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Unusual trihydroxy bile acids in the urine of healthy humans
47
Clinicu Chimicu Acta, 160 (1986) 47-53 Elsevier CCA 03597
Unusual trihydroxy bile acids in the urine of healthy humans Toshiaki Nakashima a**,Atsushi Sano a, Yoshifumi Seto a, Toshikazu Nakajima a, Yoshihiro Nakagawa a, Tadao Okuno a, Tatsuro Takino a and Takeshi Hasegawa b ’ The
Third Department of Internal Medicine and ’ Gas Chromatograph - Mass Spectrometry Laboratory, Kyoto Prefectural University of Medicine, Kyoto 602 (Japan)
Research
(Received 13 February 1986; revision 11 June 1986)
Key words: Unusual trihydroxy bile acids; Gas liquid chromatography;
Human urine
Summary The urinary bile acids of 20 adults (aged between 20 and 40 yr), 17 neonates (below 1 wk old) and 15 aged men (older than 80 yr old) who were all healthy, were analyzed by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. Frequently, three unusual trihydroxy bile acids, namely hyocholic acid, ursocholic acid and w-muricholic acid were detected as minor components. Our data further suggest that the metabolism of unusual trihydroxy bile acids in the healthy humans is related to age.
Introduction Hyocholic acid (HCA), ursocholic acid (UCA) and muricholic acid (MCA) are designated as unusual bile acids, because they were thought to be species-specific in hog [l] or rat [2] and were not usual components of human bile acids. Recently, these bile acids have been found in the urine of patients with liver diseases [3-51. Their presence in the human was believed to be related to the alteration of bile acid metabolism in liver disease. However, it seems unreasonable that bile acids which do not exist in normal liver should be synthesized by damaged liver. Therefore, further study was necessary to elucidate the physiological importance of these bile acids in the human.
* Correspondence to: Toshiaki Nakashima, Third Department of Internal Medicine, Kyoto Prefectural University, of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan. 0009-8981/86/$03.50
0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
48
We have already reported that a gas-liquid chromatography (GLC) using silicone AN-600 as the liquid phase is superior to the conventional GLC method in terms of the separation of bicinaldiol trihydroxy bile acids [6,7]. Furthermore, we have demonstrated that a GLC technique using an SE-30 capillary column, with direct inlet and solventless systems, separates (u-MCA and w-MCA [S]. Considering the fact that unusual bile acids such as HCA and MCA are bicinal-diol trihydroxy bile acids, we decided to study the bile acid composition of healthy human subjects by GLC and gas-liquid chromatography-mass spectrometry (GLC-MS). We analyzed the urinary bile acids of healthy adults, neonates and aged men by GLC and GLC-MS using an AN-600 packed column and an SE-30 capillary column, in order to clarify whether these so-called unusual bile acids occur in normal liver and whether age-related differences in the metabolism of these bile acids exist.
Materials and methods
This study included 20 human adults, aged less than 1 wk old, and 15 men older than 80 diseases or surgical operations. All subjects influence bile acid metabolism. Morning specimens of urine were collected
between 20 and 40 yr, 17 neonates, yr without previous hepato-intestinal were free of medication that might and frozen immediately.
Analysis of bile acia!s in urine Ten ml of the urine was mixed with 50 ml of l/15 mol/l phosphate buffer (pH 7.0) and applied to a Bond Elut C,, column (Analytichem Inc., CA, USA). After washing with water, bile acids were eluted from the resin with 90% ethanol, The eluate was evaporated to dryness and dissolved in acetone : methanol (10 : 1, pH 1.0) at room temperature for 72 h. After evaporating and redissolving in l/15 phosphate buffer at pH 7.0, the mixture was allowed to pass through another Bond Elut C,,. The methanol eluate was evaporated to dryness and then hydrolyzed by cholylglytine hydrolase [9]. After acidification, the hydrolyzed mixture was extracted with ethyl ether. The unconjugated bile acids were separated into the mono-, di- and tri-hydroxylated fractions on a silicic acid column [lo]. The fractions were methylated with diazomethane and acetylated by heating with acetic anhydride at 140°C for 4 h [ll]. After evaporation, the residues were dissolved in 0.1 ml of acetone containing 2 pg of cholesteryl caproate as an internal standard and subjected to GLC and GLC-MS analysis. The peak area ratio (the peak area of bile acid vs, that of the internal standard) was employed for quantitation by GLC with an AN-600 column [6]. Urine samples which produced peaks suspected of being tr-MCA or o-MCA on an AN-600 column, were further analyzed on an SE-30 capillary column.
