67

Clinica Chimica Acta, 65 (1975) 67-74 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CCA 7360

AN ENZYMIC METHOD FOR THE DETERMINATION OF THE GLYCINE/TAURINE RATIO OF CONJUGATED BILE ACIDS IN BILE

J.C.M. HAFK~NSCHEID~

and M.P.C. HECTORS

Laboratory for Clinical Chemistry, Department of Internal Medicine, Katholieke Un~~ers~teit, St. ~adboud~iekenhais, N~~~~en (The Netherlands) (Received June 9, 1975)

Summary A method is described in which the ratio of the glycine- to ~urine~onjugated bile acids (G/T ratio) in bile is determined. After pretreatment of the bile for removal of the lipids, the bile acids are deconjugated enzymically with choloylglycine hydrolase (EC 3.5.1.24). The amino acids taurine and glycine are liberated in this way, after appropriate isolation, taurine and/or glycine are then determined with ninhydrin, enabling the establishment of the G/T ratio. A nearly complete hydrolysis was obtained for 6 conjugated bile acids, while the recovery of these acids when added to hog or ox bile was quantitative. The mean G/T ratio for hog bile, ox bile and human B-bile was 6.3, 2.5 and 2.0, respectively. The amount of total, free and conjugated bile acids can be determined by this method, combined with the 3cu-hydroxysteroid dehydrogenase technique for bile acid determination described by Iwata and Yamasaki [l]. A high G/T ratio was observed in 3 cases of Crohn’s disease in the small bowel, but the extent of deconjugation in B-bile was lower than in duodenal fluid. The determination of the G/T ratio can be complement to our knowledge of the metabolism of bile salts in certain gastro-intestinal disorders.

Introduction In the liver, bile acids are synthesised from cholesterol and conjugated with glycine or taurine. The conjugated bile acids are excreted in the small bowel, where they can be deconjugated or converted to secondary bile acids. Abbreviations: GC, glycocholic acid: TC, taurocholic acid: GDC, glycodeoxycholic acid; TDC, ~urodeoxycholic acid; GCDC, ~ycochenodeoxy~boli~ acid; TCDC, taurochenodeoxycholic acid. * To whom correspondence should be addressed.

68

Most of the bile acids are absorbed in the ileum by an active transport system. The composition of the bile is governed by two moIar ratios: the G/T ratio (ratio of glycine- to t~urine-conjugated bile acids) and the ratio of cholic acid : chenodeoxycholic acid : deoxycholic acid. A large number of methods has been described for the quantitative determination of the various bile acids. Thin-layer chromatography is the most popular technique for the separation of the various bile acids, although the difficulty in separating the conjugates of deoxycholic acid from those of chenodeoxycholic acid is a disadv~tage of this method. Coupled with the enzymic determination of the individual bile acids with Scr-hydroxysteroid dehydroEC 1.1.1.50) originally genase (3a-hydroxysteroid : NAD( P)’ oxidoreductase, described by Iwata and Yamasaki [ 1 J , it is possible to determine the concentration of each of the bile acids. Gas-liquid chromatography is another technique often used for the determination of the various bile acids. A disadvantage of this method is that it cannot be used for analysing conjugated bile acids. So no information is available on the G/T ratio or the extent of deconjugation. An elevated G/T ratio is found [Z-7], in certain ileal dysfunctions. When the enterohepatic circulation is interrupted during cholestyramine therapy, the G/T ratio is also elevated [8,9]. In this case the raised G/T ratio has been explained in terms of a partial unavailability of taurine in face of a much increased demand of bile acid conjugation. The peptide link in the conjugated bile acids is very resistant to cleavage by chemical hydrolysis or proteolytic enzymes [ lO,ll] . However, the enzyme choloylglycine hydrolase (3a:,7~,12~-trihydroxy-5/3-cholan-24-oxylglycine amidohydrolase, EC 3.5,1.24), present in many intestinal microorganisms like Enterococci, C~ost~id~a and ~ucte~o~des, is able to hydrolyse bife acid conjugates [12,13]. After hydrolysis either the free bile acid or the glycine or taurine moiety can be determined. In the present paper a method is described in which the G/T ratio in bile is established by the determination of the glycine and/or taurine, which have been set free after hydrolysis of the conjugated bile acids with choloylglycine hydrolase. When the amount of liberated glycine and taurine is known exactly, the content of conjugated bile acids can be calculated. With the 3a-hydroxysteroid dehydrogenase method of Iwata and Yamasaki [ 11 the total amount of bile acids can be determined. From the difference between the total content of bile acids and the content of conjugated bile acids, the amount of non-conjugated bile acids can be calculated. Materials and methods The procedure for the determination of the G/T ratio can be divided into 4 parts. a. Pretreatment of the bile according to the principles of Ahrens and BorgStrom [14]. b. Removal of free amino acids from the bile. c. Enzymic cleavage of the conj ‘ugated bile acids and separation of the liberated amino acids.

