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Yonago Acta medica 2001;44:91–105Review Article

Internal Standard Compounds for Quantitative Determination of BileAcids by Gas Chromatography

Nobuo Yamaga, Yoshio Ogura, Kazuo Yamada, Hiromi Kohara* and KiyohisaUchida†

Department of Biochemistry and *Department of Pathological Biochemistry, Tottori UniversityFaculty of Medicine, Yonago 683-0826, and †The Cell Science Research Foundation, Osaka541-0045 Japan

Gas chromatography is well recognized as a useful tool with several advantages for theanalysis of bile acids as well as various compounds. In gas chromatographic analysis,bile acids in an analytical sample are subjected to a number of complicated proceduresinvolving many steps such as extraction, fractionation, solvolysis, hydrolysis, derivatiza-tion and injection to the gas chromatograph. These procedures result in the loss of bileacids in the analytical sample. The addition of suitable internal standard compound(s)into the analytical sample prior to the extraction of bile acids is indispensable for anaccurate determination of bile acids. There are two methods for the quantitative de-termination of bile acids in a biological sample by gas chromatography: one is thedetermination of total bile acid amounts in the sample. The other is the determinationof bile acid amounts in each fraction after group separation of bile acids in the biologicalsample using an ion exchange gel column. The addition of 7βββββ,12ααααα-dihydroxy-5βββββ-cholanoic acid or 7βββββ,12βββββ-dihydroxy-5βββββ-cholanoic acid as an internal standard compoundis useful for the former method. On the other hand, the addition of 7βββββ,12βββββ-dihydroxy-5βββββ-cholanoic acid, glyco-7ααααα,12ααααα-dihydroxy-5βββββ-cholanoic acid, tauro-7ααααα,12βββββ-dihydroxy-5βββββ-cholanoic acid and glyco-7βββββ,12ααααα-dihydroxy-5βββββ-cholanoic acid 7-sulfate is a suitablecombination as internal standard compounds for the latter method.

Key words: bile acids; biological sample; gas chromatographic analysis; internal standard compounds;quantitative determination

The use of gas chromatographyin investigation of bile acids

Gas chromatography is well recognized as auseful tool with several advantages for the analy-sis of bile acids as well as various compounds.It especially has good sensitivity and is able todetermine a number of different compounds in a

class simultaneously. In 1960, Vanden Heuveland coworkers (1960) for the first time appliedgas chromatography to the analysis of bile acidsafter converting them into methyl ester deriva-tives. Since then, gas chromatography has con-tributed greatly to the investigation of bile acidmetabolism, analyzing bile acids in several biol-ogical samples such as bile, feces, serum, urine,gallstones or tissues.

Abbreviations: αα , 7α ,12α-dihydroxy-5β-cholanoic acid; αβ, 7α ,12β-dihydroxy-5β-cholanoic acid; βα,7β,12α-dihydroxy-5β-cholanoic acid; ββ, 7β,12β-dihydroxy-5β-cholanoic acid; Glyco-αα , glyco-7α,12α-dihydroxy-5β-cholanoic acid; Glyco-αα 7-sulfate, glyco-7α ,12α-dihydroxy-5β-cholanoic acid 7-sulfate;Glyco-βα 7-sulfate, glyco-7β,12α-dihydroxy-5β-cholanoic acid 7-sulfate; Tauro-αα , tauro-7α ,12α-dihydroxy-5β-cholanoic acid; Tauro-αβ, tauro-7α ,12β-dihydroxy-5β-cholanoic acid; Tauro-ββ, tauro-7β,12β-dihydroxy-5β-cholanoic acid

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Metabolism and movementof bile acids

Bile acids in the human body are mainly com-posed of cholic acid, chenodeoxycholic acid,deoxycholic acid, lithocholic acid and urso-deoxycholic acid (Beppu et al., 1982; Yamagaet al., 1994a; Yamaga et al., 1996). Chenode-oxycholic acid and cholic acid are primary bileacids synthesized from cholesterol in the liver,and they are major catabolic products of choles-terol (Danielsson, 1973). Deoxycholic acid,lithocholic acid and ursodeoxycholic acid aresecondary bile acids converted from primarybile acids by the action of intestinal flora (Hilland Drasar, 1968; Shimada et al., 1969; Ariesand Hill, 1970). Most free bile acids are easilyconjugated with glycine or taurine in the liver.Bile containing glycine- and taurine-conjugatedbile acids is stored and concentrated in thegallbladder, and then released into the duode-num. Primary and secondary bile acids are ab-sorbed almost exclusively from the ileum, re-turn quantitatively to the liver by way of theportal circulation and are secreted into bile.This is called enterohepatic circulation of bileacids (Lack and Weiner, 1967; Dietschy, 1968;Dowling, 1972; Heaton, 1972). During entero-hepatic circulation, some parts of bile acids arenot absorbed to any significant extent from theintestine and excreted into feces (Eneroth etal., 1966; Dietschy, 1968; Tyor et al., 1971; Heaton,1972). A part of bile acids leaks into the system-ic circulation (van Berge-Henegouwen et al.,1974), and then excretes into the urine throughthe kidney.