GLC and GLC-MS GLC was performed on a Shimadzu GG7A gas chromato~aph equipped with a flame ionization detector, using a silanized glass column (3 mm i.d. x 1 m), which
49
was packed with 1.5% AN-600 coated on Gas Chrom Q (100-120 mesh). The temperature of the injection and detector chambers was kept at 290°C. The temperature of the column was increased from 220°C to 260°C at a rate of l”C/min. The flow rate of the carrier gas N, was 50 ml/mm. GLC-MS analyses were carried out on a Shimadzu GCMS 6020, equipped with SCAP 11/23 data processing system. The conditions were: column, a glass column (3 mm i.d. x 1 m) with 1.5% AN-600 coated on Gas Chrom Q (loo-120 mesh) or an open-tubular glass capillary column (0.36 mm i.d. X 20 m) coated with SE-30 (LKB, Stockholm, Sweden). Column temperature, 210-260°C for the packed column and 270-300°C for the capillary column at programming rates of l”C/min. Ion source temperature, 290°C for the packed column and 310°C for the capillary column. Electron energy, 20 eV, emission current, 60 PA and accelerating voltage, 3.5 kV. Reagents Hyocholic acid (HCA) was purchased from Calbiochem-Behring Co. (California, USA). (Y-, /3- and o-Muricholic acids (MCA) and ursocholic acid (UCA) were prepared from cholic acid by conventional methods [12]. Cholic acid, the other authentic bile acids, and cholylglycine hydrolase (Clostridium welchii acetone powder, type IV) were purchased from Sigma Co. (St. Louis, MO, USA). Cholesteryl caproate was synthesized from the reaction of cholesterol with caproyl chloride. Results GLC and GLC-MS analyses of urinary bile acids Cholesteryl caproate employed as an internal standard on AN-600 packed column, is not present in human urine and is completely separated from all other bile acids in the urine. The relative retention times (based on CA = 1.0, 25 min) were: cholesteryl caproate 0.41, HCA 1.11, UCA 1.18, cr-MCA 1.25, o-MCA 1.28 and /3-MCA 1.37, respectively. Plotting the peak area ratio (the peak area of authentic bile acid tested vs that of a fixed amount of the internal standard) against varying amounts of bile acid yielded a straight line for each bile acid and each line passed through the origin (Fig. la). To examine reproducibility of the results, authentic bile acid mixtures had been added to a single pool of human urine and their recovery rates were estimated. Recoveries of HCA, UCA, a-MCA, w-MCA and /3-MCA were 93, 90, 95, 95 and 93% respectively. Figure lb shows a typical gas chromatogram of methyl ester acetates of trihydroxy bile acids from the urine of an adult, using the AN-600 packed column. Three unusual bile acids, HCA, UCA and w-MCA, were detected. They were identified on the basis of their relative retention times and their mass spectra. The methyl ester acetates derivatives of HCA, (u-MCA, o-MCA and P-MCA had a spectrum with a base peak at m/z 386 (M - (60 x 2 - 42) and prominent peaks at m/z 253(ABCDring), m/z 313(ABCD-ring + side chain), m/z 386(M - 60 X 3) and m/z 428(M 60 x 2); that of UCA had a spectrum with a base peak at m/z 313(ABCD-ring + side
(b)
CA I”-MCA
I.
Fig. 1. a: Linear relationships between amounts of bile acid and peak area ratios (peak area against to that of cholesteryl caproate as internal standard). b: A typical gas chromatogram of methyl ester acetates of urinary bile acids in the fraction of trihydroxy bile acids separated by silicic acid column in healthy adult (case M.O., 34 yr, male), using an AN-600 packed column. I.S. denotes internal standard.
SCAN NUHBER Fig. 2. a: A typical mass chromatogram of methyl ester acetates of urinary bile acids in the fraction of trihydroxy bile acids separated by silicic acid column in healthy adult (case J.Y., 36 yr, female), using an SE-30 capillary column. HCA, UCA and o-MCA are seen. Furthermore, the 3 peaks (1, 2 and 3), possibly trihydroxy bile acids, are detected. The fragment ion of m/z 253 or m/z 368 represents the ABCD-ring and that of m/z 386, the presence of 3,6,7-triacetate (131. b: The peaking with authentic a-MCA and &MCA. The peaks of HCA, UCA, w-MCA, a-MCA and B-MCA are clearly separated with one another and do not correspond with the peaks 1, 2 and 3.
51 TABLE Ia The rate of occurrence of unusual trihydroxy bile acids in the urine of healthy humans Subiects
HCA
UCA
w-MCA
Neonates Adults Aged men
17/17 (100%) 9,‘20 (45%) 2/15 (13%)
2/17 (12%) 16/20 (80%) 7/15 (47%)
o/17 (0%) 6/20 (30%) 4/15 (27%)
TABLE Ib The concentration of unusual trihydroxy bile acids in the urine of healthy humans Subjects
Neonates Adults Aged men
No.
