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Microanalysis of bile acid in human liver tissue by selected ion monitoring
ANALYTICAL
BIOCHEMISTRY
104, 75-86
Microanalysis
(1980)
of Bile Acid in Human Liver Tissue by Selected Ion Monitoring
JIRO YANAGISAWA,* MASAHIRO IT0H.t MASATAKA ISHIBASHI,t HIROSHI MIYAZAKI,~ AND FUMIO NAKAYAMA* *Department
of
Surgery
I. Kyushu
Fduoka 812. Japan; Nippon Ka,vnku
University iResearch Compurly.
Received
Fcircult~ Loborcltorirs 3-31 Shimo.
August
of
Mrdicirrr,
3-l-I
~)~Phnrt~~acc,frtical Kita-Xu. Tokw I/5,
,Mnedashi.
Higashi-Xu.
Diriviorf. Japcrn
23. 1979
A method of microquantitative determination of bile acid in 5-30 mg of human liver tissue was developed. Bile acids were converted to their ethyl ester dimethylethylsilyl ether derivatives and analyzed by capillary gas chromatography-selected ion monitoring, using [‘H,]lithocholic (LCA), L*H,]deoxycholic (DCA). L”H,]chenodeoxycholic (CDCA). rLHI]ursodeoxycholic (UDCA). and [*H,]cholic (CA) acids as internal standards. Precision and reproducibility of the present method were tested using surgically obtained liver specimens. The results were statistically analyzed according to one-way layout and the orthogonal polynomial equation. Bile acids except LCA were determined with 2.3 to 11.4% of the coefficient of variation. Recoveries of conjugated bile acids ranged from 72.2 to 96.05 with a mean of 84.3%. The amount of bile acids present in histologically normal liver specimens (n = 10) was found to be 29.56 ? 8.62 &g liver. The relative compositions (r?) of CDCA and CA were 38.8 + 8.9 and 41.1 + 11.0. respectively.
The metabolism of bile acid has aroused much interest because of the recent recognition of its importance in bile secretion and intestinal absorption as well as in various disease states such as cholelithiasis (1,2), Crohn’s disease (3.41, bacterial overgrowth (5,6), ileal resection (7), and liver disease (8). Bile is present in wellconfined body compartments forming the enterohepatic circulation (9), i.e., liver, bile, intestinal tract, and portal blood. These compartments are relatively inaccessible to sampling. Bile requires duodenal intubation Portal blood is usually inaccessible. Intestinal content can be sampled in the form of feces but the bile acid present is a spillover of bile acid from intestinal content further modified by intestinal flora so extensively that it does not give much information on the bile acid present in enterohepatic circulation. Bile acids present in systemic circulation can be analyzed by
sampling venous blood but they are also spillover of bile acid brought to the liver via portal blood and far from representing those in the enterohepatic circulation. The liver has been relatively inaccessible to sampling. Needle biopsy is increasingly used for histological diagnosis of liver disease. However, the small amount of liver tissue obtainable limits the utility of the specimen for chemical analysis of its constitutent bile acid. Since the reports of cellular and subcellular bile acids in the rat liver by Okishio and co-workers (10,l l), several papers (12- 15) describing the analysis of bile acid in the human and rat livers have been published. In these studies, however, bile acid was identified mainly by means of gas chromatography, which lacks sensitivity and specificity for microanalysis of bile acid in complexed biological mixtures. The analysis of bile acid can be improved by the use of gas chromatography-mass spectrom75
0003-2697/80/070075-
12$02.00:0
Copyright r 1980 by Academic Pres\. Inc. All rights of reproduction m any form reserved.
76
YANAGISAWA
e&y-selected ion monitoring (gc-MS-SIM)’ with a wall-coated open tubular (WCOT) glass capillary column (16). The present study deals with the development of an analytical method for trace amounts of bile acids in biopsy specimens of human livers. MATERIALS
AND
METHODS
Lithocholic (LCA), deoxycholic (DCA), chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), and cholic (CA) acids and sodium salts of their taurine-conjugated bile acids and glycine-conjugated CDCA were purchased from Tokyo Kasei Kogyo Company, Tokyo, Japan, Applied Science Laboratories, Inc., State College, Pennsylvania, and Sigma Chemical Company Ltd., St. Louis, Missouri. Taurolithocholic acid3-sulfate (TLCAS) and glycolithocholic acid-3-sulfate (GLCAS) were kindly supplied by Dr. G. A. D. Haslewood and lithocholic acid-3-sulfate (LCAS) by Dr. R. H. Palmer. [24-‘“C]Cholic, glyco[G-“H]cholic, and [24-14C]taurocholic acids were obtained from New England Nuclear Corporation, Boston, Massachusetts. Their purities were checked by gas chromatography and thin-layer chromatography (see below). Solvents were of analytical grade and used without further purification. Pyridine was redistilled before use over P,O,. Trimethylsilyl imidazole (TMSI) was purchased from Tokyo Kasei Kogyo Company.Dimethylethylsilyl imidazole (DMESI) was prepared in our laboratory by the ’ Abbreviations used: gc-MS-SIM. gas chromatography-mass spectrometry-selected ion moniwall-coated open tubular (glass toring: WCOT, capillary column): LCA, lithocholic acid; DCA. deoxycholic acid; CDCA, chenodeoxycholic acid; UDCA. ursodeoxycholic acid: CA, cholic acid: TLCAS. taurolithocholic acid-3-sulfate; GLCAS, glycolithocholic acid-3-sulfate; LCAS. lithocholic acid-3-sulfate: TMSI. trimethylsilyl imidarole; DMESI. dimethylethylsilyl imidazole: tic. thinlayer chromatography; LSC. liquid scintillation counting; IS, internal standard.
