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Analysis of the Conjugated Bile Acid Distribution in Human Intestinal Aspirates Using Gas Liquid Chromatography * L.J

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Analysis of the Conjugated Bile Acid Distribution in Human Intestinal Aspirates Using Gas Liquid Chromatography * L.J ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 6, No. 1 Copyright © 1976, Institute for Clinical Science Analysis of the Conjugated Bile Acid Distribution in Human Intestinal Aspirates using Gas Liquid Chromatography * L.J. HAEFFNER, Ph.D., S. J. GORDON, M.D.t, S. STRUM, M.D., M. ELLIOT, B.S., AND O. D. KOWLESSAR, M.D. Department of Medicine, Division of Gastroenterology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107 ABSTRACT The conjugated bile acid pattern was evaluated in intestinal aspirates of five normal subjects and five patients known to have an abnormal bile acid dis­ tribution. The glycine/taurine (G/T) ratios for total bile acids were determined by gas liquid chromatography (GLC) and by enzymatic assay using 3-hydroxysteroid dehydrogenase (STDH). By both methods G/T ratios in normal samples ap­ proximated 3.0 as previously reported. A discrepancy between ratio values ob­ tained by the two methods was found in patient samples. It is suggested that the presence of keto bile acids in patient aspirates correlates with this discrepancy. The G/T ratios for individual bile acids were obtained by GLC. The G/T ratio for cholic acid was higher than previously reported. Recovery studies showed that a loss of taurocholic acid in the extraction procedure employed did not account for the high G/T-cholic acid. Introduction that G/T ratios for individual bile acids de­ Analysis of total conjugated bile acids of termined by paper chromatography approxi­ intestinal aspirates in normal subjects has mate the G/T ratio for total bile acids.13 revealed a G/T ratio approaching 3.0 in The present study was undertaken to com­ published studies.1’2'3,91012131415 This ratio pare the total bile acid G/T ratio of intes­ has been derived utilizing a number of tech­ tinal samples from normal subjects niques including paper chromatography or employing TLC followed by enzyme assay thin layer chromatography (TLC) followed (STDH) with that obtained by TLC by chemical determinations or enzymatic followed by GLC, a technique not pre­ assay. Furthermore, it has been suggested viously utilized for this purpose. The G/T “This work was supported in part by the following ratios for individual bile acids were also grants: Training Grant 2T01 AM5572, National In­ assessed by GLC. In addition, the same stitute of Arthritis, Metabolism and Digestive Disease, methods were applied to intestinal aspirates Bethesda, MD. Contract N00014-68-A-0516, Office of Naval Research, Project Themis and Wallace Labora­ of patients having an abnormal bile acid dis­ tories, Cranbury, NJ. tribution characterized by absent deoxy- t Address reprint requests to Susan J. Gordon, M.D. cholic acid. 15 16 HAEFFNER, ET AL. Materials and Methods solvent system propionic acid: iso-amyl acetate: water: n-propanol (30:40:10:20).7 SAMPLE COLLECTION Standards of cholic acid and glycocholic Intestinal samples were obtained from five acid were simultaneously chromatographed normal subjects and five patients previously and bile acids were detected with iodine found to have an abnormal total bile acid vapor. distribution characterized by the absence of The regions corresponding to the glycine- deoxycholic acid. After an overnight fast, a and taurine-conjugated bile acids were modified Cantor tube was positioned under removed from the plate with a scintered fluoroscopic guidance at the ligament of glass suction apparatus,“ eluted with meth­ Treitz. Thirty minutes after a test meal, anol and dried on a rotary evaporator. The consisting of one slice of buttered toast and a solutions containing glycine- and taurine- cup of tea with sugar, a duodenal sample was conjugated bile acids were brought to 10 and collected in two subjects, while in four sub­ 5 ml, respectively, with methanol. jects the tube was allowed to pass into the distal jejunum where samples were obtained. DETERMINATION OF G/T RATIO All samples were collected over ice, im­ STDH assay. Precisely 0.1 ml and 0.2 ml mediately centrifuged and 2.0 ml aliquots of the glycine- and taurine- conjugated bile were stored at —20° until processed. acid solutions, respectively, were assayed by the method of Iwata and Yamasaki,8 SAMPLE PREPARATION modified to include a blank containing A modification of the STDH assay of sample solution, buffer, NAD and water. Iwata and Yamasaki8 was used to determine The G/T ratio was calculated directly from the bile acid content of an aliquot of intes­ the change in absorbance at 340 nm (aA ) as tinal sample. Using this determination, the follows: total number of aliquots containing 4 to 5 itmoles of bile acid was further processed. AA glycine conjugates x 100 For each 2.0 ml aliquot, the proteins were ACA taurine conjugates x 25 precipitated with 20 ml of ethanol by GLC assay. Exactly 9 ml and 4 ml of the heating at 90° for ten minutes. Exactly 4.6 solutions containing the glycine- and ml N NaOH was added to the supernatant taurine-conjugated bile acids, respectively, which was then extracted with 20 ml petro­ were evaporated to dryness and reconsti­ leum ether. The ether was washed with a tuted with 1.5 ml methanol. The bile acids mixture of 1.2 ml water and 2.2 ml N NaOH were hydrolyzed with 3.5 ml of 2.7 N NaOH in 50 percent ethanol. The initial aqueous- in an autoclave for three hours (122°, 15 psi). ethanol layer and the wash were combined, The free bile acids were recovered by four adjusted to pH 1.1 and extracted se­ chloroform extractions, dried and the quentially with 35, 50, 35, 50 ml of chloro­ methyl-trifluoroacetate derivatives prepared form. The chloroform extracts of all 2.0 ml for GLC (vide infra). The G/T ratios were aliquot preparations from the same sample calculated from the areas on the chromato­ were then combined and evaporated to graph, using the formula: dryness. A methanolic solution of each sample was quantitatively applied to a thin 10/9 (GL + GD + GCDC + GC) layer plate. 5/4 (TL + TD + TCDC + TC) The glycine- and taurine-conjugated bile in which L, D, CDC, and C represent the acids were separated by TLC on Silica Gel G plates 1 mm thick (20 x 20 cm) with the * Lurex Corporation. ANALYSIS OF CONJUGATED BILE ACID DISTRIBUTION 17 areas of measurable lithocholic acid, deoxy- ica Gel G.7 The purified 14C-TC was cholic acid, chenodeoxycholic acid and brought to 10 ml in benzene: ethanol (3:2). cholic acid of the glycine (G) or taurine (T) After protein precipitation of intestinal sam­ conjugated bile acid solution. ples from one normal subject (sample 1) and one patient (sample 2), 0.2 ml of the 14C-TC CONJUGATED BILE ACID DISTRIBUTION (GLC) was added to the supernatant solutions. The GLC analyses of the methyl-trifluo- After mixing, aliquots were removed for roacetate derivatives were performed on STDH and scintillation counting and the glass columns (4 mm id x 4 ft) packed with 2 values obtained were considered to repre­ percent QF-1 on 100/200 Gas Chrom Q.° sent 100 percent. After removal of the neu­ The operating temperatures were: injection tral fats and steroids with petroleum ether, port, 260°; column oven, 245°; hydrogen aliquots were again taken for STDH and flame detector, 260°. Helium was used as the counting. Finally, aliquots of both the carrier gas at a flow rate of 60 ml per min. chloroform and aqueous layers were counted The area of each bile acid on the and STDH assays were performed on metha- chromatograph was determined by triangu­ nolic solutions of the dried chloroform ex­ lation and expressed as area per /¿I solution tracts. Radioactivity was measured by liquid injected. Four injections per sample were scintillation spectrometry using both assayed and the values averaged. The indi­ Aquasol (3.5 ml water and 15 ml Aquasol) vidual bile acids were expressed as a and PPO (5 ml ethanol and 15 ml 2,5- percentage of the total measurable bile acid diphenyloxazole-5.5 g per liter toluene). content. Results RADIOACTIVE SODIUM TAUROCHOLATE (TC) The G/T ratios from intestinal aspirates of RECOVERY the five normal subjects as determined by Exactly 1.5 mg of sodium-taurocholate- the STDH and GLC assays are reported in (Carbonyl 14C)t was purified by TLC on Sil- table I. Statistical analysis comparing results obtained by the two methods reveals a p ° Applied Science Laboratories. value of 0.05. The mean G/T ratios for intes­ tMallinckrodt. tinal samples were 3.1 ± 0.9 by STDH and T ABLE I Conjugated Bile Acids in Control Subjects: G/T Ratios Sex, Age G/T-CDC G/T-C Subject Race Sample G/T STDH G/T GLC G/T-D GLC GLC GLC LL M, 32, W Duodenal 3.6 3.7 3.4 2.2 10.5 OB (1) M, 45-, W Duodenal 2.4 2.1 1.5 1.2 5.0 (2) Distal jejunal 2.6 1.9 2.7 1.7 1.2 FH M, 23, W Distal jejunal 2.0 2.3 2.1 1.9 2.9 AS M, 21, W Distal jejunal 4.3 3.6 2.1 1.6 8.8 VW F , 26, B Distal jejunal 3.8 2.0 0.9 0.5 6.2 Mean ± S.D. 3.1 ± 0.9 2.6 ± 0.8 2.1 ± 0;9 1.5 ± 0.6 5.8 ± 3.5 STDH - indicates data obtained by the 3-hydroxysteroid dehydrogenase assay. GLC - indicates data from gas liquid chromatographs calculated by triangulation. Aliquots from the same preparations were used for both assays (G - glycine conjugated; T - taurine conjugated; D - deoxycholic acid; CDC - chenodeoxycholic acid; C - cholic acid).
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