17 20 15 *
Total amount of urinary bile acids (Mean + SE)
Concentration (mean (lowest-highest)) HCA
UCA
o-MCA ( p mol/l)
X.6+11.9 32.61 4.5 .52.8+ 12.9
8.7(t - 24.4) 0.6(t - 2.0) l&O.4 - 2.4)
t 3.5(0.2 - 15.8) l.g(O.5 -6.6)
1.6(t - 6.7) 3.8(t - 10.9)
t = trace. * An aged person (case A.M., 82 yr, female) had UCA (0.8 pmol/l), p mol/B.
a-MCA (trace) and P-MCA (5.4
chain) and prominent peaks at m/z 253(ABCD_ding), m/z 368(M - 60 x 3) and m/z 428(M - 60 x 2) [13]. There was poor separation of at-MCA and w-MCA on AN-600 column [6]. Besides, the mass spectrum of methyl ester acetates derivatives of ol-MCA is identical with that of w-MCA. In order to distinguish (w-MCA from w-MCA, the urine sample was further analyzed on an SE-30 capillary column. Figure 2 shows the mass chromato~~ of methyl ester acetates of trihydroxy bile acids from the urine of an adult, using the SE-30 capillary column. The peak of w-MCA is clearly separated from other peaks.
Theoccurrence of un~uol bile acids in bu~on urine As shown in Table Ia, unusual trihydroxy bile acids, namely HCA, UCA and w-MCA, were present in the urine of healthy adults as minor components: HCA were found in 9 (45%), UCA in 16 (80%) and w-MCA in 6 (30%) of 20 subjects. In 17 neonates, HCA was presented in all (100%) and UCA in 2 (12%) of the subjects. However, o-MCA that occurred in the urine of adults could not be found. In 15 aged men, HCA was found in 2 (13%), UCA in 7 (47%), and w-MCA in 4 (27%) of the subjects. Furthermore, an aged person had ET-MCAand @-MCA. The concentration of unusual trihydroxy bile acids in each group was shown in Table Ib.
52
Discussion Urine is preferable to serum for the analysis of minor components of bile acids, because large samples can be obtained and desalted with Bond Elut C,, [14]. We combined such purification with GLC and GLC-MS using AN-600 and SE-30 columns, and found several unusual trihydroxy bile acids, such as HCA, UCA and w-MCA in the urine of healthy humans. These findings suggest that the unusual bile acids are not confined to patients with liver diseases but also exist as minor components in the healthy state. In neonatal urine, HCA was readily found but MCA could not be detected. This pattern may be explainable by the immaturity of bile acid metabolism in neonates. Tazawa et al [15] showed that sulfate-conjugated bile acids in the urine of neonates are lower than in adults. Sulfation is an important route of metabolism for the less water-soluble bile acids, such as mono- or di-hydroxy bile acids, and neonates who lack the sulfation enzymes may have other excretory mechanisms such as polyhydroxylation. Thus the occurrence of HCA (3cq6q7a-trihydroxy cholanoic acid) in neonatal urine may suggest that HCA is an excretory hydroxylation product of CDCA (3a,7or-dihydoxy cholanoic acid) at the 6cY-position. cw-MCA and /3-MCA occurred in the urine of an aged person but not of neonates and adults. Although the precursors of these muricholic acids remain unknown, CDCA may be hydroxylated at the 6&position to produce a-MCA (3a,6P,7a-trihydroxy cholanoic acid) and further epimerized at the 7-position to produce /3-MCA (3cY,6a,7@-trihydroxy cholanoic acid). Epimerization of bile acids at the 7-position is known from conversion of CDCA to UDCA. 6P-Hydroxylation of bile acids, on the other hand, has not been demonstrated in humans until now. However, it is well known that 6fi-hydroxy bile acids such as LX-MCA and P-MCA are synthesized from CDCA in the:.rat liver [16]. These findings suggest that in aged men the bile acid metabolism may resemble that in the rat. In addition, we identified UCA and w-MCA in the urine of adults and aged men. UCA (3a,7p,12cu-trihydroxy cholanoic acid) may be biosynthesized from CA (3a,7o,l2a-trihydroxy cholanoic acid) by 7/3-epimerization as UDCA is synthesized from CDCA. w-MCA (3a[,6cq7/?-trihydroxy cholanoic acid) may be biosynthesized from UDCA (3a,7/3-dihydroxy cholanoic acid) by 6cu-hydroxylation as HCA is biosynthesized from CDCA. We conclude that so-called ‘unusual’ trihydroxy bile acids such as HCA, UCA and w-MCA are normal urinary components, that 6a-hydroxylation is a common route of metabolism of bile acids and that it operates regardless of age but mainly in the immature liver. 6/&Hydroxylation, however, is only detectable in the aged liver. To be confirm of these conclusions, the influence of intestinal bacteria on the hydroxylation of bile acids must be excluded by in vitro studies of human liver as was done by Trtilzsch et al [17] to demonstrate 6cw-hydroxylation of LCA to HDCA. References 1 Bergstriim S, Danielsson H, GBransson A. On the bile acid metabolism in the pig. Bile acids and steroids 81. Acta Chem Stand 1959; 13: 776-783.