ET AL.
method described previously (17). [6,6,7,7‘H,]LCA, [6,6,7,7,8-“H,]DCA, [l I,1 1, 12, I2-?H,]CDCA, and [ 11,l 1, 12P-2H,,]CA were prepared in our laboratory (H. Miyazaki, M. Ishibashi, and M. Itoh, in preparation) and ]11,11,12.12-ZH,]UDCA was kindly supplied by Tokyo Tanabe Company, Tokyo, Japan. Amberlite XAD-2 resin was obtained from Rohm Haas Company, Philadelphia, Pennsylvania, and Sephadex LH20 (25-100 pm) from Pharmacia Fine Chemicals AB. Uppsala, Sweden. The former was washed prior to use according to the method of Axelson and Sjovall (18). Kieselgel 60 (70-230 mesh) was purchased from Merck, Darmstadt, West Germany. Thin-luyrr c.hrornatograpk~ (tic). Thinlayer chromatography was carried out using precoated plates with kieselgel 60 (Merck). Solvent systems used were isopropyl etherisooctane-acetic acid, 10:5:5 (v/v/v) (19). for unconjugated bile acids and n-butanolacetic acid-water, 10: 1: I (v/v/v) (20), for conjugated bile acids. Gas chromutogruphy (gci. A Shimazu GC-7AG gas chromatograph equipped with a flame ionization detector was employed. The column was 2.0 m x 2.5mm glass with 1.5%’ OV-101 (Ohio Valley Specialty Chemical Co.) on Gas-Chrom Q, 80-100 mesh (Applied Science Laboratories, Inc.). The temperature of the column oven was maintained at 275°C. The flow rate of the carrier gas (nitrogen) was 40 ml min’. The temperature of the injection port and detector were 300°C. Gus cllronlcrtoRrrtp~l~-tncrss rtry -.sclrctrd iota monitoritlK.
spcctrotw
An LKB2091 gc-MS system equipped with a data processing system (Shimazu GCMSPAC90) and Van den Berg’s solventless injector was employed. The column used was WCOT, 25 m x 0.35 mm i.d., coated with SE-30 (LKB-Producktor, Stockholm, Sweden). The operating conditions were injection port temperature 285”C, column oven temperature 270°C. separator temperature
MICROANALYSIS
OF BILE
ACID TABLE
MASS
SPECTROMETRIC
DATA
OF THE DMES
ETHER
IN
HUMAN
M,
Lithocholic acid Deoxycholic acid Chenodeoxycholic acid Ursodeoxycholic acid Cholic acid
490 592 592 592 694
IMI:
[M-IS]
2.1 0.1 0.1 0.2 0.1
5.9 6.3 0.1 5.8 5.3
27o”C, ionization source temperature 290°C. flow rate of helium carrier gas I .5 ml min-‘. ionization energy 22.5 eV, acceleration voltage 3.5 kV, and trap current 100 PA. SIM was carried out using a multiple ion detector. Liyuid scintillation counting (LSC). The accuracy and reproducibility of the sample preparation procedure was checked by counting the radioactivity of the sample before and after the procedure in the scintillation cocktail (Aquasol 2, NEN). LSC was performed with a Packard liquid scintillation counter, model 3380, equipped with AAA. Liver specimens. Surgical biopsies were performed during laparotomy at the Department of Surgery 1, Kyusyu University Faculty of Medicine, for histological examination. A part of the liver tissue was used in the present investigation. Cleunup procedure. Five to 30 mg of liver tissue was rinsed with ice-cold saline, briefly dried on a filter paper, and weighed. After the addition of adequate amounts of L2H4]LCA, I’H,]DCA, [‘HJCDCA, [‘HJUDCA, and [“H,,]CA as internal standards (IS). the tissue was solubilized with 1 ml of 5%’ aqueous sodium hydroxide solution at 70-8O”C, diluted with 9 ml of distilled water, and passed through a column (8 x 120 mm) of the XAD-2 resin (21). After washing with water until neutral, the bile salts were eluted with 15 ml of ethanol. After evaporation under reduced pressure, the residue was subjected to solvolysis
77
TISSUE
I DERIVATIVES Relative
Compound
LIVER
OF BILL intensity
ACID
Other 386 443 459 488 561
