D]IGESTWE D SEASES

Bile Composition in the Pregnant Rabbit Joy Johnson, MSc and Norman Kalant, MD, PhD

To determine if there are changes in bile composition related to pregnancy, bile was collected from the hepatic duct and the gallbladder of normal and 3-week pregnant female rabbits. Bile acids were separated by thin-layer chromatography and measured by an enzymatic procedure. The relative and absolute concentra- tions of the bile acids in hepatic bile were the same for the 2 groups of animals. Glycodeoxycholic was the major bile acid, while glycochenodeoxycholic was the second most abundant. The relative concentration of glycine conjugates was higher in the gallbladder than in hepatic bile, while concentrations of litho- cholate, unconjugated dihydroxy acids and two minor unidentified acids were lower; these differences between hepatic and gallbladder bile are most readily explained by reabsorption on unconjugated mono- and dihydroxy acids through the gallbladder wall. The differences were more marked in pregnant animals; this may be due to bUiary stasis and increased time for reabsorption.

Although the relat ionship between preg- nancy and cholelithiasis is controversial, there is considerable evidence that the inci- dence of gallstones in women is higher than that in men and dur ing the child-bearing years it is higher in mul t iparous than in nulliparous women (I). I t is well estab-

From the Lady Davis Institute for Medical Re- search of the Jewish General Hospital and the De- partment of Experimental Medicine, McGill Uni- versity, Montreal.

Supported by a grant from the Medical Research Council of Canada.

Address for reprint requests: Norman Kalant, MD, Lady Davis Institute of the Jewish General Hospital, 3755 Cote Ste Catherine Road, Montreal, PQ.

lished that bile salts and phospholipids, part icularly lecithin, are responsible for keeping cholesterol in solution in the bile (2, 3). Theoretical ly, an increase in choles- terol content beyond the solubilizing capac- ity of bile could lead to the precipi ta t ion of cholesterol a n d ul t imately .to the format ion of gallstones (4). Early studies indicated an increase in cholesterol concentrat ion and in the cholesterol: total bile acid ratio in gal lbladder bile of pregnant women (5, 6). However, these findings were not subse- quent ly confirmed (7).

A more precise description of the solubil- ity properties and equil ibria of bile constit- uents is provided by a phase diagram (8);

Digestive Diseases, Voi. 17, No. 1 (January 1972) 1

JOHNSON & KALANT

that for conjugated bile salt-lechithin- cholesterol appears to supply a sound basis for predicting cholesterol precipitation from bile. Analytic data, reported by Large et al (7), for bile from pregnant women, when plotted on a phase diagram, fall on the border between the micellar (soluble) and crystalline (insoluble) regions for cholesterol (8). However, such phase di- agrams are based on the assumption that all bile salts behave similarly in solubility in- teractions while some studies indicate that dihydroxy bile acids form micelles at lower concentrations (2) and have a greater solvat- ing capacity for cholesterol (9) than do tr ihydroxy acids. It is therefore conceivable that an increase in the relative concentra- tion of trihydroxy acids, without a change in the total acid concentrat ion (and thus with no change in the phase diagram), m i g h t favor the precipitation of choles- terol. T h e present study was undertaken to determine whether pregnancy is associated with such changes in the relative concentra- tions of the various bile acids in gallbladder and hepatic bile.

MATERIALS AND METHODS Sampling of Bile

T w o groups of hea l thy whi te New Zealand female rabbi ts were used. N o n p r e g n a n t (control) an imal s weighed 4-5 kg. P regnan t rabbi ts of the same initial weight were s tudied in their fou r th week of pregnancy. W h i l e in the laboratory, the rabbi ts were m a i n t a i n e d on P u r i n a rabbi t chow a n d tap water ad l ib i tum. Prior to expe r imen ta t ion , they were fasted overn igh t and anesthet ized the nex t day w i t h pentobarb i ta l . T h e cystic duct was c lamped and the ga l lb ladder bile collected by aspi ra t ion into a syringe. T h e cystic duc t c l amp was left in place and t he c o m m o n b i l e d u c t was cannu la t ed w i t h

pol?ethylene t u b i n g for collection of hepat ic bile over an interval of 1 hour . Samples of bile were used immedia te ly or kept frozen un t i l ready for use; p re l imina ry e x p e r i m e n t s indicated tha t no change in bile acid composi t ion occurred with storage for several weeks.

