Individual Serum Bile Acids Research Articles

Diagnostic sensitivity of fasting and postprandial serum bile acids determined by different methods

The diagnostic sensitivities of serum bile acids determined by three different methods in the fasting and in the postprandial state were compared in 43 patients with cirrhosis of the liver. When a method with high analytical sensitivity (capillary gas-liquid chromatography, GLC, or radioimmunoassay, RIA) was used, the serum concentrations of bile acids exhibited similar diagnostic sensitivities in the fasting state (GLC, 98%; RIA, 93%) and in the postprandial state (GLC, 95%; RIA, 93%). By contrast, when an enzymatic method with limited analytical sensitivity was employed, the diagnostic sensitivity of fasting serum bile acids was lower (79%) than that of postprandial serum bile acids (93%). The measurement of individual serum bile acids by GLC did not add any further diagnostic information. The results of this study demonstrate that the diagnostic sensitivity of serum bile acids strongly depends on the analytical method used.

Analysis of the Individual Bile Acids in Serum by High Performance Liquid Chromatography

Analysis of the Individual Bile Acids in Serum by High Performance Liquid Chromatography

Serum bile acid composition in patients with cystic fibrosis

Serum bile acid composition was examined in detail using capillary column gas chromatography and mass spectrometry in 10 children with cystic fibrosis (CF) and 4 healthy children. The mean total bile acid concentrátion in fasting serum of CF patients was 2.33 ± 0.84 / gmmol/l, slightly lower than but not statistically significantly different from healthy controls (mean 2.86 ± 0.98 μmol/l) and appeared to show no relationship to the degree of exocrine pancreatic insufficiency. Analysis of individual serum bile acids in these children showed that cholic acid represented less than 10% of the total bile acids. Chenodeoxycholic acid was the predominant serum bile acid; the mean concentration in CF patients was 0.98 ± 0.51 / gmmol/l, lower than for the healthy controls (1.69 ± 0.84 μmol/l). Concentrations of lithocholic acid, 3/gb-hydroxy-5-cholenoic, ursodeoxycholic and 3/gb,7α,12α-trihydroxy-5/gb-cholanoic acids in fasting serum samples of the CF patients were not significantly different from the healthy control sera but were higher than those normally found in adults. Measurements of fecal bile acid excretion indicated an increased loss of primary bile acids in patients with CF consistent with an impairment of the enterohepatic circulation of bile acids.

The diagnostic value of fasting individual serum bile acids in anicteric alcoholic liver disease: relation to liver morphology.

The aim of the present study was to evaluate the diagnostic value of measuring individual serum bile acids in patients with suspected alcoholic liver disease. A highly accurate and specific mass-fragmentographic technique with high sensitivity was used. Anicteric patients with fatty liver (n = 10) and liver cirrhosis (n = 9) were compared with healthy controls (n = 27). The measurement of serum bile acids did not discriminate patients with fatty liver from controls. In general, an increased serum level of cholic acid indicated more serious liver disease. Determination of chenodeoxycholic acid and deoxycholic acid did not add any further diagnostic information.

Enzymatic fluorimetric determination of the individual bile acids in blood serum

An enzymatic fluorimetric method is described for the determination of total bile acids (cholic acid and deoxycholic acid), primary bile acids (cholic and chen acids and individual bile acids in serum without prior separation of the acids. Total and primary bile acids are determined by equilibrium procedures by conver of the 3α- and 7α-hydroxy bile acids to 3-oxo and 7-oxo bile acids by α-NAD +, in the presence of 3α- and 7α-hydroxysteroid dehydrogenase (HSD), respectively, and measurement of the generated NADH fluorimetrically. Chenodeoxycholic acid is determined with 7α-HSD in the presence of cholic and deoxycholic acids by a differential kinetic procedure, and cholic and deoxycholic acids are calculated by difference. Interferents are removed by treatment of serum with Sachrom rein. Only 1.00 ml of serum is required. Low cost, simplicity and reliability are the main features of the method. The recovery of bile acids added to serum averaged 103% (range 83–122%). The method is suitable for routine use in small clinical laboratories.

A highly sensitive determination of individual serum bile acids using high-performance liquid chromatography with immobilized enzyme

We have developed a highly sensitive method for the simultaneous determination of individual bile acids in serum using high-performance liquid chromatography (HPLC) combined with immobilized 3α-hydroxysteroid dehydrogenase. Both the HPLC column and the immobilized-enzyme column are suitable for use with alkaline solutions necessary in working with this enzyme system. With this method we were able to determine simultaneously fifteen different serum bile acids.

