Identification of bile alcohols in serum from healthy humans

IDENTIFICATION FROM HEALTHY

OF BILE ALCOHOLS HUMANS

IN SERUM

Toshihito Hiraoka,a Kenji Kihira,b Daisaku Kosaka,a Kohda,a Takahiko Hoshita,b and Coro Kajiyamaa

Tadahiro

First Department of internal Medicine” and Institute of Pharmaceutical Science,b Hiroshima University School of Medicine, Kasumi l-2-3, Minami-Ku, Hiroshima, 734 Japan Corresponding author: Toshihito Hiraoka Received January 14, 1987 Rewed October 4, 1987

ABSTRACT Bile alcohol glucuronides present in human serum were isolated by ion exchange chromatography on piperidinohydroxypropyl Sephadex LH-20. Following hydrolysis with f3glucuronidase, the bile alcohols were analyzed by a combination of gas-liquid chromatography and mass Bile alcohols identified were 27-nor-5$spectrometry. cholestane-3a,7a,l2a,24,25-pentol, 27-nor-5$-cholestane-3a, 7o1,12cr,24,25,26_hexol, SB-cholestane-3a,7a,l2a,24,25-pentol, 5&cholestane-3a,7ol,l2@,24,26-pentol,5$-cholestane-%,7a, 12o1,25,26-pentol, and 5&cholestane--3a,7ct,l2~,24,25,26hexol. The bile alcohol compositionin serum was similar to that in urine but not to that in bile. The concentrationof total bile alcohols in serum was 59.5 k 24.6 rig/L..

INTRODUCTION

Bile alcohols had long been supposed to occur only in primitive vertebrates such as fishes and amphibians as evolutionary

precursors

of mammalian bile acids (1).

Interest in bile alcohols increased when these compounds were

found

in both healthy

and diseased

humans

as

intermediatesand side products of normal pathways in bile

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acid biosynthesis (2-12). In 1981, Karlaganis -et al (6) found a number of bile alcohols in human urine. The most abundant bile alcohol in human urine has been identified as 27-nor-5&cholestane3cr,7cr,12a,24,25_pentol (6,9).

In addition to the C26 bile

alcohol, several C26 and C27 bile alcohol carrying the cholic acid type of nuclear structure and the sterol side chain possessingtwo or three hydroxyl groups at C-24 - C-27 are also excreted in human urine as the minor constituents of bile alcohols (8,9). Recently, Kuroki -et al (10) and Une -et al (11) have found that 27 bile alcohols are present, though in trace amounts, in human gallbladder bile.

The bile alcohol

composition in the bile is quite different from that in the urine. The major bile alcohol in the bile was identifiedas 58-cholestane-3or,7c,l2a,26-tetrol. The compositions of bile alcohols in the glucuronide fraction are different between urine and bile in both healthy humans and the patients with cerebrotendinousxanthomatosis. However, the major site of bile alcohol formation is thought to be the liver. It would be interesting to establish whether the bile alcohols in urine derived through blood after formation in liver or modified by renal enzymes. However, the serum bile alcohols

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have never been previouslyexamined. The reason for the difference in bile alcohol profiles between the urine and the bile is not known.

A possible

explanationis renal metabolism of bile alcohols. Metabolic reactions of steroids have been demonstratedto occur in the kidney,

e.g., the I-hydroxylation of 25-hydroxychole-

calciferol and the sulfation of bile acids. To investigate the role of the kidney in bile alcohol metabolism

and the metabolic origin of urinary bile

alcohols, the present study was undertaken in which serum from healthy persons was analyzed for bile alcohols.

MATERIALS AND MFTHODS Subjects. The study was conducted on seven healthy volunteers (6 males and 1 female, 32.6 + 6.9 years old). Reference compounds. 27-Nor-S&cholestane-3cr,701,12cr,24,25pentol (9), 27-nor-5~-cholestane-3ff,7~,12~,24,25,26-hexol (lo), 5S-cholestane-3a,7a,lZcr,24,25-pentol (13), Secholestane-3ol,7ol,l2c(,24,26-pentol (14),5S-cholestane-3a,7a, 12a,25,26_pentol (15), and 5B-cholestane-3cc,7c,l2cx,24,25,26hexol (16) were synthesized according to the methods reported previously, Isolation _of bile alcohols. Serum (20 mL) diluted with 6 vol of 0.1 M sodium hvdroxide was stirred for 30 min at 64'C and then passed through a Sep-Pak Cl8 (Waters Associates, Milford, MA). Following a wash with 10 mL of water, the extracted materials were eluted with 10 mL of methanol. The eluent was evaporated to dryness and the resulting residue was dissolved in 90% ethanol, and the solution was passed through a column of piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20)(17). The eluant and an additional wash with 8 mL of 90% ethanol were collected to give a fraction of neutral compounds. Elution with 8 mL of 0.2 M formic acid