53 2 Hsia SL, Elliott WH, Matschiner JT, Doisy EA Jr, Thayer SA, Doisy EA. Bile acids, XIII. Further ~nt~butions to the condition of mu~cholic acids. J Biol Chem 1960, 253: 1963-1967. 3 Amuro Y, Endo T, Higashino K, Uchida K, Yamamura Y. Serum, fecal and urinary bile acids in patients with mild and advanced liver cirrhosis. Gastroenterol Jpn 1981; 16: 506-513. 4 Ammo Y, Hayashi E, Endo T, Higashino K, Kishimoto S. Unusual trihydroxylated bile acids in urine of patients with liver cirrhosis. Clin Chim Acta 1983; 127: 61-67. 5 Szczepanik PA, Steilaard F. Detection of atypical bite acids in disease states and their identification by gas chromatography-mass spectrometry-computer technique. In: Paumgartner G, Stiehl A. Gerok W, eds. Biological effects of bile acids. Lancaster: MTP, 1979: 287-298. 6 Nakashima T, Ban Y, Kuriyama K, Takino T. An improved gasliquid chromatographic method using silicon AN-600 column for separation of bile acids: Its application on analysis of bile acids in rat bile. Japan J Pharmacol 19’79; 29: 667-670. 7 Nakashima T, Hasegawa T, Sano A, Nakagawa Y, Seto Y, Okuno T, Takino T. Analysis of trihydroxy bile acids in human materials with GC-MS. J Kyoto Pref Univ Med 1984; 93: 927-932. 8 Murata T, Takahashi S, Ohnishi S, Hosoi K, Nakashima T, Ban Y, Kuriyama K. Characterization of bile acid methyl ester acetated derivatives of rat bile using solventless glass capillary gas chromatography and electron impact and ammonia chemical ionization mass spectrometry. J Chromatogr 1982; 239: 571-583. 9 Roseleur OJ, Van Gent CM. Alkaline and enzymatic hydrolysis of conjugated bile acids. Clin Chim Acta 1976; 66: 269-272. 10 Ikawa S. Group separation of bile acids and salt by silicic acid column chromatography. Anal Biochem 1978; 85: 197-208. 11 Tsuda K, Sato Y, Ikekawa N, Tanaka S, Higashikuze H, Ohsawa R. Studies on bile acids and bile alcohols. II. Separation of bile acids by gas-liquid chromato~aphy. Chem Pharm BulI (Tokyo) 1965; 13: 720-723. 12 Hsia SL. Hyocholic acid and muricholic acid. In: Nair PP, Kritchevsky D, eds. The bile acids, Vol 1. New York: Plenum Press, 1971: 95-120. 13 Szczepanik PA, Hachey DL, Klein PD. Characterization of bile acid methyl ester acetate derivatives using gas-liquid chromatography, electron impact, and chemical ionization mass spectrometry. J Lipid Res 1976; 17: 314-334. 14 Setchell KDR, Worthington J. A rapid method for the quantitative extraction of bile acids and their conjugates from serum using commercially available reverse phase octadecylsilane bonded silica cartridges. Clin Chim Acta 1982; 125: 135-144. 15 Tazawa Y, Konno T. Sulfated and non-sulfated bile acid in urine of infants with obstructive jaundice. Tohoku J Exp med 1979; 129: 55-63. 16 Gleim H, Triilzsch D, Roboz J, Dressier K, Czygan P, Hutterer F, Schaffner F, Popper H. Mechanism of cholestasis. 5. bile acids in normal rat livers and in those after bile duct ligation. Gastroenterol 1972; 63: 837-845. 17 Trhlzsch D, Roboz, J, Greim H, Czygan P, Rudick J, Hutterer F, Schaffner F. Popper H. Hydroxylation of taurolith~holate by isolated human liver microsomes. Identification of metabolic product. B&hem Med 1974; 9: 158-166.