ESTERS
(%)
[M-29]’ 100.0 100.0 6.8 100.0 100.0
ETHYL
(57) (3) (76) (19) (67)
ions
257 (30) 384 (8, 385 (100) 459 (15) 383 (33)
215 255 255 383 253
(53) (60) (20) (24) (26)
according to the method described by Palmer and Bolt (22). After solvolysis the residue was dissolved in 5% aqueous sodium hydroxide solution. Hydrolysis of glycine and taurine conjugates was performed in a sealed glass tube at 120°C for 7 h. After acidification to pH 1 with concentrated hydrochloric acid, the bile acids were extracted three times with IO-ml volumes of ethyl acetate. The combined ethyl acetate extract was evaporated to dryness and the residue was derivatized to ethyl ester-DMES ether as described below. Deri\,utizcction. To each authentic bile acid or hydrolyzed samples, 0.5 ml of 5% (w/v) hydrogen chloride ethanol solution was added and allowed to stand for 60 min at room temperature. After evaporation under a stream of nitrogen, the residue was transferred onto a silica gel column (6 x 50 mm, silica gel 60) with a small volume of n-hexane-ethyl acetate, 9:l (v/v), mixture and washed with 10 ml of the solvent (23). The bile acid ethyl ester derivatives were eluted with 10 ml of ethermethanol, 9: 1 (v/v), mixture. After evaporation to dryness under reduced pressure, the residue was treated with 5’0 ~1 of DMESI and allowed to stand for 15 min. Excess silylating reagent was removed on a Sephadex LH-20 column (6 x 60 mm) prepared in the solvent system, n-hexane-chloroform-methanol, 10: 10: 1 (v/v/v), mixture (24). The DMES ether derivatives of bile acid ethyl esters were recovered in the first
78
YANAGISAWA
ET AL.
TABLE DEUTERIUM
CONTENTS
2
OF DEUTERIUM-LABELED
BILE
ACIDS
‘H contents Deuterium-labeled
bile acid
Measured
L’H,]Lithocholic acid I’HJDeoxycholic acid [‘H,]Chenodeoxycholic acid (‘H,JUrsodeoxycholic acid 1’HJCholic acid “Measured
as the methyl
ion”
(M-TMSOH]+ [M-2 x TMSOH]: [M-2 x TMSOH]? [M-2 x TMSOH]+ IM-2 x TMSOH]’ ester-TMS
ether
‘H”
‘H,
‘H,
‘H,,
YH,
-
-
16.0 -
71.0
12.9
-
6.1
0.1 -
0.4 2.2
38.6 79.5 77.0
55.3
0.3
0.5
1.4 3.0 24.4
16.6 19.4 73.4
RESULTS AND DISCUSSION Gas Chromatography -Mass Spectrometry
Gas chromatographic separation of bile acids on a glass capillary column was found to be superior to that on a packed column, even though various liquid phases and derivatives were available for the latter. The dimethylethylsilyl (DMES) ether derivatives of bile acid ethyl esters gave, as reported earlier (16), sharp, symmetrical peaks and eluted in regular order according to the number of hydroxyl groups. Furthermore, excellent resolution can be obtained using a glass capillary column coated with SE-30.
IN AMBERLITE
COLUMN
1.7 -
LIVER
-
Propriety of Deuterium-Lahelrd Compounds as Internal Standards
When [‘HJCDCA was used as an internal standard for the determination of bile acids in human liver, the analytical errors for UDCA and LCA were found to be large in comparison to those for CDCA and CA. This is probably due to an error
3
OF RADIOISOTOPE-LABEI.ED TO SOLUBILIZED HUMAN
XAD-2
‘H,
The mass spectrometric data of the DMES ether derivatives of bile acid ethyl esters are listed in Table 1. All bile acids except CDCA showed the characteristic fragment ions of [M-29]+ formed by the elimination of the ethyl group at the silicon atom, while the CDCA derivative yielded the [M-dimethylethylsilanol-29]+ ion as a prominent peak (25). The appearance of a prominent ion at the high mass region seems to be of great advantage in avoiding interference when biological materials are to be thus enhancing reliability in analyzed, quantitation.