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in 90% ethanol gave a glucuronidefraction. The glucuronide fraction was hydrolyzedenzymaticallywith 5,000 units of Bglucuronidase (E.G.3.2.31,Sigma Chemical Co., Type H-l) in 0.1 M sodium acetate buffer (10 mL, pH 5.0). After a 48 h incubation period, the mixture was extracted with a Sep-Pak Cl8 cartridge as described above. The methanol eluate was evaporated to dryness. The resulting residue was dissolved in 5 mL of 90% ethanol and passed through a column of PHPLH-20. The eluant and an additional wash with 5 mL of 90% ethanol were collected and evaporated to give a fraction of deconjugated bile alcohols. Analysis-of bile alcohols. Bile alcohol TMS ethers prepared as described previously (9) were analyzed by means of gasliquid chromatography (GLC) and gas-liquidchromatographymass spectrometry (GLC-MS) using a selected ion monitoring method. Identificationand quantitationof individualbile alcohol were carried out by direct comparison of the retention times and peak areas with those of reference compounds as external standards. GLC was carried out on a OV-1 capillary column (0.35 mm x 25 m)in a Shimadzu GC-8A gas chromatographequipped with a flame ionization detector and Van den Berg's solventlessinjector. GLC-MS was carried out on a Shimadzu model QP-1000 gas chromatograph mass spectrometer equipped with a data processing system. Operating conditionswere as follows: column, same column as for GLC; column temperature,285OC; ion source temperature, 250"Cjonizing energy, 70 eV; and trap current, 60 HA. For selected ion monitoring the following ions were used: m/z 321 for 27-nor-5g-cholestane-3o,7o,l2o,24,25-pentol, 27nor-5&cholestane-3a,7a,l2a,24,25,26-hexol,5&cholestane301,7a,12cr,24,25-pentol, and 5$-cholestane-3u,7a,12a,24,26pentol; m/z 439 for 5$-cholestane-3cr,7c(,f2cr,25,26-pentol; m/z 219 for 58-cholestane-3~,7~,12~,24,25,26_hexol.

RESULTS AND DISCUSSION

Bile alcohols in serum were analyzed for the first time after fractionationby the mode of conjugation. By direct comparison of gas chromatographicproperties

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RRTh !I&/Lc

%a

ile alcohols excreted as glucuronides. RT were described relative to the TMS ether of methyl cholate (1.00). CYean * SD 40 of total bile alcohols and the values in parenthesesindicate % f total bile alcohols in urine excreted as glucuronides (16). e!Somers of hydroxyl groups at C24 and/or C25 in the side chain.

4

1.72 27-~or-S8-=holeatsne-3~,7~,12~,24,25-~e~tol~1.80 32.2jzl2.6 54.0(48.7) 27-nor-58-cholestane-3cr,701,12or,24,25,26-hexol 2.53 4.M 2.8 7.7( 9.4) 58-cholestane-3cr,7c,lZa,24,25-pentol 2.11 6.8zk3.4 11.4(15.9) 5@-cholestane-3cr,7cr,12a,24,26-pentol 2.07 5.4* 3.0 9*1(12,0) 58-cholestane-3a,7c,l2o,25,26-pentol 2.35 1O.Ozk7.7 16.8(11.0) 5&cholestane-3or,7a!,12a,24,25,26-hexol 3.17 0.6* 0.5 l.O( 3.0) Total 59.5zk24.6

Bile alcohols a

Table 1. Serum bile alcohols of healthy humans

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and mass spectra with reference compounds the following six bile alcohols were identifiedin the glucuronidefraction of serum from healthy humans: 27-nor-5&-cholestane-3a,7a,12a, 24,25-pentol,27-nor-SB-cholestane-3a,7a,12a,24,25,26-hexol, S&cholestane-3a,7a,l2~,24,25-pentol, 5&cholestane-3ol,7or, 12or,24,26-pentol, .%-cholestane-3a,7a,12,25,26-pentol,and 5@-cholestane-3a,7a,l2a,24,25,26-hexol

(Table 1). For

quantitativeanalysis,selected ion monitoring method was employed.

