63
Clinica ~~irn~~aActa, 64 (1975) 63-68 0 Elsevier Scientific Pu~Iis~ing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7336
DETERMINATION OF SERUM BILE ACIDS BY GLASS CAPILLARY GAS-LIQUID CHROMATOGRAPHY
Summary Bile acids were extracted from serum samples by chromatography on Amberlite XAD-2 and, after alkaline or enzymic hydrolysis, purified by chromatography on aluminium oxide. The quantitation was carried out by gasliquid chromatography with an OV-101 glass capillary column using their methyl ester trimethylsilyl derivatives. The mean total amount of cholic, chenodeoxy~holic and deoxycholic acids in a group of healthy fasting women was 2.14 @mol/l, in a group of fasting pregnant women at 8-12 weeks of gestation 1.13 pmol/l and at 38-41 weeks of gestation 2.10 ymolll. In patients with cholestasis of pregnancy the total bile acid levels varied from 6 to 86 E.tmol/l.
Introduction Serum bile acids have been determined by means of gas-liquid chromatography over the last ten years [l--5]. Difficulties to quantitate bile acids by means of GLC have also been reported [I], For the separation of individual bile acids by GLC, their trifluoroacetate derivatives have been used. These derivatives are, however, easily destroyed during the GLC process depending on the individu~ properties of the columns used, and this makes the use of these derivatives in quantitative work difficult. To overcome these difficulties we have determined serum bile acids by GLC with glass capillary columns, which allows the use of more stable trimethylsilyl ethers of bile acid methyl esters. Here we wish to report results of bile acid analyses of serum samples from healthy non-pregnant and pregnant subjects as well as from patients with intrahepatic eholestasis of pregnancy. * Address
correspondence to: Dr. T. Laatikainen, Department of Obstetrics and Gynecology, sity Central Hospital, Ha~m~inkatu 2. SF-00290 Helsinki 29, Finland.
Univer-
61
Material
and methods
5-ml blood samples were drawn after overnight fasting from 15 healthy women, 21-39 years of age. They had not taken any contraceptive pills. Samples were also collected from 15 healthy pregnant women at 8-12 weeks of gestation after overnight fasting, and from 18 healthy pregnant women in labor at 38-41 weeks of gestation after at least 8 h fasting. Furthermore, blood samples were collected from 18 patients with intrahepatic cholestasis of pregnancy at 32-38 weeks of gestation after overnight fasting. All these patients had pruritus and elevated levels of serum aminotransferases, which disappeared after labor. Determinations of serum Au-antigen revealed negative results in every case. Blood samples were allowed to coagulate and the serum samples were stored at -18°C until analyzed. Unconjugated, and taurine or glycine conjugated bile acids were purchased from Calbiochem (Los Angeles, U.S.A.), Schwartz-Mann Research Labs (New York U.S.A.) and from Sigma Chemicals Co. (St. Louis, U.S.A.). Lithocholic acid was kindly provided for us by Professor T. Miettinen, Helsinki. The enzyme N-choloylglycine hydrolase (EC 3.5.1.23) was purchased from Schwarz-Mann Research Labs. Purification and hydrolysis of serum bile acids was carried out in all essential details as described by Makino and Sjiivall [6]. The procedure was briefly as follows: l-ml serum samples were diluted with 9 ml of 0.1 M NaOH and bile acids extracted by chromatography on Amberlite XAD-2. Bile acids were eluted from the column with ethanol and subjected to alkaline hydrolysis for 5 h in 15% NaOH in the ethanol at 120°C under pressure in Teflon containers. After acidification of the mixture, bile acids were extracted with ethyl acetate and converted to their methyl ester derivatives with diazomethane in ether/ methanol (5 : 1, v/v). Before methylation 2.5-10 1.18 of 7-ketodeoxycholic acid was added to the samples as an internal standard. The bile acid methyl esters were then purified by chromatography on a 200 mg column of aluminium oxide (Woelm, activity grade I). Cholesterol and impurities were eluted with 1% ethyl acetate in toluene and bile acid methyl esters with acetone/methanol (1 : 1, v/v). Enzymic hydrolysis of bile acid conjugates as an alternative to alkaline hydrolysis was carried out as described by Nair and Garcia [ 71. After Amberlite XAD-2 chromatography bile acids were dissolved in 2.0 ml of a 0.1 M acetate buffer, pH 5.6 25 ~1 of the choloylglycine hydrolase preparation, 0.5 ml of 0.2 M EDTA and 0.5 ml of 0.2 M /3-mercaptoethanol were added and the mixture incubated at 37°C for 