Clinicu Chimicu Acta, 160 (1986) 47-53 Elsevier CCA 03597
Unusual trihydroxy bile acids in the urine of healthy humans Toshiaki Nakashima a**,Atsushi Sano a, Yoshifumi Seto a, Toshikazu Nakajima a, Yoshihiro Nakagawa a, Tadao Okuno a, Tatsuro Takino a and Takeshi Hasegawa b ’ The
Third Department of Internal Medicine and ’ Gas Chromatograph - Mass Spectrometry Laboratory, Kyoto Prefectural University of Medicine, Kyoto 602 (Japan)
Research
(Received 13 February 1986; revision 11 June 1986)
Key words: Unusual trihydroxy bile acids; Gas liquid chromatography;
Human urine
Summary The urinary bile acids of 20 adults (aged between 20 and 40 yr), 17 neonates (below 1 wk old) and 15 aged men (older than 80 yr old) who were all healthy, were analyzed by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. Frequently, three unusual trihydroxy bile acids, namely hyocholic acid, ursocholic acid and w-muricholic acid were detected as minor components. Our data further suggest that the metabolism of unusual trihydroxy bile acids in the healthy humans is related to age.
Introduction Hyocholic acid (HCA), ursocholic acid (UCA) and muricholic acid (MCA) are designated as unusual bile acids, because they were thought to be species-specific in hog [l] or rat [2] and were not usual components of human bile acids. Recently, these bile acids have been found in the urine of patients with liver diseases [3-51. Their presence in the human was believed to be related to the alteration of bile acid metabolism in liver disease. However, it seems unreasonable that bile acids which do not exist in normal liver should be synthesized by damaged liver. Therefore, further study was necessary to elucidate the physiological importance of these bile acids in the human.
* Correspondence to: Toshiaki Nakashima, Third Department of Internal Medicine, Kyoto Prefectural University, of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan. 0009-8981/86/$03.50
0 1986 Elsevier Science Publishers B.V. (Biomedical Division)
48
We have already reported that a gas-liquid chromatography (GLC) using silicone AN-600 as the liquid phase is superior to the conventional GLC method in terms of the separation of bicinaldiol trihydroxy bile acids [6,7]. Furthermore, we have demonstrated that a GLC technique using an SE-30 capillary column, with direct inlet and solventless systems, separates (u-MCA and w-MCA [S]. Considering the fact that unusual bile acids such as HCA and MCA are bicinal-diol trihydroxy bile acids, we decided to study the bile acid composition of healthy human subjects by GLC and gas-liquid chromatography-mass spectrometry (GLC-MS). We analyzed the urinary bile acids of healthy adults, neonates and aged men by GLC and GLC-MS using an AN-600 packed column and an SE-30 capillary column, in order to clarify whether these so-called unusual bile acids occur in normal liver and whether age-related differences in the metabolism of these bile acids exist.
Materials and methods
This study included 20 human adults, aged less than 1 wk old, and 15 men older than 80 diseases or surgical operations. All subjects influence bile acid metabolism. Morning specimens of urine were collected
between 20 and 40 yr, 17 neonates, yr without previous hepato-intestinal were free of medication that might and frozen immediately.
Analysis of bile acia!s in urine Ten ml of the urine was mixed with 50 ml of l/15 mol/l phosphate buffer (pH 7.0) and applied to a Bond Elut C,, column (Analytichem Inc., CA, USA). After washing with water, bile acids were eluted from the resin with 90% ethanol, The eluate was evaporated to dryness and dissolved in acetone : methanol (10 : 1, pH 1.0) at room temperature for 72 h. After evaporating and redissolving in l/15 phosphate buffer at pH 7.0, the mixture was allowed to pass through another Bond Elut C,,. The methanol eluate was evaporated to dryness and then hydrolyzed by cholylglytine hydrolase [9]. After acidification, the hydrolyzed mixture was extracted with ethyl ether. The unconjugated bile acids were separated into the mono-, di- and tri-hydroxylated fractions on a silicic acid column [lo]. The fractions were methylated with diazomethane and acetylated by heating with acetic anhydride at 140°C for 4 h [ll]. After evaporation, the residues were dissolved in 0.1 ml of acetone containing 2 pg of cholesteryl caproate as an internal standard and subjected to GLC and GLC-MS analysis. The peak area ratio (the peak area of bile acid vs, that of the internal standard) was employed for quantitation by GLC with an AN-600 column [6]. Urine samples which produced peaks suspected of being tr-MCA or o-MCA on an AN-600 column, were further analyzed on an SE-30 capillary column.