TABLE RECOVERY ACID ADDED
-
‘H,
derivatives
2.5 ml of effluent. Solvent was evaporated to dryness under reduced pressure. The residue was redissolved in 5% (v/v) pyridine-n-hexane solution and injected to gc-MS.
TABLE
(%)
CHOLIC TISSUE
CHROMATOGRAPHY
Bile acid
Recovery (%) i SD (n = 3)
124-“C]Cholic acid [24-‘“ClTaurocholic acid Glyco[G-JH]cholic acid
95.6 2 1.2 96.5 ? 0.5 94.0 -e 2.9
COMPLETENESS LABELED
4
OF HYDROLYSIS OF RADIOISOTOPECONJUGATED CHOLIC ACID ADDED
Completeness of hydrolysis (%) i SD (n = 4)
Bile acid [24-‘“ClTaurocholic GlycolG-“Hlcholic
acid acid
94.0 IT 3.5 87.6 i- 7.0
MICROANALYSIS
0.01
0.03
OF BILE
ACID
IN HUMAN
LIVER
79
TISSUE
0.05
O.liJ
I 0
I 1.0
I ?.O
I
LTR '
(.n-"'rJ)
40
I 0
I 1 0
1 2.0
1 4.0
DCA
(
I
I 0.5
1 LOCAL 1 .I!
b
0.25
I 0
0.25
I 0
Cl.?5
I
0 20
,j)
1 UDCA(.lO-' 1.0
q)
I
1 CP 1.0
q)
I 0.5 of
.lil-"
31
I 05
Fmounts
1O-9
blip
( 10-B
ac>di
FIG. I _ Calibration curves of bile acids. The weight ratio of a component relative to the corresponding deuterated internal standard is plotted on the abscissa and the peak height ratio of the component to the internal standard is plotted on the ordinate. (0) LCA (,,I/; 4611465): (r‘,) DCA (I>I/: 5631568): (A) CDCA (/n/z 4591463); (B) UDCA (rni; 5631567); (0) CA (r,r/,l 6651668).
in SIM as well as the difference in recovery of individual bile acids during the cleanup procedure. Thus, instead of a single IS, the five deuterium-labeled analogs corresponding to each bile acid were prepared and used as IS. The deuterium contents in these compounds were measured as methyl ester-TMS ether derivatives and are summarized in Table 2. No loss of deuterium atoms in these compounds was found to occur during alkaline hydrolysis, confirming that the deuterated bile acids can be added before the sample preparation.
The recovery of “H- or 13C-labeled bile acid added to the solubilized liver tissue corresponding to about 50 mg of fresh liver tissue was studied in the Amberlite XAD-2 coiumn chromatographic extraction procedure. Triplicate runs with 0.1 FCi of ‘%or 0.5 $Zi of “H-labeled bile acid were made. The radioactivity was measured with the liquid scintillation counter before and after Amberlite XAD-2 column chromatography. As shown in Table 3, taurine- and glycineconjugated and unconjugated CA were quantitatively recovered. Radioisotope-
80
YANAGISAWA
ET AL (e)
[‘I 567J
568
I\
..,
j
-'
I
1
I
I
0
5
10
15
RETENTION
TlME(min)
FIG. 2. Selected ion recording of an authentic mixture of the DMES ether derivatives of LCA (a), DCA (b), CDCA (c), UDCA (d). and CA (e) ethyl esters. Monitoring ions were selected as follows: m/z 4611465 for LCA/]‘H,]LCA, 5631568 for DCA/]‘H,]DCA, 4591463 for CDCA/]“H1]CDCA, 5631567 for UDCA/]?H,]UDCA. and 6651668 for CA/(‘H:,]CA. Monitoring ions were changed at the positions indicated by the asterisks.
labeled compounds added to human liver were subjected to alkaline hydrolysis. After neutralization, an aliquot was applied to a
silica gel G plate (0.25 mm thick, Merck) and developed with isopropyl ether-isooctane-acetic acid, 10:5:5 (v/v/v). The
1
,
I
I
0
5
10
15
RETENTION FIG. 3. Selected ion recording from human liver tissue (Patient
TiME(mio)
of the DMES ether 1. Table 9): (a) LCA,
derivatives (b) DCA,
of bile acid ethyl esters in extract (c) CDCA, (d) UDCA. and (e) CA.