69

d. Determination of glycine and/or taurine. ad a. In a test tube, 6 ml of a mixture of ethanol/ether/n-heptane (1 : 1 : 1) is added to 2 ml bile. After vigorous shaking and phase separation, the upper layer is discarded and 4 ml upper phase of a ethanol/ether/n-heptane/water (1 : 1 : 1 : 1) mixture are added to the lower phase. After shaking and removal of the upper layer, the procedure is repeated. Finally, 5 ml ethanol (96%) are added to the lower phase and the solution is heated for 2 min until boiling. This coagulates the proteins. After centrifugation the isolated supernatant is evaporated to dryness and the residue is dissolved in 10 ml ethanol (50%). ad b. An aliquot of this solution (1 ml) is placed on to a column (5 X 1 cm) of Dowex AG 50 W-X 2 (50-100 mesh) in the H+ form to remove the free amino acids. The elution is carried out with 20 ml ethanol (50%). The inorganic cations, most of the pigment and all of the amino acids except taurine are retained [ 151. The eluate is evaporated to dryness and dissolved in 5 ml 0.1 M acetate buffer, pH 5.7, containing 14.2 PM fl-mercaptoethanol. ad c. To 2 ml of this solution 2 ml enzyme solution are added (4 mg choloylglycine hydrolase are suspended in 2 ml of the above-mentioned acetate buffer). After incubation overnight at 37°C and centrifugation, 1 ml of the supernatant is diluted to 10 ml with water and 3 X 1 ml is taken for the determination for the sum of glycine and taurine in triplicate. Another 1 ml of the supernatant is placed onto a column (2 X 1 cm) of Dowex 50 W-X 8 (100-200 mesh) in the H’ form for the separation of the glycine and taurine according to the method of Sorbo [16]. The taurine is collected by elution with water until 5 ml has been collected from the column and 3 X 1 ml of this eluate is taken in triplicate for the determination of taurine. ad d. The amino acids were determined according to the method of Sorbo [16] with a slight modification. To 1 ml solution obtained for the determination of the sum of glycine and taurine or of taurine alone, 2 ml ninhydrin reagent are added (25 ml citrate buffer (0.2 M; pH 5.7), 25 ml ninhydrin stock solution and 0.1 ml stannous chloride solution). After heating in an oil bath for 20 min at 100°C the solution is cooled, 5 ml water are added and the absorption is read at 570 nm in a spectrophotometer. By subtracting the taurine content from the glycine + taurine content the glycine content can be calculated and so the G/T ratio is known. The amount of conjugated bile acids can be established from the content of glycine and taurine. The ninhydrin stock solution is prepared by dissolving 3 g ninhydrin in 100 ml ethanol (96%) and the stannous chloride solution by dissolving 4.5 g and diluting to 10 ml with water. SnClz * 2 Hz0 in 1.7 ml HCl (concentrated) The total content of bile acids was determined after pretreatment of the bile as described under (a) with 3a-hydroxys-teroid dehydrogenase according to the method of Turnberg and Anthony-Mote [17]. The thin-layer chromatography was performed with 2 different solvent systems for free and conjugated bile acids according to Hofmann [18]. The bile acids were revealed by spraying the plates with 10% phosphomolybdic acid in ethanol [ 181. Choloylglycine hydrolase from Clostridium welchii was obtained from Sigma and 3a-hydroxysteroid dehydrogenase from Pseudomonas testosteroni

