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2340

THE ~DR#LYSIS OF BILE ACID CONJDGATES W. T. Beher, S. Stradnieks, G. R. Beher and G. J. Lin Lipid Metabolism Laboratory, Henry Ford Hospital Detroit, Michigan 48202 ABSTRACT

Received 6-30-78

Studies were made of a) the relationship of bile acid structure and analytical recoveries (measured by 3-hydroxysteroidoxidoreductase) following vigorous alkaline hydrolysis of bile acid conjugates and b) the relationship of structure and hydrolysis time of taurine- and glycine bile acid conjugates in a reaction catalyzed by glycocholic acid hydrolase. Alkaline hydrolysis resulted in good recoveries of hydroxy and 7 and 1% oxo-bile acids but poor recoveries of 3-oxo-bile acids. Borohydride reduction of the 3-oxo-acids prevented these losses. Complete enzymatic hydrolysis of glycine conjugated bile acids was about five times more rapid than that of taurine conjugates. Hydrolysis of conjugates containing 0x0 groups was slow. Borohydride reduction of oxoacids corrected this and did not inhibit enzymatic hydrolysis. It was concluded that both vigorous alkaline and enzymatic hydrolysis are satisfactory in bile acid assays if borohydride reduction is instituted before the hydrolytic step. However, due to the presence of possible enzyme inhibitors and solubility difficulties, strong alkaline hydrolysis is preferable to enzymatic hydrolysis in fecal bile acid determinationsat this time.

The determination of fecal bile acids is difficult since the extracts used for this purpose can contain 28 or more bile acids of different structure and a variety of impurities. Several chemical methods have been developed for the assay. However, almost all suffer inaccuracies due, in part, to destruction of the acids during alkaline hydrolysis of bile a&d

conjugates, a step which is necessary in fecal bile acid anal-

ysis because small amounts of conjugates may be present in feces obtained from normals and major amounts are sometimes found in the feces of abnormal and drug treated subjects. This difficulty is well known and studies (1,2) suggest that oxo-bile acids are especially prone to destruction. To achieve an accurate assay, these losses must be prevented or at least minimized. There are at least two possible approaches; a) reduction of oxo- to hydroxy-bile acids before hydrolysis; this approach assumes that oxo-acids are responsible for most of the losses, b) enzymatic hydrolysis of bile acid conjugates present in fecal extracts. To study the practicality of these approaches, we have investigated 1) the effects of bile acid structure on the quantitative recovery of free and ~oZwne 32, Number 3

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T1813tOXDI

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TDEOXDI

conjugated acids following alkaline hydrolysis, 2) the effects of oxobile acid reduction prior to alkaline hydrolysis on bile acid recoveries, and 3) the influence of conjugated bile acid structure on the rate of enzymatic hydrolysis using glycocholic acid hydrolase (3,4).