MICROANALYSIS
OF
BILE
ACID
completeness of hydrolysis was calculated from the radioactivity of unconjugated bile acid. As shown in Table 4, hydrolysis was found to be fairly complete, i.e., up to 87%. Consequently, these results suggested that these deuterium-labeled unconjugated bile acids could be used as convenient IS for the determination of conjugated bile acids in the present procedure, as the degree of hydrolysis could be compensated. Since only a minute amount of nonlabeled component was found to be present in the deuterated bile acids used, i.e., 0.3% these compounds may be added to approximately 10 times the bile acids of interest as IS and carriers. TABLE REYRODUCIBILITY:
5
CHENODEOXYCHOLIC
LEVELS
IN HLIMAN I. ANAL
LIVER
YTITAI.
ACID
TISSUE
DATA
Found
Sample
t&g
A B C D E
7.54 7.81 7.51 7.63 7.37 II.
liver) 7.36 7.68 7.81 7.48 7.52
ANALYSIS
7.41 7.34 7.15 7.21 7.62 or
Mean
2 SD
7.44 7.61 7.69 7.44 7.50
2 O.OY it 0.24 ? 0.16 ? 0.21 -c_ 0.13
VARIANCE
Source
L’
Sample preparation Error (selected ion monitoring)
0. II306
“’4
0.037015
0.30774
10
0.030774
Total
0.45580
14
10, 0.05)
= 3.478
P(4. S: residual ,f‘: number
Fu 1.203
sum of squares of degrees of freedom,
.f, : .fm?,l<.*wpam~,,m .f; : .f,,r,,, V: unbiased variance F,,: observed value following F distribution variance ratio ( Vsam,~, PrPPalatlWli V,,,,,, ) F(j’, J;,(Y): Density function of F distribution withf’, and fi degrees of freedom
IN HUMAN
LIVER
TABLE C~EFFICIENTOF
VARIATION
AND
SELECTED
Bile acid Lithocholic acid Deoxycholic acid Chenodeoxycholic acid Ursodeoxycholic acid Cholic acid ” Coefficient ’ Coefficient * Statistically
Calihrtrtion
81
TISSUE
of variation of variation significant
6 IN SAMPLE
ION
PREPARATION
MONITORING
CV;,” (‘V )
CV,.” (7)
12.1” I.5 0.6
7.4 9.3 2.3 Il.4 3.9
1.3 during sample preparation. for selected ion monitoring. at 5% level.
Clirr-1,s
Figure 1 shows the calibration curves for five kinds of bile acids. The weight ratio of a component relative to the corresponding IS was plotted on the abscissa and the peak height ratio of the component to the IS was plotted on the ordinate. There were good linearities in the range of 20-400 pg for LCA, 0.05-l ng for UDCA, 0.2-4 ng for DCA, and OS- 10 ng for CDCA and CA. Figure 2 illustrates the representative selected ion recording of an authentic bile acid mixture. The four channels in the multiple ion detector were initially focused on ml: 4611465 for LCA and 5631568 for DCA. The monitored ions were changed to ml,: 4591463 for CDCA, 5631567 for UDCA, and 6651668 for CA. The gain in the amplifier for the recording of each endogenous compound was adjusted to IO-fold that for IS. Figure 3 shows the typical selected ion recording obtained by analyzing the liver extract. The peaks of LCA. DCA, CDCA, UDCA, and CA in the selected ion recording corresponded to approximately 0.04. 1.4, 7.0. 0.4. and 10.0 ng. respectively. Statistic& Analysis c?f‘Accuracy and Precision qf‘thr Present Method
The accuracy and precision of the present method as applied to the determination of
82
YANAGISAWA
ET AL.
TABLE RECOVERY
7
OF CONJUGATED
BILE
ACID
ADDED"
Sample
IX,, + ml Bile
acid"
(n = 0. I. 2. 3)
Amount (f&g
added liver)
Amount found t&g liver)
Recovery I%) + SD
0 0
0.165 0.133
0.171 0.149
0.163 0.150
0.089 0.089
0.250 0.218
0.220 0.201
0.244 0.227
80.5
k 20.2
Estimated value (,?,,I k confidence limit (95%) (&g/g liver)
0 178 -c 0.024
TLCA E Flx,,,
+ ?ri, I
0.178 0.178
0.321 0.315
0.314 0.327
0.314 0.314
91.3
i
3.0
G "IX,,,.