Some parts of bile acids, especially second-ary bile acids in the blood and urine are found astheir sulfates (Palmer, 1967; Makino et al.,1973; Back, 1974), glucosides (Marschall et al.,1988; Marschall et al., 1989), glucuronides(Palmer,1967; Stiehl, 1974; Alme and Sjövall,1980), and/or N-acetylglucosaminides (Marschallet al., 1988; Marschall et al., 1989; Takikawa etal., 1982; Yamaga et al., 1994a). The formationof unusual bile acids and the ratios of variousconjugated bile acids offer useful informationon hepatobiliary and gastrointestinal diseases

(Garbutt et al., 1969; Neale et al., 1971; Takikawaet al., 1983a, 1983b).

Derivatizations of bile acids forgas chromatography

For gas chromatographic analysis the compoundsanalyzed are required to be volatile. Therefore,nonvolatile compounds should be convertedinto volatile derivatives quantitatively. It is truein the case of bile acids since they are polarcompounds having a carboxyl group and somehydroxyl groups in their molecules. Usually,the carboxyl group of bile acids is methylatedwith diazomethane or hexafluoroisopropylatedwith hexafluoroisopropanol (Imai et al., 1976).The hydroxyl group is acetylated with aceticanhydride (Roovers et al., 1968) or trifluoro-acetylated with trifluoroacetic anhydride (Endoet al., 1979). In addition, the hydroxyl group issilylated with both hexamethyldisilazane andtrimethylchlorosilane (Makita and Wells, 1963),N-trimethylsilylimidazole (Karlangnis andPaumgartner, 1979; Amuro et al., 1983) or di-methylethylsilylimidazole (Miyazaki et al., 1977;Arimoto et al., 1982). In these situations, directconversion of original conjugated bile acidsinto volatile derivatives has not been succeededup to the present.

Fractionation and gas chro-matographic analysis by difference in

conjugated bile acid forms

As mentioned above, bile acids in human andanimal bodies are present as a nonamidatedform (free form) and forms conjugated withglycine, taurine, sulfuric acid, glucuronic acidand/or others. Therefore, in gas chromato-graphic analysis, bile acids must be convertedinto volatile derivatives after carrying out anumber of complicated procedures. Bile acidsextracted from an analytical sample are con-verted into free bile acids by solvolysis withhydrochloric acid (Back, 1937; van Berge-Henegowen et al., 1976; Alme et al., 1977;Yamaga et al., 1994a), followed by alkaline

Internal standards for GC of bile acids

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for gas chromatographic analysis. Outline ofcommon procedures for systematic analysis ofbile acids in sample is shown in Fig. 1. By theseprocedures, the information on original conju-gated bile acids is available.

Glucosides, glucuronides and N-acetyl-glucosaminides of bile acids (particular bileacid conjugates) are present in only their formsor double conjugated forms with glycine ortaurine. When bile acid extract from biologicalsample was fractionated with PHP-LH-20 gelor DEAP-LH-20 gel column, these particularbile acid conjugates are not fractionated inde-pendently. No fractions are restricted where theparticular bile acid conjugates are fractionated.For example, bile acid N-acetylglucosaminidesare fractionated into both nonamidate fractionand glycine-conjugate fraction (Yamaga andKohara, 1994). There are no reports describingthe quantitative determination of bile acid gluco-sides, bile acid glucuronides and/or bile acid N-acetylglucosaminides in systematic analysis. Themethod for their analysis is referred to the individ-ual reports (Takikawa et al., 1982; Marschall etal., 1988, 1989; Yamaga and Kohara, 1994).

hydrolysis with 2 M sodium hydroxide solution(Nair and Garcia, 1969; Campbell et al., 1975;Yamaga et al., 1997) or enzymatic hydrolysis(Nair and Gordon, 1967; Cantafora et al., 1979),and free bile acids are converted into volatilederivatives. Bile acid derivatives are analyzedby gas chromatography.