30 min. The bile acids were then extracted three times with 10 ml ethyl acetate, methylated, and purified by chromatography on aluminium oxide as described above. Gas-liquid chromatography. Glass capillary columns were prepared in all essential details as described by Grob [8,9]. A 50-m OV-101 column with an internal diameter of 0.3 mm was installed into the Varian 2100 gas chromatograph. The carrier gas was nitrogen. The split ratio was 1 : 10 and the flow through the column 2 ml/min, and the additional flow of nitrogen into the detector 40 ml/min. Bile acid methyl esters were converted to their trimethylsilyl ether derivatives prior to GLC. The quantitation of bile acids was carried
65
out by comparing their peak height responses to that of the internal standard. The differences in peak height responses between bile acids were taken into account in these calculations by testing firstly the peak height responses of the appropriate reference bile acid and internal standard mixtures. Replicate quantitations were also carried out by comparing peak areas of bile acids to that of the internal standard. Peak area measurements were carried out during GLC using Infotronics Model CRF-204 automatic digital integrator. Gas chromatography-mass spectrometry of bile acids in some samples from normal subjects and from patients was carried out with an LKB-9000 instrument using a packed 1% SE-30 column for GLC. Results Fig. 1 shows that an OV-101 glass capillary column completely separates methyl ester trimethylsilyl ethers (Me-TMS) of cholic (C), chenodeoxycholic (CDC), deoxycholic (DC) and lithocholic (LC) acids. The presence of the first three of these was confirmed in the serum samples from healthy subjects and
C
Fig. 1. Glass capillary gas chromatograms of trimethylsilyl ethers of bile acid methyl esters. A 50-m OV-101 column programmed 8k/min from 150% to 255%. A: reference cholic (C). chenodeoxycholic (CDC), deqxycholic (DC) and lithocholic (LC) acids, and the internal standard (S). B: a Serum sample from a healthy female. C: a serum sample from a patient with cholestasis of Pregnancy. CS. cholesterol.
TABLE
1
SERUM
BILE
Grttu~
8-12
A:
ACID
CONCENTRATIONS
15 non-pregnant
weeks
of gestation.
IN HEALTl-IY
NON-PREGNANT
Group
Cholic acid
Chenodeoxycholie
A
0.58 *- 0.13 0.37 + 0.05 0.91 ’ 0.16
0.98 ’ 0.17 0.34 i 0.05 0.74 1 0.12
C
PREGNANT
FEMALES
healthy females, 21-.39 years of age. Group B: 15 healthy pregnant females at Group C: 18 healthy pregnant females in labor at 3X-41 weeks of gestation.
The values are expressed in i.rmol/l as mean i S.E. and are not corrected
B
AND
acid
fox mathi,dulogical
Deoxycholic
acid
losses.
Tot&
bile acids
’ 0.21 ’ 0.10 2.10 ’ 0.28
0.57 ’ 0.09 0.42 + 0.06 0.45 ’ 0.08
2.14 1.13
from patients by GC-MS, and their peaks were located in glass capill~y gas chromatograms by injecting small amounts of reference bile acid Me-TMS with the samples, after which a symmetrical increase of the peak to be measured was found. No impurities disturbing the bile acid and internal standard Me-TMS peaks were found when 1 ml of water as a method blank was analysed. 5-10 pg of glycocholic acid and taurodeoxycholic acid were added into 1 ml samples of a serum pool. The samples were analysed as described above. lJsing alkaline hydrolysis the recoveries varied from 63 to 89 (mean 76) ‘%and
TABLE
II
RILE ACID CONCENTRATION CHOLESTASIS OF PREGNANCY
(~motjl)
IN
THE
SERUM
OF
PATIENTS
WITH
INTRAHEPATIC
The simultaneous measurements of total and conjugated serum bilirubin (i.lmul/l), aminotransferases ASAT and ALAT and aIkaIine phasphatase (U/l) are given for comparison. Upper limits in URc~mplicated pregnancy: Bilintbin: total
Clinica ~~irn~~aActa, 64 (1975) 63-68 0 Elsevier Scientific Pu~Iis~ing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7336
DETERMINATION OF SERUM BILE ACIDS BY GLASS CAPILLARY GAS-LIQUID CHROMATOGRAPHY
Summary Bile acids were extracted from serum samples by chromatography on Amberlite XAD-2 and, after alkaline or enzymic hydrolysis, purified by chromatography on aluminium oxide. The quantitation was carried out by gasliquid chromatography with an OV-101 glass capillary column using their methyl ester trimethylsilyl derivatives. The mean total amount of cholic, chenodeoxy~holic and deoxycholic acids in a group of healthy fasting women was 2.14 @mol/l, in a group of fasting pregnant women at 8-12 weeks of gestation 1.13 pmol/l and at 38-41 weeks of gestation 2.10 ymolll. In patients with cholestasis of pregnancy the total bile acid levels varied from 6 to 86 E.tmol/l.