+ 30, I
0.267 0.267
0.361 0.384
0.409 0.372
0.394 0 377
85.3
t
6.3
0 0
1.56 I.62
1.78 1.98
1.72 1.78
0.52 0.52
2.02 2.02
2.14 1.05
2.40 2.15
76. I ? 28. I
1.03 1.03
2.66 2.66
2.67 2.64
2.55 2.63
86.9
ic
4.3
1.55 1.55
2.97 3.23
3.26 3.31
3.20 3.37
96.0
t
8.9
0 0
7.54 7.37
7.36 7.52
7.41 7.62
3.02 3.02
10.31 9.82
10.29 10.09
9.99 9.67
84.7
e
8.5
6.04 6.04
12.84 II.76
Il.50 II.85
12.43 12.46
77.3
e
8.5
9.06 9.06
15.22 IS.60
14.60 15.05
14.59 15.08
834+
A Blx,,,,) C Dw<,,,
+ (1111
IX,,,
= 0.1ss*
I.76
lO.l84P]
_t 0.26
TDCA
$x,,,,
+ 3a,,J
IX,,,,
= 1.74*
9.06
(7.06,**]
k 0.80
TCDCA E FlXm"
C DLL,
+ 2N< II)
+ (I,,)
IX,,
(8.86$'*j
4.2
0 0
0.158 0.156
0.166 0.171
0.144 0.165
0. I I I 0. I I I
0.260 0.241
0.253 0.250
0.249 0.258
82.8
t
6.2
0.222 0.222
0.387 0.354
0.353 0.335
0.370 0.345
88.9
t
8.4
0.333 0 333
0.471 0.507
0.475 0.453
0.469 0.481
94.9
t
5.3
0 0
x.42 8.93
8.76 8.48
8.42 8.99
0.162
t 0.019
TUDCA
c,(X,,, + ui 1
3.20 3 20
10.83 10.84
10.79 II.20
10.97 II.24
72 2 t
6.2
E Flx#,,
6.40 6.40
14.54 13.50
13.96 13.08
14.09 13.39
79 6 f
8.3
9.60 9.60
16.90 17.52
16.69 17.50
16.89 17.15
87.9
3.6
IX,,,
= 0.160*
9.69
(0.190)**1
5 0.92
TCA + ?U,)
2
IX<,,
= 8.67*
(10.28)'*]
MICROANALYSIS
OF
BILE TABLE
ACID
IN HUMAN
LIVER
7 (Conrinrccd)
Sample Bile
acid”
IX, + ml (II = 0. 1. 2. 3)
Amount C&g
added liver)
U 0
Amount found I @gig liver)
Recovery I’-‘:) + SD
7.54 7.37
7.36 7.52
7.41 7.62
5.10 5.10
11.60 11.2
II.88 IO.90
11.52 11.82
78.8
t
7.3
1,)
10.70 10.20
17.04 16.93
16.13 16.22
16.08 17.0x
89.5
2
4.5
+ 3ri’, 1,)
IS.30 15.30
20.02 19.03
20. I3 19.91
21.03 3l.05
82.1
r
4.1
A B lx’w,) I J IX,,,,,
+
K L IX,,,.,,
+
O’rn)
83
TISSUE
Estimated 2 confidence C&g
8.91
value limit liver)
(,?,I 195~6)
-c 0.x.5
GCDCA
M , N (X,,,,,
k’,
IX,..,>
= 7.47s
(8 86)*“]
jq = x4.3*** fl All amounts were calculated as their free form. b Abbreviations used: TLCA, taurolithocholic acid: TDCA. taurodeoxycholic acid; TCDCA, taurochenodeoxycholic TUDCA, tauroursodeoxycholic acid; TCA. taurocholic acid: GCDCA. glycochenodeoxycholic acid. * The mean ofthe total amounts ofindividual bile acid in original liver tissue without correction. ** The mean corrected by dividing x,, by I?. *** The mean ofrecoveries of individual bile acids added.
acid:
bile acids in human liver tissue were investiand significantly high error during sample gated. The liver specimen used was re- preparation in LCA may be due to the moved from the patient suffering from minute amount present in comparison to hepatolithiasis. The tissue seemed to be that of other bile acids and interference normal in appearance as well as in histology. from adjacent peaks which could not be The solubilized tissue was divided into 17 removed adequately by the present cleanaliquots equivalent to 25 mg each of fresh up procedure. For the recovery experiment, to three liver tissue. After the addition of IS, five samples were subjected to cleanup pro- groups of two samples each, known amounts cedure. The others were allotted to six of taurine-conjugated bile acids were added, groups of two each for the recovery experiand to the other three groups of two samples ment. SIM was carried out in triplicate each, glycochenodeoxycholic acid was for each sample. added. Table 7 shows analytical data, reReproducibilities were investigated by coveries of conjugated bile acids added, analyzing five samples in triplicate by SIM. estimated values of each endogenous bile The result was analyzed according to one- acid, and 95% confidence limits. The reby dividing the way layout (26), where the analytical errors covery was calculated were divided into two sources of sample amount recovered by that of bile acid added. preparation and measurement of SIM. The estimated values and their confidence Table 5 shows the analytical data and the limits were obtained as an index of precision analysis of variance in CDCA. For other according to the orthogonal polynomial bile acids, the same statistical analyses were equation (26). The relatively low recoveries done and the results are summarized in observed were thought to be attributable Table 6. Almost all of the variance in the to the incomplete hydrolysis of conjugated analysis of bile acids except LCA was bile acids in addition to the losses during thought to be attributable to the measurethe present cleanup procedure. As shown ment of SIM, because the errors during in this table, the recoveries for DCA and sample preparation were negligible. The UDCA improved with increases of the relatively large coefficient of variation (CV) amount added, indicating a small sample
84
YANAGISAWA
ET AL.