GLC and GLC-MS GLC was performed on a Shimadzu GG7A gas chromato~aph equipped with a flame ionization detector, using a silanized glass column (3 mm i.d. x 1 m), which
49
was packed with 1.5% AN-600 coated on Gas Chrom Q (100-120 mesh). The temperature of the injection and detector chambers was kept at 290°C. The temperature of the column was increased from 220°C to 260°C at a rate of l”C/min. The flow rate of the carrier gas N, was 50 ml/mm. GLC-MS analyses were carried out on a Shimadzu GCMS 6020, equipped with SCAP 11/23 data processing system. The conditions were: column, a glass column (3 mm i.d. x 1 m) with 1.5% AN-600 coated on Gas Chrom Q (loo-120 mesh) or an open-tubular glass capillary column (0.36 mm i.d. X 20 m) coated with SE-30 (LKB, Stockholm, Sweden). Column temperature, 210-260°C for the packed column and 270-300°C for the capillary column at programming rates of l”C/min. Ion source temperature, 290°C for the packed column and 310°C for the capillary column. Electron energy, 20 eV, emission current, 60 PA and accelerating voltage, 3.5 kV. Reagents Hyocholic acid (HCA) was purchased from Calbiochem-Behring Co. (California, USA). (Y-, /3- and o-Muricholic acids (MCA) and ursocholic acid (UCA) were prepared from cholic acid by conventional methods [12]. Cholic acid, the other authentic bile acids, and cholylglycine hydrolase (Clostridium welchii acetone powder, type IV) were purchased from Sigma Co. (St. Louis, MO, USA). Cholesteryl caproate was synthesized from the reaction of cholesterol with caproyl chloride. Results GLC and GLC-MS analyses of urinary bile acids Cholesteryl caproate employed as an internal standard on AN-600 packed column, is not present in human urine and is completely separated from all other bile acids in the urine. The relative retention times (based on CA = 1.0, 25 min) were: cholesteryl caproate 0.41, HCA 1.11, UCA 1.18, cr-MCA 1.25, o-MCA 1.28 and /3-MCA 1.37, respectively. Plotting the peak area ratio (the peak area of authentic bile acid tested vs that of a fixed amount of the internal standard) against varying amounts of bile acid yielded a straight line for each bile acid and each line passed through the origin (Fig. la). To examine reproducibility of the results, authentic bile acid mixtures had been added to a single pool of human urine and their recovery rates were estimated. Recoveries of HCA, UCA, a-MCA, w-MCA and /3-MCA were 93, 90, 95, 95 and 93% respectively. Figure lb shows a typical gas chromatogram of methyl ester acetates of trihydroxy bile acids from the urine of an adult, using the AN-600 packed column. Three unusual bile acids, HCA, UCA and w-MCA, were detected. They were identified on the basis of their relative retention times and their mass spectra. The methyl ester acetates derivatives of HCA, (u-MCA, o-MCA and P-MCA had a spectrum with a base peak at m/z 386 (M - (60 x 2 - 42) and prominent peaks at m/z 253(ABCDring), m/z 313(ABCD-ring + side chain), m/z 386(M - 60 X 3) and m/z 428(M 60 x 2); that of UCA had a spectrum with a base peak at m/z 313(ABCD-ring + side
(b)
CA I”-MCA
I.
Fig. 1. a: Linear relationships between amounts of bile acid and peak area ratios (peak area against to that of cholesteryl caproate as internal standard). b: A typical gas chromatogram of methyl ester acetates of urinary bile acids in the fraction of trihydroxy bile acids separated by silicic acid column in healthy adult (case M.O., 34 yr, male), using an AN-600 packed column. I.S. denotes internal standard.
SCAN NUHBER Fig. 2. a: A typical mass chromatogram of methyl ester acetates of urinary bile acids in the fraction of trihydroxy bile acids separated by silicic acid column in healthy adult (case J.Y., 36 yr, female), using an SE-30 capillary column. HCA, UCA and o-MCA are seen. Furthermore, the 3 peaks (1, 2 and 3), possibly trihydroxy bile acids, are detected. The fragment ion of m/z 253 or m/z 368 represents the ABCD-ring and that of m/z 386, the presence of 3,6,7-triacetate (131. b: The peaking with authentic a-MCA and &MCA. The peaks of HCA, UCA, w-MCA, a-MCA and B-MCA are clearly separated with one another and do not correspond with the peaks 1, 2 and 3.
51 TABLE Ia The rate of occurrence of unusual trihydroxy bile acids in the urine of healthy humans Subiects
HCA
UCA
w-MCA
Neonates Adults Aged men
17/17 (100%) 9,‘20 (45%) 2/15 (13%)
2/17 (12%) 16/20 (80%) 7/15 (47%)
o/17 (0%) 6/20 (30%) 4/15 (27%)
TABLE Ib The concentration of unusual trihydroxy bile acids in the urine of healthy humans Subjects
Neonates Adults Aged men
No.