However no information on the originalconjugated forms of bile acids is obtained fromthe gas chromatogram. In order to solve thisproblem, crude bile acid extract from theanalytical sample is first subjected to columnchromatography with an ion exchange gel suchas diethylaminohydroxypropyl-Sephadex-LH-20 (DEAP-LH-20) (Alme et al., 1977) or piperi-dinohydroxypropyl-Sephadex- LH-20 (PHP-LH-20) (Goto et al., 1978), fractionating into fivefractions; each containing neutral compounds,nonamidated bile acids, glycine-conjugated bileacids, taurine-conjugated bile acids and sulfatedbile acids. Each of all the fractions is subjectedto solvolysis with hydrochloric acid, followedby hydrolysis with enzyme or 2 M sodium hy-droxide solution, and then free bile acids thusobtained are converted into volatile derivatives

Fig. 1. Outline of procedures for systematic analysis of bile acids in a sample.

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iii) Internal standard compound(s) should bestable. That is, during the analysis, theymust not decompose to other compounds.

iv) After the fractionation with PHP-LH-20gel column or DEAP-LH-20 gel column,the characteristic internal standard com-pound(s) should be transferred into each ofall fractions. For this reason, four internalstandard compounds consisting of non-amidated, glycine-conjugated, taurine-conjugated and sulfated forms, are neces-sary for the quantitative and qualitativedetermination of bile acids in each fractionafter group separation.

v) The peak(s) of internal standard com-pound(s) must not pile up on the peaks ofbile acids and the peaks of other mixedcompounds in the biological sample.

vi) The retention time of the internal standardcompound peak in each fraction must notbe largely distant from those of bile acidpeaks. Preferably, the peak of the internalstandard compound should appear in therange between the first and the last bileacid peaks detectable on the gas chromato-gram.

vii) Some linear relationship should be keptbetween the peak high or area and theweight of each bile acid to the internalstandard compound.

When the selected internal standard com-pound(s) satisfy the requirements above men-tioned, the area of an internal standard com-pound peak on the gas chromatogram is regard-ed as the amount (weight) of the internal stan-dard compound added to the analytical sample,irrespective of the recovery rate during all theprocedures. There is no occasion for consider-ing the recovery ratio. It is only required to ob-tain linear calibration curves between the peakarea ratios and the weight ratios of each bileacid to the internal standard compound.

Up to now, some kinds of unique bile acidssuch as 5β-cholanoic acid (Campbell et al., 1975),

Indispensability ofinternal standard compounds

A part of bile acids in an analytical sample,especially in a small volume sample, is certain-ly lost during procedures such as extraction,fractionation and other treatments. The usualmethod for correcting the loss of bile acidsduring analytical procedures is based on eitheradding an internal standard compound to thesample in the course of the systematic analysis(Fig. 1) or performing a recovery test usinglabeled and non-labeled bile acids under a con-dition similar to the analytical condition. Forgas chromatographic analysis, the latter methoddoes not ensure that the recovery from abiological sample will be the same as that froman artificial sample that has been prepared withlabeled or non-labeled bile acids. Moreover,recovery may differ from assay to assay. There-fore, the use of internal standard compounds isentirely indispensable for correcting the loss ofbile acids.

The minimum requirements forselecting internal standard compoundfor gas chromatography of bile acids

The minimum requirements for selecting a suit-able internal standard compound for the deter-mination of bile acids by gas chromatographyare as follows.

i) Internal standard compound(s) must beadded to the analytical sample before theextraction of bile acids. Then, the peak ofcharacteristic internal standard compoundmust appear together with peaks of bileacids in the analytical sample on the samegas chromatogram.

ii) It is not necessary for internal standard com-pound(s) to be bile acid(s). However, it isbetter for them to be non-natural 5β-cholanoic acid homologues with somehydroxyl groups.