Separation and determination of bile acids by high-performance liquid chromatography using immobilized 3α-hydroxysteroid dehydrogenase and an electrochemical detector

A high-performance liquid chromatographic method using an immobilized 3α-hydroxysteroid dehydrogenase column and an electrochemical detector was developed for the determination of individual bile acids in serum and bile. Bile acids in the eluate from a Radial-Pak A column reacted with NAD in the enzyme column to generate NADH, which was monitored by a voltammetric detector after mixing with phenazine methosulphate solution. Each bile acid was measurable at the 20 pmole level at the highest sensitivity of the detector. The mean recoveries and reproducibilities of bile acids were 86.7-104.6% [coefficient of variation (C.V.) = 0.3-8.7%] within-assay and 83.8-103.4% (C.V. = 1.6-7.0%) between-assay.

Response of total and individual serum bile acids to endogenous and exogenous bile acid input to the enterohepatic circulation

The response of total nonsulfated serum bile acids, cholylglycine, and chenodeoxycholyl species was examined every 20 min for 3 h in 6 subjects. Noncaloric feeding led to a progressive decline or no change in bile acids, while there was a progressive rise in response to a standard liquid meal. After reaching a peak at 60 min, total bile acids declined progressively but cholylglycine and chenodeoxycholyl species remained elevated. Continuous infusion of cholecystokinin led to significantly greater levels most probably due to more rapid enterohepatic recirculation. Oral administration of 250 mg of chenodeoxycholic acid with water resulted in a rise in total bile acids and chenodeoxycholyl species, but not cholylglycine, indicating the rise was due to the administered bile acid and not gallbladder contraction. Administration of a meal and chenodeoxycholic acid simultaneously caused no greater rise of total serum bile acids or cholylglycine than either stimulus alone. Peak response and area under the curve were compared for each patient. The increase for chenodeoxycholyl species was additive for the two stimuli, suggesting that free chenodeoxycholic acid, when administered with a meal, decreased the absorption of endogenous conjugated bile acids. This study is compatible with the thesis that serum bile acids accurately reflect enterohepatic cycling and that administration of chenodeoxycholic acid with a meal may decrease its efficacy because exogenous chenodeoxycholic acid may compete with endogenous bile acids for absorption.

Serum and urinary bile acids in patients with primary biliary cirrhosis.

Serum concentrations and daily urinary excretions of unsulphated and total cholic (C) and chenodeoxycholic (CDC) acid were determined by radioimmunoassay in 15 patients with primary biliary cirrhosis. Thirteen patients had increased fasting serum bile acid concentrations; two of them had an increase of C only. An increase of C and CDC in serum was always followed by an increased urinary excretion of C and CDC. The individual serum bile acids were separated by gas-liquid chromatography. 3 beta-hydroxy-5-cholenoic acid was increased in nine patients, and its serum concentration was correlated to the total serum bile acid concentration. Deoxycholic (DC) and lithocholic (LC) acid were found in most patients, but their serum concentrations were not correlated to the total serum bile acid concentration. Minor bile acids comprised an average of 2% of the total concentration.

Individual serum bile acids in patients with primary hyperlipoproteinemias

Fasting serum concentrations of the individual bile acids were measured by gas chromatography in 27 patients with primary hyperlipoproteinemia (8 type IIa, 7 type IIb and 12 type IV) and in 14 healthy subjects. Total serum bile acid levels were 1618 ± 244 ng/ml (SE) in type IIa, 1296 ± 251 ng/ml in type IIb and 1569 ± 263 ng/ml in type IV hyperlipoproteinemia. These values did not differ significantly from values in the control group (1505 ± 200 ng/ml). Serum levels of cholic acid were significantly higher in patients with type IIa (551 ± 78 ng/ml) than in those with type IIb (190 ± 57 ng/ml, P < 0.01) and type IV (240 ± 57 ng/ml, P < 0.02), while intermediate values were recorded in the control group (384 ± 49 ng/ml). Ursodeoxycholic acid was found in larger amounts in hyperlipidemic patients than in controls. No significant differences with respect to other bile acids were observed between the groups examined. According to the current concepts on the enterohepatic circulation of bile acids, the findings support the hypothesis that the intestinal absorption of cholic acid may differ in various types of hyperlipoproteinemia.