Materials ~4C-Cholic acid was obta ined f rom the New

Eng land Nuclear Corporat ion. ~C-Taurochol ic acM was synthesized from the labeled cholate; a~C- labeled glycocholic and glycodeoxycholic acids were

synthesized f rom the appropr ia te acid and uC- glycine. Syntheses were carried ou t by the procedures of N o r m a n (10). T h e labeled com- p o u n d s were purif ied by prepara t ive thin-layer ch romatography . O the r bile acids used as reference s tandards du r ing ch roma tog raphy were obta ined commercia l ly and used wi thout fu r the r purifica- tion. ¢~-Hydroxysteroid dehydrogenase, used for the enzymat ic de t e rmina t ion of bile acids was obta ined f rom W o r t h i n g t o n Biochemicals.*

Measurement of Cholesterol Aliquots of bile were extracte(t on a s team ba th

with 40 volumes of c h l o r o f o r m - m e t h a n o l (2:1). T h e mix tu r e was then filtered and the filtrate evaporated to dryness in vacuo. T h e residue was dissolved in 2-3 ml of water and extracted with pe t ro l eum e ther (bp 50-80°C) . T h e extract was made up to 50 mi f rom which 3 al iquots of 5.0 ml each were taken for cholesterol de te rmina t ion (11).

Measurement of Bile Acids T h e procedure consisted of extract ion, separation

by th in- layer ch romatography , and m e a s u r e m e n t by an enzymat ic procedure. After extract ion of lipids with pe t ro l eum e ther (bp 60-80°C) , the bile was deprote inized wi th 10 vo lumes of absolute ethanol. T h e precipi ta ted pro te ins were removed by filtra- tion and washed three t imes wi th 10 ~olumes of e thanol . T h e combined filtrate and washings were dried and the residue was taken up in a suitable small vo lume of e thano l (usually 1.0 3.0 ml ) . Al iquots (15-45 p, liter) were taken for thin- layer c h r o m a t o g r a p h y on silica gel G by the me thod of Gregg (12) , us ing a solvent system of iso- octane: isopropyl ether:glacial acetic acid:isopropyl alcohol (2:1:1:1). Before use the silica gel was washed twice with nonredist i l led m e t h a n o l and once wi th redisti l led methanol , then dr ied for 12 horn's at t00°C. T h e plates were prepared from a s lurry of 30 g of washed silica gel and 58 ml of 10% e thanol (13) mixed in a homogenizer for 20 seconds.

T h i s procedure allowed good separa t ion of

*Freehold, NJ.

2 Digestive Diseases, Vol. 17, No. 1 (January 1972)

BILE COMPOSITION IN PREGNANCY

unconjugated bile acids as well as of glycolitho- cholic and glycocholic acids, all of which were eluted individually. However it did not permit separation of glycodeoxycholic from glycocheno- deoxycholic acid, and it did not resolve taurine conjugates. To separate these acids it was necessary to elute them, hydrolyze, and rechromatograph the unconjugated acids. Thus, from the first TLC plate, two eluates, one containing glycodeoxycholic and glycochenodeoxycholic, and the other contain- ing unresolved taurine conjugates, were evaporated to dryness and hydrolyzed for 3 hours in 2 ml of 5 N NaOH in sealed ampoules, in an autoclave at 21 pounds pressure and 255°F. Each hydrolysate was dilute with an equal volume of water and acidified to pH 1 with 10 N HC1. The free bile acids were extracted into chloroform, evaporated to dryness, dissolved in ethanol and chromatographed as be- fore.

After each chromatographic development, stand- ard bile acid mixtures applied to the sides of each plate were stained for guidance in eluting the unknown sample. Appropriate areas of the powder were transferred from the plate to Teflon-stoppered tubes and extracted by constant shaking for 16 hours at 60°C. The taurine conjugates were eluted with absolute ethanol while other acids were eluted with chloroform:methanol (2:1). An area of pow- der between cholic and glycolithocholic acids was removed and treated as a blank. The eluates were filtered under vacuum, evaporated to dryness under nitrogen, then dissolved in methanol.

Portions were taken for quantitat ive measure- ment of bile acids by a slight modification of the enzymatic procedure of Iwata and Yamaski (14). To 2.0 ml of 2.5 /Lmoles/ml NAD in 0.1M sodium pyrophosphate buffer pH 9.5, were added 0.15 ml of 0.02 M hydrazine hydrate and 0.5 ml of 1% B-hydroxysteroid dehydrogenase. Methanolic bile acid solution, 0.5 ml, was added, the mixture incubated at 40°C for 30 minutes in a water bath and absorbance readings taken at 340 m/z.