Although

enzymatic hydrolysis of bile acid conjugates, employing this enzyme, has been used in several assays, little is known of the influence of conjugated bile acid structure on the ease of hydrolysis. MATERIALS All chemicals were reagent grade and all solvents were distilled before use. 6a-hydroxy-3-oxo-; 7crhydroxy-3-oxo-; 12u-hydroxy-3-oxoand 7a,l2a;dihydroxy-3-oxo-5B_cholan-24-oic acids were synthesized from the corresponding hydroxy-acids by a specifically catalyzed oxidation at C-3 by 3-hydroxysteroid oxidoreductase coupled with nicotinamide adenine dinucleotide (5). Other free bile acids were purchased from Steraloids (Wilton, NH, USA). The glycine conjugates of 3,6-dioxo-; 3,7-dioxo-; 3,12-dioxo-; 3,7,12-trioxo-; 7a-hydroxy-3-oxo-; and 12a-hydroxy-3-oxo58-cholan-24-oic acids were synthesized from the corresponding free acids according to Lack -et al (6). Other glycine conjugates were purchased from Calbiochem (San Diego, CA, USA). The taurine conjugates of 3-0x0-; 3,6-dioxo-; 3,7-dioxo-; and 3,12-dioxo-5S-cholan-24-oic acids were synthesized according to Lack -et al (6). Other taurine conjugates were purchased from Calbiochem. The purity of the acids was determined by thinlayer chromatography using 200 p thick silica gel G coated plates (E. Merck, Darmstadt, PRG). When free bile acids were applied to the plates they were developed with ethyl acetate:iso-octane:acetic acid 10:10:2 (v/v). When conjugated bile acids were applied they were developed with chloroform:methanol:water:acetic acid 65:25:4:2 (v/v). When necessary, acids were purified using preparative silica gel G coated plates (1 mm) and the same solvents. After purification, the acids exhibited a single spot when examined by thin-layer chromatography. Nicotinamide adenine dinucleotide solution (NAD) was prepared by dissolving 45 mg of NAD (Sigma Chemical Co., St. Louis, MO, USA) in water and diluting to 100 ml. Pyrophosphate buffer (0.1 M pH 10) was prepared by dissolving 44.61 g of sodium pyrophosphate decahydrate in 900 ml of distilled water. The pH of the solution was adjusted to 10 by addition of 0.1 M hydrochloric acid and the resulting solution diluted to one liter with water. Hydrazine hydrate solution contained 12.5 g of 99-100% hydrazine hydrate (Eastman Kodak Co., Rochester, NY, USA) diluted to 90 ml with 0.1 M pH 10 pyrophosphate buffer. The pH of the solution was adjusted to 10 with 6 M hydrochloric acid. It was then diluted to 100 ml with pyrophosphate buffer. 3-Hydroxysteroid oxidoreductase (mixture of EC 1.1.1.50 and EC 1.1.1.51) solution (3HSD) was prepared by dissolving 0.5 mg of 3HSD (Sigma) in 1 ml of pyrophosphate buffer (0.1 M, pH 10). Glycocholic acid hydrolase (EC 3.5.1.24) solution was prepared by placing 100 mg (600 units) of crude C. perfringens (acetone pwd; Sigma) in a PotterElvehjem homogenizer together with 20 ml of water and 0.4 ml of thioacetic acid solution (0.23 ml of the reagent diluted to 100 ml with water). The mixture was homogenized for three minutes and was then

transferred to ultracentrifuge tubes. The tubes were centrifuged at 50,000 x g for 30 mins, Five ml aliquots of the supernatant were transferred to heap walled tubes, quickly frozen in liquid nitrogen and stored at -20 F. The stock solution contained 30 units of glycocholic acid hydrolase per ml. One unit is defined to release 1 u mole of glytine from glycocholate in five mins. at 37'C and pH 5.6. PROCEDURES A.

Effect of Alkaline Hydrolysis on Recoveries of Free and Conjugated Bile Acids 1)

Hydrolysis --of Bile Acids

Five-tenths ml of bile acid solution (0.22 u moles of bile acid dissolved in 0.1 M pH 10 pyrophosphate buffer) was placed in a nickel crucible. Two ml of 2 M aqueous sodium hydroxide was added and the crucibles placed in an autoclave for three hrs at 15 psi (250'F). Blanks which contained pyrophosphate buffer only were treated in the same way. After cooling, the samples were transferred to glass-stoppered tubes and acidified (pH 1) with 12 M hydrochloric acid. Each solution was extracted three times with three ml of diethyl ether. (Extraction studies showed that 95 to 100% of each free bile acid and 0 to 2% of each glycine or taurine conjugate was extracted by this procedure). The combined ether extracts which contained free bile acids were washed with water and evaporated to dryness. Five-tenths ml of pyrophosphate buffer and 0.2 ml of 0.048 M sodium borohydride solution (19 mg of sodium borohydride dissolved in 10 ml of 0.1 M pH 10 pyrophosphate buffer) was then added to each tube. The solations were allowed to stand for one hr to effect reduction of bile acids containing 3-0x0- groups (5,7), (reduction of these acids is necessary since 3HSD is used in quantification). Following reduction, 0.2 ml of 12 M hydrochloric acid was added to each tube to destroy excess sodium borohydride. Four-tenths ml of 6 M sodium hydroxide was then added to each tube to neutralize hydrochloric acid. The solutions were next diluted to exactly 2 ml. One ml of NAD solution, 1.2 ml of hydrasine hydrate solution and 0.3 ml of 3-hydroxysteroid oxidoreductase solution was added to each tube, After thorovghly mixing the content of each tube, they were incubated for 30 mins, at 37 C. The absorbance of the incubated solutions was determined at 340 nm.