Prediction of the bile acid composition of bile from serum bile acid analysis during gallstone dissolution therapy

The relationship between the relative concentrations of individual bile acids in fasting serum and bile collected simultaneously was determined in 28 patients with gallstones. Seven of these patients were being treated with chenodeoxycholic acid (15 mg/kg/day). The relative concentrations of deoxycholic acid, chenodeoxycholic acid, and ursodeoxycholic acid were found to be higher in serum than in bile, while the reverse was true for cholic acid. A highly significant correlation (P < 0.001) was found between the relative proportions of each of deoxycholic acid, cholic acid, chenodeoxycholic acid and ursodeoxycholic acid in fasting serum and bile. Each correlation was defined by linear regression analysis, and regression equations were used to predict the bile acid composition of bile from the relative concentrations of the individual bile acids in serum. Although some error was obtained between predicted and actual values in individual patients, in all 7 gallstone dissolution patients treated with chenodeoxycholic acid, both the actual and predicted cholic acid concentrations in bile were < 20 moles %, and the chenodeoxycholic acid concentrations were > 70 moles %. This latter value almost always indicates biliary cholesterol unsaturation in nonobese patients. Other studies in T-tube patients confirmed that changes in biliary bile acid profiles produced by chenodeoxycholic acid treatment could be readily followed via the serum. Serum bile acid analysis is therefore a useful alternative to bile analysis for the biochemical monitoring of patients treated with chenodeoxycholic acid, and avoids duodenal intubation for bile collection.

Individual serum bile acid concentrations in normo- and hyperlipoproteinemia as determined by mass fragmentography: relation to bile acid pool size.

Combined gas-liquid chromatography-mass spectrometry with specific ion monitoring (mass fragmentography) has been used for assay of cholic acic (C), chenodeoxycholic acid (CD), and deoxycholic acid (D) in human serum. Deuterium-labeled C and D were used as internal standards. The relative standard deviation of duplicate samples was 3, 4, and 7% for C, CD, and D, respectively. The variation within the same individual in the fasting state was small, while the day-to-day variation was greater, especially for the dihydroxy bile acids. In normal control subjects (n = 24), the fasting serum concentration of C averaged 184 +/- 24 ng/ml (mean +/- SEM), and that of CD and D 526 +/- 62 and 407 +/- 44 ng/ml, respectively. Patients with type IIa hyperlipoproteinemia (n = 32) displayed low values of serum bile acids, with a C concentration of 121 +/- 11 ng/ml (P less than 0.01 vs. controls). A similar pattern was seen in patients with a type IIb lipoprotein pattern (n = 10). Subjects with type IV hyperlipoproteinemia (n = 32) showed serum bile acid levels within the normal limits. No relationship to age, sex, or body weight was seen in any of the patient subgroups. Bile acid kinetics were determined with an isotope dilution technique using 14C-labeled C and CD under steady state conditions in control subjects and patients with type IIa and type IV hyperlipoproteinemia. The serum concentration of C correlated significantly to its pool size in control subjects and in patients with type IIa hyperlipoproteinemia but not in patients with type IV. The serum level of CD was not related to CD pool size in any of the subgroups. The data obtained are discussed in relation to present concepts of the enterohepatic circulation. It is suggested that the intestinal content of C in the fasting state is proportional to the total C pool size. The possibility of a defective intestinal uptake of C in some patients with type IV hyperlipoproteinemia is raised.

Serum bile acids in hepatobiliary disease.

We review the estimation of total and individual serum bile acids to detect the presence and nature of hepatobiliary disease. The different methods for measuring serum bile acids are discussed.

Serum bile acids in patients with hyperlipidaemia.

Individual serum bile acids were analysed by an improved gas liquid chromatography method in 12 patients with primary hyperlipidaemia. Total serum bile acid concentrations were raised in 10 subjects. Ursodeoxycholic acid was found in all 12 patients. It was present in significantly greater concentrations, accounted for a greater proportion of the total serum bile acids, and occurred more frequently than in patients with various forms of hepatobiliary disease. Patients with hyperlipidaemia had proportionately less deoxycholic acid than controls but more than patients with liver disease. There was proportionately less chenodeoxycholic acid in patients with hypercholesterolaemia, in whom the primary bile acid ratio was raised.

Fasting and postprandial serum bile acid concentrations in normal persons using an improved GLC method.

An improved GLC method was employed to study individual serum bile acids in 14 control subjects after a 12-hour fast followed by a standard fatty meal. Peripheral venous blood samples were obtained at 15-min intervals for up to 2 h in 7 subjects, and 4 h in 2 subjects. The fasting total bile acids, and cholic:chenodeozycholic acid ratio fell within a narrow range. The total bile acids and chenodeoxycholic acid increased significantly within 30 min of the meal, to reach peak values within 75-120 min. The extent of the increase was variable, and the range of values increased progressively after the meal.

Serum bile acids in patients with viral hepatitis.