Losses during the entire procedure were moni- tored by the use of the labeled bile acids previously prepared. An ethanolic solution containing approx- imately 10" dpm in less than 1 /zg of each acid was evaporated to dryness and the residue dissolved in the bile sample to be analyzed. Aliquots of the final methanolic solution used for the enzymatic meas- urement of isolated bile acids were dried in counting vials, redissolved in toluene scintillation mixture and counted in a liquid scintillation cmm- ter. Recoveries were: cholic 80-87%, glycocholic 54-65%, glycodeoxycholic 76-83% and taurocholic

65-75%; most of the loss occurred during chromat- ographic separation and elution. In each experi- ment, corrections for loss were made on the basis of the isotope recoveries, using individual recovery values for corresponding acids and an average recovery value for all other acids.

R E S U L T S

T h e c o m p o s i t i o n o f t h e b i l e s is s h o w n i n

T a b l e s 1 a n d 2. S e p a r a t i o n o f g l y c o d e o x y -

c h o l i c ( G D C ) a n d g l y c o c h e n o d e o x y c h o l i c

( G C D C ) was c a r r i e d o u t i n 4 o f t h e 6

r a b b i t s o f e a c h g r o u p ; t h e s e r e s u l t s a r e

s h o w n s e p a r a t e l y i n T a b l e 3. As e x p e c t e d ,

g l y c o d e o x y c h o l i c ( G D C ) was t h e m a j o r ac id

i n b o t h g r o u p s of a n i m a l s i n b o t h ga l l -

b l a d d e r a n d h e p a t i c b i l e . I t is o f i n t e r e s t

t h a t t h e s e c o n d m o s t a b u n d a n t a c i d w a s

g l y c o c h e n o d e o x y c h o l i c ( G C D C ) . T w o ac-

ids, c h a r a c t e r i z e d o n l y b y t h e i r R I v a l u e s

a p p e a r e d c o n s i s t e n t l y as m i n o r c o m -

p o n e m s . T h e r e w e r e n o s i g n i f i c a n t d i f fe r -

e n c e s i n r e l a t i v e c o n c e n t r a t i o n . s o f t h e ac-

ids, b e t w e e n h e p a t i c a n d g a l l b l a d d e r b i l e s

of t h e n o r m a l a n i m a l s . I n t h e p r e g n a n t

a n i m a l s , . there w e r e s i g n i f i c a n t i n c r e a s e s i n

t h e ( G D C + G C D C ) f r a c t i o n o f t h e ga l l -

b l a d d e r b i l e , c o m p a r e d w i t h t h e h e p a t i c

b i l e . T h e i n c r e a s e c o u l d b e a c c o u n t e d f o r

b y d e c r e a s e s i n t h e r e l a t i v e c o n c e n t r a , t i o n s

of g l y c o l i t h o c h o l i c , l i t h o c h o l i c ( L i C ) a n d

t h e u n c o n j u g a t e d d i h y d r o x y ac ids . T h e in-

c rease i n ( G D C + G C D C ) a p p e a r e d to b e

d u e to a n i n c r e a s e i n e a c h c o n s t i t u e n t , b u t

t h e d a t a a r e i n s u f f i c i e n t to b e c e r t a i n o f

th is .

Table 1. Bile Acid and Lipid Composition (pmoles/ml) of Gallbladder Bile in the Rabbit

Total Bile Phospho- Choles-

Acids lipid terol

Pregnant (6) 201±27 1 . 2 ± 0 . 1 1 . 7 ± 0 . 1 Nonpregnant(6) 188±31 . 5 ± 0 . 2 1 . 4 ± 0 . 3

Values represent Mean + SE

Digestive Diseases, Vol. 17, No. 1 (January 1972) 3

Table Z. Bile Composition: Percent Distribution of Bile Acids in Hepatic and Gallbladder Bile in the Rabbit

JOHNSON & KALANT

Pregnant (6) Nonpregnant (6)