2)

Control -Bile Acids

Bile acid solutions were treated as outlined above except that hydrolysis and extraction steps were omitted. Comparison of bile acid recoveries in hydrolyzed and control samples allowed one to relate bile acid structure and stability during alkaline hydrolysis. B.

The Effect of Borohydride Reduction Prior to Alkaline Hydrolysis on Recoveries of Oxo-Bile Acids

Five-tenths ml of a solution containing 0.22 u moles of oxo-bile acid dissolved in absolute ethanol was placed in a nickel crucible.

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Two-tenths ml of 0.048 M alcoholic sodium borohydride solution was added, the crucible covered and allowed to stand overnight at room temperature. The crucible's content was then evaporated to dryness and dissolved in 0.5 ml of pyrophosphate buffer (0.1 M, pH 10). Two ml of aqueous 2 M sodium hydroxide was added and the solution autoclaved at 15 psi for three hrs. The balance of the procedure was the same as outlined in A-l. A second sample of bile acid was treated in the same way except that borohydride reduction prior to pressure cooking was omitted. Comparisons of bile acid recoveries with and without borohydride reduction were determinations of the effectivenessof reduction in protecting oxo-bile acids from destruction during alkaline hydrolysis. C.

Relationship of Conjugated Bile Acid Structure and Ease of Hydrolysis Catalyzed by Glycocholic Acid Iiydrolase

Twenty-two hundredths p mole of conjugated bile acid dissolved in water, was placed in a 25 ml screw-capped test tube. Three-tenths ml of glycocholic acid hydrolase solution (various strengths), 0.2 ml of 1.86% aqueous ethylenediamine tetra-acetic acid (disodlum salt) and 0.2 ml of 0.2 M, pH 5.6 phosphate buffer was agded. The tubes were incubated, with shaking, for various intervals at 37 C after which hydrolysis was terminated by addition of 1 ml of I2 M hydrochloric acid and 5 ml of water. Free bile acids were extracted three times with three ml of diethyl ether. The combined ether extracts were evaporated to dryness and then dissolved in 0.5 ml of pyrophosphate buffer. If the conjugates before hydrolysis were 3-oxo-bile acids, the resulting free 3-oxo-bile acids were reduced and determined as outlined above (Sec. A-l). If not, borohydride reduction was omitted, the solutions made up to two ml with pyrophosphatebuffer and bile acid concentrationsdetermined as outlined above omitting borohydride reduction and subsequent work-up (Sec. A-l above). RESULTS Data obtained in studies of the effects of strong alkaline hydrolysis (2 M NaOH @ 250'F for three hrs) on recoveries of free and conjugated bile acids is shown in Table I.

Examination of this data shows several inter-

esting.points. Alkaline hydrolysis has little or no effect on the recovery of free or conjugated mono or dihydroxy bile acids. Recoveries of trihydroxy acids, on the other hand, are reduced about 10%. Oxo-bile acid recoveries are very poor in many cases; however, high losses are restricted to 3-oxo-acids. Good recoveries are obtained when 3u-hydroxy-oxo-bile acids are hydrolyzed. Perhaps the most important data in Table I is the effect of borohydride reduction in the recovery of 3-oxo-acids. Twelve hr treatment of these acids with 0.048 M ethanolic sodium borohydride completely protected them from losses incurred by alkaline hydrolysis.