Internal standards for GC of bile acids

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nordeoxycholic acid (van Berge-Henegouwenet al., 1977; Roda et al., 1978), hyodeoxycholicacid (Ali and Javitt, 1970; Karlangnis andPaumgartner, 1978; Cantafora et al., 1979), 7-ketolithocholic acid (Klaassen, 1971), 7-keto-deoxycholic acid (van Berge-Henegouwen etal., 1976) and hyocholic acid (Subbiah, 1973;Yamaga et al., 1983) have been used as an inter-nal standard compound for gas chromatograph-ic analysis. Certainly, these compounds areable to be used as an internal standard com-pound. But they have some drawbacks. Theyare sometimes found in samples from healthysubjects, neonates and some patients (Alme etal., 1977, 1978, 1980; Back and Walter, 1980;Sawada, 1981). Furthermore, oxo-5β-cholanoicacids such as 7-ketolithocholic acid and 7-keto-deoxycholic acid are easily vulnerable to de-composition during alkaline hydrolysis withsodium hydroxide solution (Lepase et al., 1978).For these reasons, it is necessary to developother internal standard compounds.

Fig. 2. Artificial bile acids chemically synthesized from cholic acid.

Internal standard compoundsfor the determination

of total bile acid amounts

Natural bile acids originally have a hydroxylgroup at the C-3α position in the steroid nucle-us. First, we chemically synthesized four iso-mers of 7,12-dihydroxy-5β-cholanoic acidlacking a hydroxyl group at C-3α position ofcholic acid; 7α ,12α-dihydroxy-5β-cholanoicacid (αα ), 7β,12α -dihydroxy-5β-cholanoicacid (βα), 7α ,12β-dihydroxy-5β-cholanoicacid (αβ) and 7β,12β-dihydroxy-5β-cholanoicacid (ββ) (Fig. 2) (Arimoto et al., 1982). Anoutline of the chemical synthesis of these arti-ficial bile acids from cholic acid is shown inFig. 3. These compounds in the derivatives ofmethyl ester dimethylethylsilyl (DMES) etherhave different retention times compared to eachother on the gas chromatogram (Fig. 4). In addi-tion, the retention times of these four compoundsare different from those of many authentic bileacids. But, only the retention time of αβ is veryclose to that of lithocholic acid (Table 1) and

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Fig. 4. Gas chromatogram of αα , αβ, βα and ββ (methyl ester DMES ether).(See Table 1 for gas chromatographic condition.)

Fig. 3. Outline of chemical synthesis of four 7,12-dihydroxy-5β-cholanoic acid isomersfrom cholic acid.

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αα

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pons

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24.1

36

20 26.7

33.3

40 46.7

ββαβ

βα

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44

25.3

59

Internal standards for GC of bile acids

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both peaks of αβ and lithocholic acid practi-cally pile up in the same gas chromatogram(Fig. 5).

Calibration curves for the quantitative deter-mination of several bile acids have a linear rela-tionship going through the origin between theweight ratio and the peak area ratio of each bileacid to the internal standard compound(s).

By way of example, the linear calibrationcurves for several bile acids are shown in Fig. 6when ββ is used as an internal standard com-pound. Then, the absolute amount of each bileacid in a biological sample can be calculated bythe following formula (Arimoto et al., 1982).

W = A × B

W: Absolute amount (weight) of each bileacid in the sample assayed.

A: Weight ratio of each bile acid to ββ, obtain-ed by multiplying the slope value of thecalibration curve by the peak area ratio ofeach bile acid peak to ββ peak from the gaschromatogram.

B: Amount (weight) of internal standardcompound added in the biological sample.

Total bile acid amounts in an analytical sam-ple can be calculated by adding the amountsof all kinds of bile acids analyzed.

Occasionally confirming the calibrationcurve is recommended.

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Weight of each bile acidWeight of ββ

Peak area of each bile acidPeak area of ββ

Fig. 5. Gas chromatogram of authentic bile acids and internalstandard compounds (methyl ester DMES ether). (See Table 1 forthe number of peaks and gas chromatographic condition.)

Fig. 6. Calibration curves of severalnatural bile acids to artificial ββ as in-ternal standard compound for quan-titative determination. (See Table 1 forthe number of peaks.)

Weight ratio:

Area ratio:

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Fig. 7. Internal standard compounds chemically synthesized for gas chromatographic analysisafter group separation.