Gal lbladder Hepatic Gal lbladder Hepatic

Glycocholic 9.2 ± 2.3 8.1 ± 2.8 6.4 ± 1.0 Glycodeoxychol ict Glycochenodeoxychol ict 69.7 ± 6.1 52.0 ± 6.1t 71.2 ± 3.2 Glycol i thochol ic 2,7 ± 0.7 5.3 ± 0.7* 4.3 ~ 0.8 Total glycine conjugates 81.7 ± 3.9 65.4 ± 5.7~: 79.8 ± 3.7 Total taur ine conjugates 5.1 ± 1.0 3.8 ± 0.8 3.0 ± 0.6 Cholic 2.9 ± 1.5 2.2 ± 0.5 2.6 ± 0.4 Ursodeoxycholic 1.5 ± 0.4 4.0 ± 0.9* 1.8 ± 0.3 Chenodeoxychol ic 0.7 ± 0.2 4.1 ± 0.8t 2.0 ± 0.3 Deoxycholic 2.0 ± 0.4 4.3 ± 0.6t 2.9 ± 0.3 Total d ihydroxy acids 4.2 ± 1.0 12.3 ± 1.g~t 6.7 ± 0.7 Lithochol ic 3 .5± 1.0 7.0 ± 1.2~L 2.8 ± 0.3 Unknown RI 0.15 1.9 ~ 0 . 3 3.5 ± 0.4t 2.1 ± 0.2 Unknown R/0.72 2.2 ± 0.5 6.9 ± 2.3 3.0 ± 0.2

6 . 3 ± 1 . 3

6 3 . 3 ± 5 . 3 5 . 3 ± 0 . 7

7 4 . 9 ± 2 . 8 2 . 0 ± 0 . 4 2 . 8 ± 0 . 6 2 . 9 ± 0 . 8 2 . 0 ± 0 . 4 4 . 7 ± 0 . 8 9 . 8 ± 1 . 6 4 . 1 ± 0 . 7 3 . 1 ± 0 . 8 3 . 8 ± 0 . 7

Values represent Mean ± SE for 6 rabbits in each group Signif icance of dif ference between values in paired samples of gal lb ladder and hepatic bile:

* P < 0.025 t P < 0.01 ~t P < 0.005

Table 3. Content of Glycodeoxycholic and Glycochenodeoxycholic Acids (Percent of total bile acids) in Hepatic and Gallbladder Bile of Rabbit

Pregnant Nonpregnant

Gal lb ladder Hepatic Gal lb ladder Hepatic

Glycodeoxycholic 53.4 ± 6.9 36.7 ± 3.7 54.7 ± 2.0 47.1 ± 6.3 Glycochenodeoxychol ic 22.5 ± 3.5 11.6 ± 3.3 17.7 ± 1.8 18.6 ± 2.6

Values are Mean ± SD of 4 determinat ions

There were no statistically significant differences between hepatic biles in the 2 groups of rabbits (Table 4) with regard to absolute concentrations of bile a.cids and cholesterol.

D I S C U S S I O N

T h e data for bile acid composition of gallbladder bile has been expressed a.s per- cent of total acids, rather than as absolute

concentrations, since the latter parameter is dependent on durat ion of bile residence in the gallbladder, a variable and un.controlla- ble [actor. On the other hand, the time element does not influence the hepatic bile; the absolute concentrations shown in Table 4, there[ore, permit direct comparison of the 2 groups of animal.s.

T h e present results are in agreement with previous reports showing GDC to be

4 Digestive Diseases, Vol. 17, No. 1 (January 1972)

BILE COMPOSITION IN PREGNANCY

Table 4. Bile Acid Composition (umoles/ml) of Hepatic Bile in the Rabbit

Non- Pregnant pregnant

Glycocholic 0.79 4- 0.2 0.51 :t: 0.1 Glycodeoxychol ic* Glycochenodeoxycholic* 5.63 4- 1.1 5.17 4- 0.5 Glycolithocholic 0.55 :t: 0.1 0.43 4- 0.04 Taurine conjugates 0.39 :t= 0.04 0.16 -4- 0.04 Cholic 0.22 :E 0.04 0.24 4- 0.04 Ursodeoxycholic 0.39 4- 0.1 0.244- 0.04 Chenodeoxycholic 0.41 4- 0.07 0.16 4- 0.04 Deoxycholic 0.47 =E 0.04 0.40 4- 0.1 Lithocholic 0.69 :/: 0.1 0.33 -4- 0.04 Rz .15 0.374- 0.1 0.26 4- 0.1 Rz .72 0.69=k 0.2 0.31 4- 0.04 Total bile acids 10.61 4- 1.1 8.21 4- 0.6 Cholesterol 0.08 =1:0.02 0.09+ 0.04

*Values represent Mean ± SE for 6 rabbits in each group

the major bile acid in the rabbit (15-17). Gregg and Poley (15) demonstrated the presence of GCDC in the bile of the rabbit; we have confirmed this finding and, in fact, found tha.t this acid is the second most important, quantitatively.

Like :the ox, another herbivore (18), the concentrations of cholesterol and phos- pholipid are low relative to that of bile acids; the amounts of cholesterol present would probably be totally soluble in the bile acid solution even in the absence of phospholipid (8).