17 20 15 *
Total amount of urinary bile acids (Mean + SE)
Concentration (mean (lowest-highest)) HCA
UCA
o-MCA ( p mol/l)
X.6+11.9 32.61 4.5 .52.8+ 12.9
8.7(t - 24.4) 0.6(t - 2.0) l&O.4 - 2.4)
t 3.5(0.2 - 15.8) l.g(O.5 -6.6)
1.6(t - 6.7) 3.8(t - 10.9)
t = trace. * An aged person (case A.M., 82 yr, female) had UCA (0.8 pmol/l), p mol/B.
a-MCA (trace) and P-MCA (5.4
chain) and prominent peaks at m/z 253(ABCD_ding), m/z 368(M - 60 x 3) and m/z 428(M - 60 x 2) [13]. There was poor separation of at-MCA and w-MCA on AN-600 column [6]. Besides, the mass spectrum of methyl ester acetates derivatives of ol-MCA is identical with that of w-MCA. In order to distinguish (w-MCA from w-MCA, the urine sample was further analyzed on an SE-30 capillary column. Figure 2 shows the mass chromato~~ of methyl ester acetates of trihydroxy bile acids from the urine of an adult, using the SE-30 capillary column. The peak of w-MCA is clearly separated from other peaks.
Theoccurrence of un~uol bile acids in bu~on urine As shown in Table Ia, unusual trihydroxy bile acids, namely HCA, UCA and w-MCA, were present in the urine of healthy adults as minor components: HCA were found in 9 (45%), UCA in 16 (80%) and w-MCA in 6 (30%) of 20 subjects. In 17 neonates, HCA was presented in all (100%) and UCA in 2 (12%) of the subjects. However, o-MCA that occurred in the urine of adults could not be found. In 15 aged men, HCA was found in 2 (13%), UCA in 7 (47%), and w-MCA in 4 (27%) of the subjects. Furthermore, an aged person had ET-MCAand @-MCA. The concentration of unusual trihydroxy bile acids in each group was shown in Table Ib.
52
Discussion Urine is preferable to serum for the analysis of minor components of bile acids, because large samples can be obtained and desalted with Bond Elut C,, [14]. We combined such purification with GLC and GLC-MS using AN-600 and SE-30 columns, and found several unusual trihydroxy bile acids, such as HCA, UCA and w-MCA in the urine of healthy humans. These findings suggest that the unusual bile acids are not confined to patients with liver diseases but also exist as minor components in the healthy state. In neonatal urine, HCA was readily found but MCA could not be detected. This pattern may be explainable by the immaturity of bile acid metabolism in neonates. Tazawa et al [15] showed that sulfate-conjugated bile acids in the urine of neonates are lower than in adults. Sulfation is an important route of metabolism for the less water-soluble bile acids, such as mono- or di-hydroxy bile acids, and neonates who lack the sulfation enzymes may have other excretory mechanisms such as polyhydroxylation. Thus the occurrence of HCA (3cq6q7a-trihydroxy cholanoic acid) in neonatal urine may suggest that HCA is an excretory hydroxylation product of CDCA (3a,7or-dihydoxy cholanoic acid) at the 6cY-position. cw-MCA and /3-MCA occurred in the urine of an aged person but not of neonates and adults. Although the precursors of these muricholic acids remain unknown, CDCA may be hydroxylated at the 6&position to produce a-MCA (3a,6P,7a-trihydroxy cholanoic acid) and further epimerized at the 7-position to produce /3-MCA (3cY,6a,7@-trihydroxy cholanoic acid). Epimerization of bile acids at the 7-position is known from conversion of CDCA to UDCA. 6P-Hydroxylation of bile acids, on the other hand, has not been demonstrated in humans until now. However, it is well known that 6fi-hydroxy bile acids such as LX-MCA and P-MCA are synthesized from CDCA in the:.rat liver [16]. These findings suggest that in aged men the bile acid metabolism may resemble that in the rat. In addition, we identified UCA and w-MCA in the urine of adults and aged men. UCA (3a,7p,12cu-trihydroxy cholanoic acid) may be biosynthesized from CA (3a,7o,l2a-trihydroxy cholanoic acid) by 7/3-epimerization as UDCA is synthesized from CDCA. w-MCA (3a[,6cq7/?-trihydroxy cholanoic acid) may be biosynthesized from UDCA (3a,7/3-dihydroxy cholanoic acid) by 6cu-hydroxylation as HCA is biosynthesized from CDCA. We conclude that so-called ‘unusual’ trihydroxy bile acids such as HCA, UCA and w-MCA are normal urinary components, that 6a-hydroxylation is a common route of metabolism of bile acids and that it operates regardless of age but mainly in the immature liver. 6/&Hydroxylation, however, is only detectable in the aged liver. To be confirm of these conclusions, the influence of intestinal bacteria on the hydroxylation of bile acids must be excluded by in vitro studies of human liver as was done by Trtilzsch et al [17] to demonstrate 6cw-hydroxylation of LCA to HDCA. References 1 Bergstriim S, Danielsson H, GBransson A. On the bile acid metabolism in the pig. Bile acids and steroids 81. Acta Chem Stand 1959; 13: 776-783.