Introduction Serum bile acids have been determined by means of gas-liquid chromatography over the last ten years [l--5]. Difficulties to quantitate bile acids by means of GLC have also been reported [I], For the separation of individual bile acids by GLC, their trifluoroacetate derivatives have been used. These derivatives are, however, easily destroyed during the GLC process depending on the individu~ properties of the columns used, and this makes the use of these derivatives in quantitative work difficult. To overcome these difficulties we have determined serum bile acids by GLC with glass capillary columns, which allows the use of more stable trimethylsilyl ethers of bile acid methyl esters. Here we wish to report results of bile acid analyses of serum samples from healthy non-pregnant and pregnant subjects as well as from patients with intrahepatic eholestasis of pregnancy. * Address
correspondence to: Dr. T. Laatikainen, Department of Obstetrics and Gynecology, sity Central Hospital, Ha~m~inkatu 2. SF-00290 Helsinki 29, Finland.
Univer-
61
Material
and methods
5-ml blood samples were drawn after overnight fasting from 15 healthy women, 21-39 years of age. They had not taken any contraceptive pills. Samples were also collected from 15 healthy pregnant women at 8-12 weeks of gestation after overnight fasting, and from 18 healthy pregnant women in labor at 38-41 weeks of gestation after at least 8 h fasting. Furthermore, blood samples were collected from 18 patients with intrahepatic cholestasis of pregnancy at 32-38 weeks of gestation after overnight fasting. All these patients had pruritus and elevated levels of serum aminotransferases, which disappeared after labor. Determinations of serum Au-antigen revealed negative results in every case. Blood samples were allowed to coagulate and the serum samples were stored at -18°C until analyzed. Unconjugated, and taurine or glycine conjugated bile acids were purchased from Calbiochem (Los Angeles, U.S.A.), Schwartz-Mann Research Labs (New York U.S.A.) and from Sigma Chemicals Co. (St. Louis, U.S.A.). Lithocholic acid was kindly provided for us by Professor T. Miettinen, Helsinki. The enzyme N-choloylglycine hydrolase (EC 3.5.1.23) was purchased from Schwarz-Mann Research Labs. Purification and hydrolysis of serum bile acids was carried out in all essential details as described by Makino and Sjiivall [6]. The procedure was briefly as follows: l-ml serum samples were diluted with 9 ml of 0.1 M NaOH and bile acids extracted by chromatography on Amberlite XAD-2. Bile acids were eluted from the column with ethanol and subjected to alkaline hydrolysis for 5 h in 15% NaOH in the ethanol at 120°C under pressure in Teflon containers. After acidification of the mixture, bile acids were extracted with ethyl acetate and converted to their methyl ester derivatives with diazomethane in ether/ methanol (5 : 1, v/v). Before methylation 2.5-10 1.18 of 7-ketodeoxycholic acid was added to the samples as an internal standard. The bile acid methyl esters were then purified by chromatography on a 200 mg column of aluminium oxide (Woelm, activity grade I). Cholesterol and impurities were eluted with 1% ethyl acetate in toluene and bile acid methyl esters with acetone/methanol (1 : 1, v/v). Enzymic hydrolysis of bile acid conjugates as an alternative to alkaline hydrolysis was carried out as described by Nair and Garcia [ 71. After Amberlite XAD-2 chromatography bile acids were dissolved in 2.0 ml of a 0.1 M acetate buffer, pH 5.6 25 ~1 of the choloylglycine hydrolase preparation, 0.5 ml of 0.2 M EDTA and 0.5 ml of 0.2 M /3-mercaptoethanol were added and the mixture incubated at 37°C for 30 