It has been reported tha.t administration of estrogen and progesterone, to simulate the high levels of these hormones associated with pregnancy, produces cholesterol gall- stones in female rabbi, ts (19). The present results do not demonstrate any significant changes in tile composition of gallbladder or hepatic bile in pregnancy which might explain that observation.

Direct comparison of bile acid composi- tion.s of hepatic and gallbladder bile has

not been reported previously. The major differences are a decrease in relative con- centration of unconjugated mono- and dihydroxy acids, and a corresponding in- crease in that of conjugated acids. Ostrow has shown absorption of bile sal.ts through the wall of the gallbladder, the rate being much higher for unconjugated than for conjugated acids (20). The differences be- tween gallbladder and hepatic bile could thus be explained by absorption from the gallbladder. These differences are much more marked in ,the pregnant animals; this may be the result of stasis, which is said to occur in pregnancy (21), and consequent increase in the time available for such reabsorption.

R E F E R E N C E S 1. Horn G: Observations on aetiology o[

cholelithiasis. Br Med J 2:732, 1956 2. Isaksson B: On the dissolving power of

lecithin and bile salts for cholesterol in human bladder bile. Acta Soc Med Upsal 59:296, 1953-54

3. Hofmann AF: The function of bile salts in fat absorption. The solvent properties of dilute micellar solutions of conjugated bile salts. Biochern J 89:57, 1963

4. Juniper K Jr: Physicochemical character- istics of bile and their relation to gallstone formation. Am J Med 39:98, 1965

5. Riegel C, Ravdin IS, Johnston CG, et ah Studies of gallbladder function; composi- tion of gallbladder bile and calculi in gallbladder disease. Surg Gynecol Obstet 62:933, 1936

6. Potter MG: Observations of gallbladder and bile during pregnancy at term. J Am Med Assoc 106:1070, 1936

7. Large AM, Johnston CG, Katsuki T, et ah Gallstones and pregnancy: the composition of gallbladder bile in the pregnant woman at term. Am J Med Sc 239:713, 1960

8. Hofmann AF, Small DM: Detergent prop- erties of bile salts. Annu Rev Med 18:333, 1967

Digestive Diseases, Vol. 17, No. 1 (January 1972) 5

JOHNSON & KALANT

9. Neiderhiser DH, Roth HP: Cholesterol sol- ubilization by solutions of bile salts and bile salts plus lecithin. Proc Soc Exp Biol Med 128:221, 1968

10. Norman A: Preparat ion of conjugated bile acids using mixed carboxylic acid anhy- drides: bile acids and steroids. Arkiv Chemi 8:331, 1955

11. Zak B, Dickerman RG, Whi te EG, et al: Rapid estimation of free and total choles- terol. Am J Clin Pathol 24:1307, 1954

12. Gregg JA: New Solvent systems for thin- layer chromatography of bile acids. J Lipid Res 7:579, 1966

13. Sinclair HB, Lehrfeld J: Use of ethanol- water absorbent slurries in coating of thin- layer chromatographic plates. Chem An- alyst 57:117, 1966

14. Iwata T, Yamaski K: Enzymatic determina- tion and thin-layer chromatography of bile acids in blood. J Biochem (Tokyo) 56:424, 1964

15. Gregg JA, Poley JR: Excretion of bile acids in normal rabbits. Am J Physiol 211:1147, 1966

16. Linstedt S, Sjovall J: On the formation of dioxycholic acid from cholic acid in the rabbit; bile acids and steroids. Acta Chem Scand 11:421, 1957

17. Okamura S, Okamura T: Uber die gallen- sauere der kaninchengalle. Z Physiol Chem (Medical Hoppe-Seyler's) 188:11, 1930

18. Nakayama F, Miyake H: Species differ- ences in cholesterol-complexing micromo- lecular fractions in bile in relations to gallstone formation. J Lab Clin Meal 67:78, 1966

19. Imamoglu K, Wangensteen SL, Root HD, et al: Production of gallstones by pro- longed administration of progesterone and estradiol in rabbits. Surg Forum 10:246, 1960

20. Ostrow JD: Absorption of bile salts by the healthy and injured gallbladder. J Lab Clin Med 72:999, 1968

21. Rothman MM: Anatomy and physiology of the gallbladder and bile ducts, Chap 107, Gastroenterology, Second edition. Edited by HL Bockus. Philadelphia, WB Saun- ders, 1965

6 Digestive Diseases, Vol. 17, No. 1 (January 1972)