53 2 Hsia SL, Elliott WH, Matschiner JT, Doisy EA Jr, Thayer SA, Doisy EA. Bile acids, XIII. Further ~nt~butions to the condition of mu~cholic acids. J Biol Chem 1960, 253: 1963-1967. 3 Amuro Y, Endo T, Higashino K, Uchida K, Yamamura Y. Serum, fecal and urinary bile acids in patients with mild and advanced liver cirrhosis. Gastroenterol Jpn 1981; 16: 506-513. 4 Ammo Y, Hayashi E, Endo T, Higashino K, Kishimoto S. Unusual trihydroxylated bile acids in urine of patients with liver cirrhosis. Clin Chim Acta 1983; 127: 61-67. 5 Szczepanik PA, Steilaard F. Detection of atypical bite acids in disease states and their identification by gas chromatography-mass spectrometry-computer technique. In: Paumgartner G, Stiehl A. Gerok W, eds. Biological effects of bile acids. Lancaster: MTP, 1979: 287-298. 6 Nakashima T, Ban Y, Kuriyama K, Takino T. An improved gasliquid chromatographic method using silicon AN-600 column for separation of bile acids: Its application on analysis of bile acids in rat bile. Japan J Pharmacol 19’79; 29: 667-670. 7 Nakashima T, Hasegawa T, Sano A, Nakagawa Y, Seto Y, Okuno T, Takino T. Analysis of trihydroxy bile acids in human materials with GC-MS. J Kyoto Pref Univ Med 1984; 93: 927-932. 8 Murata T, Takahashi S, Ohnishi S, Hosoi K, Nakashima T, Ban Y, Kuriyama K. Characterization of bile acid methyl ester acetated derivatives of rat bile using solventless glass capillary gas chromatography and electron impact and ammonia chemical ionization mass spectrometry. J Chromatogr 1982; 239: 571-583. 9 Roseleur OJ, Van Gent CM. Alkaline and enzymatic hydrolysis of conjugated bile acids. Clin Chim Acta 1976; 66: 269-272. 10 Ikawa S. Group separation of bile acids and salt by silicic acid column chromatography. Anal Biochem 1978; 85: 197-208. 11 Tsuda K, Sato Y, Ikekawa N, Tanaka S, Higashikuze H, Ohsawa R. Studies on bile acids and bile alcohols. II. Separation of bile acids by gas-liquid chromato~aphy. Chem Pharm BulI (Tokyo) 1965; 13: 720-723. 12 Hsia SL. Hyocholic acid and muricholic acid. In: Nair PP, Kritchevsky D, eds. The bile acids, Vol 1. New York: Plenum Press, 1971: 95-120. 13 Szczepanik PA, Hachey DL, Klein PD. Characterization of bile acid methyl ester acetate derivatives using gas-liquid chromatography, electron impact, and chemical ionization mass spectrometry. J Lipid Res 1976; 17: 314-334. 14 Setchell KDR, Worthington J. A rapid method for the quantitative extraction of bile acids and their conjugates from serum using commercially available reverse phase octadecylsilane bonded silica cartridges. Clin Chim Acta 1982; 125: 135-144. 15 Tazawa Y, Konno T. Sulfated and non-sulfated bile acid in urine of infants with obstructive jaundice. Tohoku J Exp med 1979; 129: 55-63. 16 Gleim H, Triilzsch D, Roboz J, Dressier K, Czygan P, Hutterer F, Schaffner F, Popper H. Mechanism of cholestasis. 5. bile acids in normal rat livers and in those after bile duct ligation. Gastroenterol 1972; 63: 837-845. 17 Trhlzsch D, Roboz, J, Greim H, Czygan P, Rudick J, Hutterer F, Schaffner F. Popper H. Hydroxylation of taurolith~holate by isolated human liver microsomes. Identification of metabolic product. B&hem Med 1974; 9: 158-166.