70

from Worthington Biochemical Corporation. Cholic acid was purchased from Merck, GC from Carl Roth, Karlsruhe, TC from Fluka and GDC, TDC, GCDC and TCDC from Calbiochem. All other reagents were analytical grade. Results From preliminary experiments it was observed can be separated and determined very well according

that glycine and taurine to the method of Sijrbo

1161. The relationship between the enzymic cleavage of GC and TC with choloylglycine hydrolase and the incubation time is shown in Fig. 1. Complete hydrolysis for GC is obtained after 2 h, but an incubation time of about 18 h is necessary for TC. Table I shows that 6 different conjugated bile acids are hydrolysed nearly quantitatively by choloylglycine hydrolase. Only GCDC gives a somewhat lower value than loo%, perhaps caused by impurities of the bile acid. Although the various bile acids have different K, values for the enzyme [ 121, it is revealed that a stoichiometric conversion of the conjugated bile acids can be obtained. Fig. 2 shows that mixtures of GC and TC can be separated and determined with this method. From these results it can be concluded that the conjugated bile acids occurring most frequently can be determined quantitatively after cleavage of the peptide bond by determination of their respective amino acids. In Table II the G/T ratio of ox, hog and human B-bile is given. The range for the G/T ratio in ox bile is somewhat smaller than that in hog bile. From the difference between the total amount of bile acids and the conjugated bile acids, the content of free bile acids can be calculated. About 75 to 84% of the total amount of bile acids is conjugated in these 3 different types of bile. In Table III it can be seen that the recovery of conjugated bile acids added to hog bile is quantitative. Identical results were obtained with ox bile.

0

1

2

3

4

5

18 time

Fig. 1. Cleavage of glycocholic acid and tauacholic incubation time. l-----+, glycocholic acid; X -X,

(

h

)

acid with choloylglycine hydrolase in relation to the taurocholic acid.

71

TABLE

I

CLEAVAGE After

OF

CONJUGATED

hydrolysis,

glycine

BILE

or taurine

ACIDS

was

WITH

CHOLOYLGLYCINE

determined.

All

solutions

MeZUl

S.D.

C.V.

(mmwu

(mmol/l)

(%)

HYDROLASE

contained

2.0

mm01

II*

of

the

(%)

GC

1.87

0.09

4.8

18

93.5

1.82

0.08

4.4

22

91.0

GCDC

1.67

0.12

7.2

13

83.5

TCDC

1.86

0.14

7.5

13

93.0

GDC

1.91

0.14

7.3

8

95.5

TDC

2.08

0.08

3.8

12

104.0

of determinations.

.// ‘i

05

x

0.4

.’

/

/

./

\

E

acid.

Hydrolysis

TC

* Number

bile

:, 6 0.2 T

x

0.:

.

/

0.1

x

\

/

,

1.0 1.0

0.5 1.5

F," s L 1

x

,\

1.5 0.5 ,

~.

'8

I mmoles,l

Fig.

2.

Determination

mixture

of both

TABLE

II

G/T

RATIO

of

bile

AND

acids.

BILE

G/T

glycocholic

lA,

ACID

acid

I.> I

and

taurocholic

glycocholic

acid:

CONCENTRATION

IN BILES Bile acid

ratio

Mean

acid

S.D.

Range

ll*

as glycine

X ---X.

Total

and

taurocholic

OF

VARIOUS

taurine,

respectively,

in a

acid.

ORIGIN

concentration

(mmol/l)

Conju-

Non-con-

gated

jugated

n*

Ratio conjugated total (%)

Hogbile

6.3

0.9

4.1-7.8

13

139.0

46.0

3

75.0

Ox bile

2.5

0.3

1.9-3.2

13

185.0 77.2

63.3

13.9

3

84.0

2.0

0.6

1.5-3.4

12

43.6

36.4

8

83.4

Human

B-bile* * * Number * * Bile

of determinations

collected

from

various

in a specimen. normal

individuals.

7.2

72 TABLE

III

RECOVERY

OF STANDARD

BILE ACIDS

ADDED

TO HOG BILE

The stated amount of bile acid was added to bile, and the glycine and/or taurine content was determined. All concentrations are expressed in mmoI/l. Added Bile GC TC GC+TC GDC TDC GDC + TDC GCDC TCDC GCDC + TCDC GC + GDC GC + TDC GC + GCDC GC + TCDC TC + GDC TC + TDC TC + GCDC TC + TCDC

0.37 -

Found

Recovered

-

-

0.76 0.75 0.78 0.71 0.72 0.73 0.79 0.68 0.69 0.81 0.80 0.82 0.76 0.82 0.81 0.73 0.71

0.49 0.41 0.45 0.38 0.38 0.38 0.34 0.24 0.29 0.44 0.44 0.42 0.37 0.40 0.38 0.38 0.33

(%)

88.4 96.1 95.1 94.7 95.8 97.4 111.3 111.2 104.5 100.0 98.7 101.3 102.7 98.7 107.8 97.3 101.4 100.1 f i 6.0)

Mean (i S.D.)