Total and individual serum bile acid concentrations were studied in 18 patients with viral hepatitis. The initial high values returned to the normal range at approximately the same time as the conventional liver function tests. Chenodeoxycholic acid was the predominant bile acid initially, but cholic acid predominated as the disease resolved. All patients had proportionately less doexycholic acid than controls. It is concluded that the measurement of serum bile acid does not provide added information of value in the management of patients with viral hepatitis.

Individual Bile Acids in Portal Venous and Systemic Blood Serum of Fasting Man

The serum concentrations of cholic acid (C), chenodeoxycholic acid (CD), and deoxycholic acid (D) were determined in peripheral venous and portal venous blood from 10 otherwise healthy patients undergoing elective cholecystectomy. A highly specific and accurate gas chromatographic-mass spectrometric technique was used. Peripheral venous serum contained 0.49 +/- 0.16 (mean +/- SEM) mumole per liter of C, 1.55 +/- 0.32 mumoles per liter of CD, and 1.44 +/- 0.57 mumoles per liter of D. Arterial serum, obtained from 5 of the subjects, did not show any differences in bile acid concentrations compared to venous serum. In contrast, the portal venous content of each bile acid was several-fold greater, 6.14 +/- 1.20 mumoles per liter of C, 8.40 +/- 1.84 mumoles per liter of CD, and 6.18 +/- 2.27 mumoles per liter of D. The hepatic uptake of C was estimated to be about 90%, whereas that of CD and D was lower, about 70%. This difference in hepatic uptake between the individual bile acids was reflected in the relative composition of the total bile acids, which was 30:39:31 (C:CD:D) in portal venous serum and 13:50:37 in peripheral serum. Compared to common duct bile obtained simultaneously, the portal vein contained a greater proportion of CD. The relevance of the data obtained to our present concept of the enterohepatic circulation of bile acids is discussed, and it is suggested that the higher fasting level of CD compared to C in peripheral serum results from the combination of a lower fractional hepatic extraction and a higher portal venous input to the systemic circulation.

Elevations in Skin Tissue Levels of Bile Acids in Human Cholestasis: Relation to Serum Levels and to Pruritus

To define the relationship of bile acid retention to the pruritus of cholestasis, we quantified individual bile acids in serum, acetone swabs of skin, and skin tissue in 13 patients with cholestasis undergoing laparotomy and in 8 controls. There was no consistent relationship between pruritus and concentrations of either total or individual bile acids in serum. Skin tissue concentrations of bile acids were elevated in patients with cholestasis, were linearly related to serum levels, and did not differentiate between those patients with and those without pruritus. Concentrations of bile acids on the skin surface, which were lower than those reported by others, did not correlate with pruritus, and were decreased by simple soap and water washing. These data indicate that the pruritus of cholestasis is not directly related to the skin tissue concentration of any of the major bile acids, although a relationship to a particular molecular form of bile acids could not be excluded.

Relative concentrations of individual nonsulfated bile acids in the serum and bile of patients with cirrhosis.

The relative concentrations of individual nonsulfated bile acids were determined in samples of serum and bile obtained simultaneously from 16 patients with biopsy-proven alcoholic cirrhosis. A highly significant (P less than 0.001) correlation was found between the fasting relative concentrations of each of the three major bile acids (cholic, chenodeoxycholic, and deoxycholic) in serum and bile. This relationship persisted after manipulation of bile acid pools produced by feeding of individual bile acids. We conclude that the relative concentrations of individual nonsulfated bile acids in serum accurately reflect those in bile and that measurement of individual serum bile acids may be used for screening and serial determination purposes. In particular, low levels of biliary deoxycholate can be reliably predicted by serum measurements.

Dynamics of the Enterohepatic Circulation of the Glycine Conjugates of Cholic, Chenodeoxycholic, Deoxycholic, and Sulfolithocholic Acid in Man

Highly sensitive and specific radioimmunoassays for cholylglycine, chenodeoxycholylglycine, deoxycholylglycine, and sulfolithocholylglycine have been used to study the kinetics of the enterohepatic circulation of these conjugated bile acids in 8 healthy subjects. Venous blood samples were collected over a 32-hr period, during which time the subjects ate three meals. Serum levels of cholylglycine and chenodeoxycholylglycine rose after each meal, and reached their maximum level within 30 to 60 min. A second chenodeoxycholylglycine peak occurred 2 to 3 hr after the first two meals in all subjects; a second peak was also found for cholylglycine in 3 of the 8 subjects. Serum deoxycholylglycine levels also rose postprandially; the peak level generally occurred 30 min later than that of cholylglycine. Serum sulfolithocholylglycine levels did not alter significantly after meals. The data indicate that the dynamics of the enterohepatic circulation of individual serum bile acids differ both quantitatively and qualitatively.