min. The bile acids were then extracted three times with 10 ml ethyl acetate, methylated, and purified by chromatography on aluminium oxide as described above. Gas-liquid chromatography. Glass capillary columns were prepared in all essential details as described by Grob [8,9]. A 50-m OV-101 column with an internal diameter of 0.3 mm was installed into the Varian 2100 gas chromatograph. The carrier gas was nitrogen. The split ratio was 1 : 10 and the flow through the column 2 ml/min, and the additional flow of nitrogen into the detector 40 ml/min. Bile acid methyl esters were converted to their trimethylsilyl ether derivatives prior to GLC. The quantitation of bile acids was carried
65
out by comparing their peak height responses to that of the internal standard. The differences in peak height responses between bile acids were taken into account in these calculations by testing firstly the peak height responses of the appropriate reference bile acid and internal standard mixtures. Replicate quantitations were also carried out by comparing peak areas of bile acids to that of the internal standard. Peak area measurements were carried out during GLC using Infotronics Model CRF-204 automatic digital integrator. Gas chromatography-mass spectrometry of bile acids in some samples from normal subjects and from patients was carried out with an LKB-9000 instrument using a packed 1% SE-30 column for GLC. Results Fig. 1 shows that an OV-101 glass capillary column completely separates methyl ester trimethylsilyl ethers (Me-TMS) of cholic (C), chenodeoxycholic (CDC), deoxycholic (DC) and lithocholic (LC) acids. The presence of the first three of these was confirmed in the serum samples from healthy subjects and
C
Fig. 1. Glass capillary gas chromatograms of trimethylsilyl ethers of bile acid methyl esters. A 50-m OV-101 column programmed 8k/min from 150% to 255%. A: reference cholic (C). chenodeoxycholic (CDC), deqxycholic (DC) and lithocholic (LC) acids, and the internal standard (S). B: a Serum sample from a healthy female. C: a serum sample from a patient with cholestasis of Pregnancy. CS. cholesterol.
TABLE
1
SERUM
BILE
Grttu~
8-12
A:
ACID
CONCENTRATIONS
15 non-pregnant
weeks
of gestation.
IN HEALTl-IY
NON-PREGNANT
Group
Cholic acid
Chenodeoxycholie
A
0.58 *- 0.13 0.37 + 0.05 0.91 ’ 0.16
0.98 ’ 0.17 0.34 i 0.05 0.74 1 0.12
C
PREGNANT
FEMALES
healthy females, 21-.39 years of age. Group B: 15 healthy pregnant females at Group C: 18 healthy pregnant females in labor at 3X-41 weeks of gestation.
The values are expressed in i.rmol/l as mean i S.E. and are not corrected
B
AND
acid
fox mathi,dulogical
Deoxycholic
acid
losses.
Tot&
bile acids
’ 0.21 ’ 0.10 2.10 ’ 0.28
0.57 ’ 0.09 0.42 + 0.06 0.45 ’ 0.08
2.14 1.13
from patients by GC-MS, and their peaks were located in glass capill~y gas chromatograms by injecting small amounts of reference bile acid Me-TMS with the samples, after which a symmetrical increase of the peak to be measured was found. No impurities disturbing the bile acid and internal standard Me-TMS peaks were found when 1 ml of water as a method blank was analysed. 5-10 pg of glycocholic acid and taurodeoxycholic acid were added into 1 ml samples of a serum pool. The samples were analysed as described above. lJsing alkaline hydrolysis the recoveries varied from 63 to 89 (mean 76) ‘%and
TABLE
II
RILE ACID CONCENTRATION CHOLESTASIS OF PREGNANCY
(~motjl)
IN
THE
SERUM
OF
PATIENTS
WITH
INTRAHEPATIC
The simultaneous measurements of total and conjugated serum bilirubin (i.lmul/l), aminotransferases ASAT and ALAT and aIkaIine phasphatase (U/l) are given for comparison. Upper limits in URc~mplicated pregnancy: Bilintbin: total