TABLE RECOVERY
Bile Lithocholic
acid
OF BILL
Amount C&g
Sample
acid-3-sulfate
Taurolithocholic acid-3-sulfate
Glycolithocholic acid-3-sulfate
ACID
8
SULFAI
added liver)
I E ADDED”
Amount (pig/g
found liver)
Recovery
(%)
+ SD
A B
I.526 1.526
1.328 i 0.038 (N : 3) 1.354 -+ 0.007 (?I = 3)
87.9 r
A B
2.126 2.126
1.580 + 0.026 1.496 + 0.006
(n = 3) (n = 3)
72.4 f 2.3 (86.1
k 2.7)*
A B
2.016 2.016
1.672 -t 0.035 (n = 3) 1.574 -t 0.026 (n = 3)
80.5 5 3.0 (95.9
+ 3.6)*
” All amounts were calculated as their free form. * Value corrected by the correction factor of 0.84.
size is partly responsible for low recovery. Considerably narrow ranges of confidence limits were suggestive of satisfactory precision of the present method even for bile acid in minute amounts. If a large amount of sample is used and analyzed by SIM, the accuracy and precision should be greatly improved. The recovery of all bile acids added ranged from 72.2 to 96.0% with a mean of 84.3%. The statistical analysis by one-way layout indicated no significant differences among individual bile acids. When the mean of original samples was
8. (see Table
B11.t Acius
IN HISTOI
OCKALLY
1.9
7)
divided by that of recoveries, 0.84, the value obtained coincided closely with the estimated value. These results seem to verify the adequacy of 0.84 as a correction factor. Since the value obtained by the present method seems to be underestimated as shown in Table 7, the amounts of bile acids in the human liver tissue should be corrected by dividing the value by the factor of 0.84. Thus. the present method permits analysis of each bile acid in minute amounts, i.e., 5-30 mg of liver tissue is satisfactory for the purpose.
TABLE
9 PROVEF~
NORMAL
L1w.R
‘I’lSSut
Bile acid\ (w’yg IlverY’
Patlent NO.
Age
Sex
I
16 4x 44 67 51 55 47 79 70 62
IM F M F M F F M M
3 4 s 6 7 x 9 IO
E CONJLIGA
Clinical dlagnocl, Ga,tnc Gartnc Gastric Gastric Gastric Gastric Gastric Gastric Gastric Gastric Mean
” Figures m parsntkes
LCA
ulcer ulcer ulcer cancer cancer cancer cancer cancer cancer cancer + SD
reprewnf
percentage
DCA
0 OS (0 2) I.22 (3.91 0.0s (0 2) 0.33 (0.9) 0 50 (1.X) 0.28 (1.2) 0.47 , 1.0) 0.15 (0.X1 0.69 12.3) 04?11 II
I.60 (7.51 7.35 123 51 0.83 (4.01 ? 24ll4.1) 3.18 ill.41 2.33 ,10.3) 9.54 (?O.Kl 3.3x 11X.0) 7.62 (23 I) 5.43 (13 71
0.42 t 0.35 (1.3 1 II)
4 65 z 2.x7 (14.8 t 6 9)
ofeach
CDCA X.64 X.1’) 9.89 15.78 12.81
140.6) (26 I) 147.7) (42.5) (46.0)
5 21 (23.1) 20.05 8 05 13.76 Ii.‘)8
(43.71 (42.X) (45.21 00.2)
Il.44 t 4.37 (3R.R + X.91
Me aud m total bde acid\ of liver.
