determining bile acids in serum. The bile acids are first liberated from serum protein by dialysis at alkaline pH and then measured fluorometrically after the following enzymic reaction. Bile acids are converted to 3-oxo bile acids with 3alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) with concomitant reduction of NAD+ to NADH. The hydrogen in the generated NADH is transferred by diaphorase (EC 1.6.4.3) to resazurin to yield resorfin, the fluorophore. Only 100 microliter of serum is required and 40 determinations can be done per hour. The CV for 20 replicate determinations in serum with a mean bile acid concentration of 9.8 mumol/liter was 2.6%. The CV for day-to-day variation for another serum on 27 successive days was 3.0% (mean concentration, 10.0 mumol/liter). We applied this method to 826 sera from various diseases; 29% exceeded the upper limit of normal, 10 mumol/liter, and abnormally high values (greater than 20 mumol/liter) were almost exclusively limited to sera from hepatobiliary and enteric disorders.