The G/T ratio in various human biles is shown in Table IV. The mean G/T ratio varied from 1.2 to 3.8 in 3 human B-biles collected from 3 normal individuals. About 82.0% of the bile acids has been conjugated. Biles 4 and 5 were colfected from 2 patients suffering from Crohn’s disease in the small bowel. The G/T ratio in these biles was raised to 11.3 and 12.4 indicating a high content of glycine-conjugated bile acid. The percentage of conjugated bile acid is normal in these biles. The G/T ratio in duodenal fluid collected from a patient suffering from Crohn’s disease in the small bowel is also high (14.0), but in this case only 31.2% of the bile acids is conjugated. TABLE

IV

G/T RATIO Bile

AND BILE ACID

CONCENTRATION

G/T ratio Meall

IN VARIOUS

S.D.

Range

n*

HUMAN

BILES

Bile acid concentration Total

Conjugated

(mmoI/l>

Non-conjugated

1,*

Ratio conjugated total (%)

1

2 3 4 5 Duodenal fluid _-

3.8 2.2 1.2 12.4 11.3

0.4 0.2 0.3 1.9 2.2

14.0

2.7

* Number of determinations.

3.14.4 1.82.5 l.O1.7 9.7-14.6 9.0-13.2 11.8-17.0

6 6 5 6 4

52.0 90.0 20.8 29.7 17.9

42.1 73.9 17.2 25.9 15.1

9.9 16.1 3.6 3.8 2.8

5 5 4 5 5

80.7 82.0 82.6 87.2 84.2

3

7.7

2.4

5.3

4

31.2

73

Discussion Many gastro-intestinal disorders are associated with disturbances in the bile acid metabolism. One of the parameters in studying this metabolism is the ratio of the glycine-conjugated to the taurine-conjugated bile acids. Normally a G/T ratio of 2.0-3.0 is found in bile. In this paper a method is described in which the G/T ratio can be determined without knowledge of the concentration of the various individual bile acids. The enzyme choloylglycine hydrolase is capable of splitting the conjugated bile acids nearly quantitatively. This is in contrast to the findings of Nair et al. [12], who did not observe a complete hydrolysis. However, if the incubation is carried out for 18 h as in our experiments, a complete deconjugation can be obtained. Average recoveries of 100.1 and 96.0%, respectively, were obtained, when various conjugated bile acids were added alone or in combination to hog or ox bile. So it seems that the bile acids do not influence each other in the enzymic cleavage. Combined with the determination of the total content of bile acids, the concentration of the free and conjugated bile acids can be determined. In normal human biles, about 80--85% of the bile acids are conjugated. A high G/T ratio was observed in 3 patients suffering from Crohn’s disease in the small bowel. The breath test determined according to Fromm and Hofmann [19] and Sherr et al. [20], in which [l-l “C] glycocholic acid was given orally to the patients, was positive. An indicanuria was also observed in these patients indicating that an overgrowth of bacteria in the small intestine may be present. These bacteria are capable of splitting the conjugated bile acids. As taurine is the limiting factor, it is possible that most of these free bile acids are conjugated with glycine after reabsorption in the ileum. So it can be argued that the G/T ratio is elevated. In the duodenal fluid of the patient suffering from Crohn’s disease in the small bowel only 31.2% of the bile acids were conjugated. This is in contrast to the B-biles of the 2 other patients suffering from the same disease, where a normal conjugation percentage was observed. This difference can be explained by the fact that in B-bile practically all bile acids are conjugated and that deconjugation occurs in the small bowel by the bacteria present there. So in duodenal fluid the G/T ratio may be equal to that in B-bile for these patients, but the degree of deconjugation may be higher. Thin-layer chromatography confirmed these differences. It seems that the experiments described here provide good information about the G/T ratio in bile or duodenal fluid and about the extent of deconjugation. The method can be complementary to other methods which are used in the study of the bile salt metabolism.

Acknowledgement The authors specimens.

are indebted

to Dr C. Avila for collecting

the human

bile

74

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New

taurine ratio of conjugated bile acids in bile.

A method is described in which the ratio of the glycine- to taurine-conjugated bile acids (G/T ratio) in bile is determined. After pretreatment of the...
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