The ions those are relatively intense and

characteristic for the bile alcohols were chosen as the monitoring ions according to the mass spectra reported. The ion at m/z

321 was

used

for 27-nor-5B-cholestane-

3a,7a,12o,24,25_pentol (6), 27-nor-5@-cholestane-3c1,7cr,12a, 24,25,2&hexol(lO), 5~-cholestan~-3~,7~,12~,24,25-pentol (lo), and 5B-cholestane-3cr,7o,l2~,24,26-pentol (16).The ion at m/z 439 was used for 5B-cholestane-3a,7c,l2o,25,26-pentol (9).

The ion at m/z 219 was used for SB-cholestane-

3o,7a,120r,24,25,2Ghexol (16).

The average concentration

of serum bile alcohols found in the glucuronidefraction was 59.5 f 24.6 Ftg/L.In the neutral (unconjugated)

fraction no

bile alcohols were detected by the method employed here. The serum bile alcohol profile was essentially same=

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the urinary bile alcohol profile that has been reported in the previous paper (16). In both biological fluids most or all of the bile alcohols are present as the glucuronoconjugates. Previously identified as

predominant bile alcohol in

the urine, 27-nor-5B-cholestane-3a,7a,12a,24,25-pentol constitutedan average of 54% of the total bile alcohols in the serum. serum

All of the other bile alcohols found in the

in the present

investigationwere

previously

identified as the urinary bile alcohols. The similarity of the bile alcohol profiles between the serum and urine may synthesis of urinary bile alcohols in

indicate that the

healthy humans is a function of the liver rather than the kidney.

Renal metabolism of bile alcohols is possible but

less likely.

The bile alcohol glucuronidesin serum may be

merely transferred

into urine by filtration without any

modificationsby renal enzymes in the healthy humans.

REFERENCES

1. Hoshita, T. In: Sterols and Bile Acids.(Danielsson,H. and Sjovall, J., Editz),lsevier/North Holland Biochemical Press. Amsterdam. (1985),pp 279-299. 2. Setoguchi, T., Salen, G., Tint, G.S.,and Mosbach, E.H. J. CLIN. INVEST.2, 1393 (1974). 3. Yasuhara, M., Kuramoto, T., Hoshita, T., Itoga, E., and Kito, S. STEROIDSZ, 333(1978).

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4. Kibe, A., Nakai, S., Kuramoto, T., and Hoshita, T. J.

LIPID RES.2, 594 (1980). 5. Hoshita, T., Yasuhara, M., Une, M., Kibe, A., Itoga, E. Kito,S., and Kuramoto,T. J. LIPID RES. -21 1015 (1980). 6. Karlaganis,G.,Alme, B., Karlaganis, V., and Sjovall, J., J. STEROID BIOCHEM.14, 341 (1981). 7. Wolthers, B.G.,Volmer, M., van der Molen,J.,Koopman, A.E.J.,and Waterreus, R.J. CLIN. CHIM. :& 8d7eJga38::;83). 8. Ludw&ohn, H., Henning, H.V.,Sziedat, A., Matthaei, D., Spiteller, J., Reiner, J., and Egger, H.J. EUR. J. CLIN. INVEST.13, 91 (1983). 9. Kuwabara, M., Ushiroguchi, T., Kihira, K., Kuramoto, T., and Hoshita, T. J. LIPID RES.25, 361 (1984). 10. Kuroki,S., Shimazu, K., Kuwabara, M., Une, M., Kihira, K., Kuramoto, T., and Hoshita, T. J. LIPID RES.26, 230 (1985). 11. Une, M., Shinonaga, Y., Matoba, N., Kuroki, S., Kihira, K ., and Hoshita, T. J. LIPID RES. 27, 1318 (1986). 12. Dayal, B., Tint, G.S.,Toome, V., Batta, A.K.,Shefer, S., and Salen, G. J. LIPID RES.26, 298 (1985). 13. Hoshita, T. J. BIOCHEM.(Tokyo)z, 176 (1962). 14. Cross, A.D. J. CHEM. SOC. 2817 (1961). 15. Okuda, K., Hoshita, T., and Kazuno, T., J. BIOCHEM. (Tokyo)5l, 48 (1962). 16.Hiraoka, T., Kihira, K., Kajiyama, G., Kuramoto, T., and Hoshita,T. J. LIPID RES.2, 895 (1987). 17. Goto, J., Hasegawa, H., Kato, H. and Nambara, T. CLIN. CHIM. ACTA87, 141 (1978). 18. Karlaganis, G., Karlaganis, V., and Sjovall, J. J. LIPID RES.2, 693 (1984).

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