Yamaga and coworkers (1983) adopted ββ as aninternal standard compound for the quantitativedetermination of total bile acids in urine by gaschromatography. Other workers used tauro-7α,12α-dihydroxy-5β-cholanoic acid (Tauro-αα ) as an internal standard compound for thesame project (Ghoos et al., 1983). These syn-thesized compounds (αα , βα, ββ and Tauro-ααexcept αβ, because the peak of αβ piles up onthat of lithocholic acid) are adequate internalstandard compounds for the quantitative deter-mination of bile acids by gas chromatography.However, as far as only one kind of internalstandard compound is used, the analysis aftergroup separation of bile acids can not attainaccurate data in all fractions, since ββ transfersonly into the nonamidate fraction after an ionexchange gel column chromatography. On theother hand, Tauro-αα transfers only into thetaurine-conjugate fraction in a similar meaningas above. Accordingly, adequate internal stan-dard compounds must be added into other frac-tions except the nonamidate fraction in the caseof ββ and tauro-conjugate fraction in the case ofTauro-αα on half way just after group separa-tion in systematic analysis.

Internal standard compoundsfor the determination of

group separated bile acids

Yamaga and coworkers (1987) have chemicallysynthesized four internal standard compoundsexactly transferred into each conjugate fractionby group separation. They are four different com-pounds using 7,12-dihydroxy-5β-cholanoic acidisomers; 7β,12β-dihydroxy-5β-cholanoic acid(ββ) for the nonamidate fraction, glyco-7α,12α-dihydroxy-5β-cholanoic acid (Glyco-αα ) for theglycine-conjugate fraction, tauro-7β,12β-dihydroxy-5β-cholanoic acid (Tauro-ββ) forthe taurine-conjugate fraction and glyco-7α ,12α-dihydroxy-5β-cholanoic acid 7-sulfate(Glyco-αα 7-sulfate) for the sulfate fraction(Fig. 7). An artificial sample composed of thesefour compounds is fractionated into each frac-tion with PHP-LH-20 gel column, and theneach internal standard compound appears as apeak in each corresponding fraction coincidingwith the internal standard compound by gaschromatographic analysis. However, when

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Fig. 8. Gas chromatogram of sulfate fraction fromhuman urine with ββ, Glyco-αα , Tauro-ββ and Glyco-αα 7-sulfate. Cholesterol DMES ether and αα methylester DMES ether. (See Table 1 for gas chromato-graphic condition.)

human urine with Glyco-αα 7-sulfate added isanalyzed in the same way, αα and cholesterolappear as peaks on the same gas chromatogramhaving almost the same retention time in sulfatefraction in some biological samples. This evid-ence indicates that cholesterol sulfate in urine isfractionated into the sulfate fraction when theextracts from urine using Amberlite XAD-2 orSepak C18 are fractionated with PHP-LH-20 gelcolumn, and then cholesterol is detected in sul-fate fraction (Fig. 8). In fact, cholesterol sulfateis present in biological samples, especially inurine (Winter and Bongioanni, 1968: Muskiet etal., 1983). Therefore, it becomes necessary ei-ther to remove cholesterol from the solvolysateby extraction with hexane after solvolysis of thesulfate fraction or to select another sulfated can-didate from 7,12-dihydroxy-5β-cholanoic acid

isomers except αα . Finally, the combination ofββ, Glyco-αα , Tauro-αβ and Glyco-βα 7-sulfate(Fig. 7) is most suitable as an internal standardcompound in gas chromatographic analysisafter group separation.

Figure 9 shows the result of an artificialsample containing ββ, Glyco-αα , Tauro-αβ andGlyco-βα 7-sulfate as four internal standardcompounds being analyzed by the systematicanalysis shown in Fig. 1. Four internal standardcompounds are well fractionated into individualcorresponding fraction. Besides, Fig. 10 showsthe results that bile acids in human urine withand without the four internal standard com-pounds were analyzed by the same method de-scribed above. When both profiles from theanalysis of a urinary sample with and withoutfour internal standard compounds were com-pared, neither peaks of bile acid nor of non-bileacid compound piling up on the peak of the in-ternal standard compound are detected on thegas chromatogram in any fraction. Moreover,the profile from the analysis of the urinary sam-ple with the above combination of internal stan-dard compounds lends a helpful suggestion. Itenables us to judge from the gas chromatogramwhether the fractionation with PHP-LH-20 gelcolumn is perfect or not, as the different fourinternal standard compounds were used. Forexample, the peaks of different internal stan-dard compounds more than two are detected inone fraction when the fractionation is incom-plete. Furthermore, the peak of an internal stan-dard compound in each fraction acts as a pecu-liar indicator for the quantitative determinationof each bile acid in its fraction and for the iden-tification of bile acid peaks by agreement withthe relative retention time of individual bileacids to the internal standard compound appear-ing in its fraction (Table 1). The amount of each internal standard com-pound, 1–5 µg in 1 mL of serum and in 5 mL ofurine, added in a biological sample should beenough in the analysis, though it depends on theconcentration of bile acids in the biologicalsample.