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TABLE I Effect on Alkaline Hydrolysis** on Bile Acid Recoveries % loss during alkaline hydrolysis*

-56-cholan-24-oic acid

No BH 4 reduction

BH i reduction***

prior to hydrolysis

prior to hydrolysis

2 f 1' 7+1 0+2 7*1 13 ir 1 722 0+1 10 f 2 44 f 5 51 f 6 43 f 4 39 + 1 38 + 5 94 + 2 33 i: 6 43 * 1

5+1 Ok2 2t1 0+1 0+2 4*1 2*1 1+1 0+1 1*1 3+_2

3a-hydroxy3a,7a-dihydroxy3a,lZa-dihydroxy3a,ba,7a-trihydroxy3a,7a,lZu-trihydroxy3a-hydroxy-7-oxo3a-hydroxy-12-oxo3a-hydroxy-7,12-dioxo7a-hydroxy-3-oxobet-hydroxy-3-oxolPa-hydroxy-3-oxo7a,lZa-dihydroxy-3-oxo3-0x03,6-dioxo3,12-dioxo3,7,12-trioxo-N-(Z-sulfoethyl)-amide 3a-hydroxy3a,7a-dihydroxy3a,lZa-dihydroxy3a,7a,lZa-trihydroxy3-0x03,6,-dioxo3,7-dioxo3,12-dioxo3,7,12-trioxo-N-(carboxymethyl)-amide 3a-hydroxy 3a,7a-dihydroxy3a,lZa-dihydroxy3a,7a,lZa_trihydroxy3-0x03,12-dioxo3,7,12-trioxo-

of 520 O?l 7+_1 10 + 1 27 f 2 79 z!z 5 40 ? 3 30 f 1 28 I! 2

c

421 O&l 10 + 4 021 O&l

2+1 7*1 5+1 7fl 25 * 2 28 f 3 29 f 2

021 0+1 Ok1

of

* 10 determinations in each case ** 2 M NaOH; 250'F for 3 hrs *** Reduced 12 hrs with 0.048 M ethanolic sodium borohydride Standard deviation t

Table II presents the results of studies on the hydrolysis of bile acid conjugates by glycocholic acid hydrolase. The data shows that the enzyme hydrolyzes glycine conjugates (-N-(carboxymethyl)-amides), about five times faster than taurine conjugates (-N-(2-sulfoethyl)-amides). However, with the exception of certain oxo-bile acid conjugates, sufficient enzyme concentration results in rapid hydrolysis of both types of conjugate. It is interesting that the time required for complete conjugate hydrolysis positively correlates with the degree of nuclear hydroxylation. The same type of relationship occurs with increases in the number of nuclear 0x0 groups. Further examination of the data shows that while conjugated hydroxybile acids are easily hydrolyzed, it is much more difficult to hydrolyze 0x0-conjugates. Reduction of oxo-conjugateswith sodium borohydride results in easily hydrolyzed compounds. TABLE II Hydrolysis of Bile Acid Conjugates Catalyzed by Glycocholic Acid Hydrolase (EC 3.5.1.24)

-5b-cholan-24-oicacid

Minutes

Units/ml -N-(Z-sulfoethyl)-amideof 3o-hydroxy3a,7a-dihydroxy3o,f2a-dihydroxy3a,7a,l2a-trihydroxy3-0x03,7,-dioxo3,3.2-dioxo3,7,X2-trioxo-

3.7 3.7 3.7 3.7 7.5 7.5 7.5 7.5

(5 10

The hydrolysis of bile acid conjugates.

355 2340 THE ~DR#LYSIS OF BILE ACID CONJDGATES W. T. Beher, S. Stradnieks, G. R. Beher and G. J. Lin Lipid Metabolism Laboratory, Henry Ford Hospita...
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