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Internal standards for GC of bile acids

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Sulfate fraction

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Taurine-conjugate fraction

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espo

nse

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Fig. 9. Gas chromatograms of nonamidate, glycine-conjugate, taurine-conjugate and sulfate fractions fromartificial sample added ββ, Glyco-αα , Tauro-αβ, and Glyco-βα 7-sulfate. (See Table 1 for gas chroma-tographic condition.)

Views on the future

It is inevitable that in analytical experiment anumber of complicated procedures result in theloss of the compound analyzed as mentionedabove. Therefore, the use of internal standardcompound(s) is indispensable for the correctionof the data obtained by quantitative analysis.Very few investigators have described what com-pound(s) have been adopted as internal standardcompounds and many investigators are apt toomit the description of whether internal stan-dard compound(s) were used or not in gas chro-matographic analysis. The cause may be basical-ly that there have been very few investigationson internal standard compounds in the past; but

on the other hand there seems to be an increas-ing trend that acquisition of experimental datais too far reaching for consideration in the experi-mental method. If so, the authors themselves im-ply no reliability in the data they have obtained.

It is important to obtain data by appropriateexperimental methods, and then, discussion isto be established based on reliable analyticaldata.

This article comprehensively described thenecessity of suitable internal standard com-pounds and their importance in gas chromatog-raphy at the present time as a general review.At the present time, in the quantitative determi-nation of bile acids using gas chromatography,either ba or bb is the most suitable internalstandard compound for the determination oftotal bile acid amounts in the biological sample.

αβ←Tauro-αβββ

αα← Glyco-αα βα← Glyco-βα 7-sulfate

20 26.7

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Nonamidate fraction Taurine-conjugate fraction

Glycine-conjugate fraction Sulfate fraction

Fig. 10. Gas chromatograms of nonamidate, glycine-conjugate, taurine-conjugate and sulfatefractions from human urine with and without four internal standard compounds. (See Table 1 for gaschromatographic condition.)

30.5

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41.8

7235.6

3637

.240

40.6

22

25.0

91

40.5

26

35.5

56

37.1

9130.4

8028

.744

21.5

0023

.195

24.3

0926

.481

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86

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12

33.9

22

45.2

22

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75

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52

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20

34.3

70

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36

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2028

.478

31.6

5733

.963

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46

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27

34.3

79

30.0

2430

.586

32.8

7334

.348 34

.868

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18

29.9

31

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59

30.5

1532

.773

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82 42.5

13

35.4

35

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13

39.6

97

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70

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76

23.6

22

28.2

73

31.1

1032

.817

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61

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5120 26

.7

33.3

40 46.7

23.5

9324

.077

32.7

6634

.842

31.0

7030

.783

27.2

92 28.2

47

46.9

77

20 26.7

33.3

40 46.7

20 26.7

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40 46.7

20 26.7

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40 46.7

(αβ)

(ββ)

(αα)

(βα)

withoutTauro-αβ

withoutββ

withoutGlyco-αα

withoutGlyco-βα 7-sulfate

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withTauro-αβ

withββ

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withGlyco-βα 7-sulfate

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On the other hand, the combination of bb,Glyco-aa, Tauro-ab and Glyco-ba 7-sulfate isthe the most suitable internal standard com-pound for the accurate determination of bileacid amounts in each fraction after group sepa-ration of bile acids in the biological sample us-ing the ion exchange gel column. However, it isnot because all problems in the accurate deter-mination of bile acids using gas chromatog-raphy were completely solved. When unknownbile acid(s) and new bile acid form(s) conjugat-ing with other substance(s) are discovered, thedevelopment of new internal standard com-pound(s) and new systematic analyses may be-come necessary. It has already been requestedthat new systematic analyses for glucoside,glucuronide and N-acetylglucosaminide of bileacids should be developed without delay in-cluding new internal standard compound(s).

Acknowledgments: We sincerely thank Ms. YumikoUyama of the Department of Biochemistry, TottoriUniversity Faculty of Medicine for her help in cleri-cal works.

References

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Received April 2, 2001; accepted May 10, 2001

Corresponding author: Nobuo Yamaga