LJDCA
CA
049 (2.31 0.57 (1 81 ?.16(104) 2.81 (7.61 0 57 (2.0) 0.88 I3 9) 1.08 12.4) 0.45 (2 4) 1.12 13.7) LOX I2 7)
IO 4’) 14.00 7 79 12.93 10.79 13.88 I4 71 6 76 7.22 20.76
(49.3) (44.7) I37 6) (34.91 13X.7) (61.51 (32 I, 136.0) (23.71 (52.31
1.12 IO.7X (3 9 e 2.X)
I I .Y3 f 4.27 (41.1 + 11.0,
Total 2, 27 31 33 20.72 37 w 27.X 22.58 45.85 1x.7’, 3u.41 79 70 2’3.56 4 X.62
MICROANALYSIS
OF BILE
ACID
Bile Acid Sulfate Conjugate As it was difficult to obtain the radioisotope-labeled bile acid sulfate conjugate, the recovery experiment for investigating the loss of the sulfate during the extraction procedure was carried out using the known amounts of TLCAS, GLCAS, or LCAS added to liver extract equivalent to 30 mg of fresh tissue. Prior to addition, the level of endogenous LCA in the liver tissue was determined by SIM. It was found to be 17.0 rig/g liver, which was negligible as compared to the amounts of bile acids added. Table 8 shows amounts of bile acid sulfate conjugates added and their recoveries. When the recoveries of TLCAS and GLCAS were corrected with the factor of 0.84. they were 86.2 and 95.855, respectively. Some losses found in Table 8 may have occurred during the Amberlite XAD-2 column chromatographic extraction and solvolysis. Preliminary experiments showed the amounts of bile acid sulfate conjugates present in histologically normal liver tissue were relatively low in comparison to the total amount of bile acids. Therefore, no correction was made for bile acid sulfate conjugate. The investigation on the sulfated bile acid present in human liver will be published elsewhere.
Table 9 summarizes the amounts of bile acids present in livers of 10 patients, confirmed to have no hepatobiliary abnormality by clinical and histological examinations. The mean and standard deviation of the total bile acids in normal liver tissue was 29.59 2 8.62 pgig liver. The relative compositions (5%) of primary bile acids, CDCA and CA, agreed well among the patients and were 38.8 2 8.9 and 41.1 ? 11.0, respectively. However, those of secondary bile acids differed considerably. The levels of CDCA and CA were comparable to those reported by Greim et 111. (13.15). Though the values obtained were the sum of bile
IN HUMAN
LIVER
TISSUE
85
acids present in the hepatocyte, serum contained in the specimen, and bile present in the bile canaliculi, the results would aid diagnosis df the disease state present. If a larger analytical error is allowed or the quantitation of only primary bile acids is required, it is possible to use smaller sample sizes, probably as little as I mg of liver tissue, i.e.. well within the range of needle biopsy specimens. The present method seems to open up a wider area of application such as profiling the bile acid composition of liver tissue and studing bile acid metabolism in various liver diseases where needle biopsy is indicated. ACKNOWLEDGMENTS The authors are indebted to Dr. W. Tanaka. Research Laboratories of Nippon Kayaku Company, for his encouragement throughout this work. to Dr. G. A. D. Haslewood. Biochemistry and Chemistry Department, Guy’s Hospital Medical School. London. Great Britain, for providing samples of sulfate esters of taurolithocholic and glycolithocholic acids. to Dr. R. H. Palmer. College of Physicians and Surgeons of Columbia University. Department of Medicine, New York. for sulfate ester of lithocholic acid. and to Research Laboratories of Tokyo Tanabe Company for deuterated ursodeoxycholic acid.
REFERENCES 1. Admirand. W. H.. and Small, D. M. (1968) .I. C/in. Iu1,csr. 47, 1043- 1052. 2. Vlahcevic. Z. R.. Bell. C. C.. Jr., Buhac. 1.. Farrar. J. T.. and Swell. L. (19701 G;trrtr.oc~rrftvdo~?. 59, 165- 173. 3. Meihoff. W. E.. and Kern. F.. Jr. (196X) .1. Cl;,,. In\~esr. 37, 26 I-267. 4. Hofmann. A. F. (1967) (;tl\tro~lt/(,~.~)l~,t?. 52, 751-7.57. 3. Donaldson, R. M.. Jr. t 1970) AC/I.LIII. Irlfcru. \Ic,c!. 16, 191-:I?. 6. Gracey. M. (1971) C;rrr 12. 403-410. 7. Hofmann, A. F. (1972, .4rc,/r. /rz/rrrr. .%lcjtl. 130, 597-60.5. 8. Makino. I.. Nakagawa, S.. and Mashimo. K. (1969) (;‘(~sfro(,nt(.r~/~h’?’ 56, lO33- 1039. 9. Heaton. K. W. (1972) Bile Salts in Health and Disease. pp. SX-81. Churchill Livingstone. Edinburgh/London. IO. Okishio. T., and Nair, P. P. i 1966) Bicrc$rt,,ni.t!r\ 5. 3663-3668.
86
YANAGISAWA
11. Okishio, T.. Nair, P. P.. and Gordon, M. (1967) Biochem. J. 102, 654-659. 12. Greim, H., Triilzsch, D., Roboz, J., Dressler. K., Czygan, P., Hutterer, F.. Schaffner, F., and Popper, H. (1972) Gastroenrerolog~ 63, 837-845. 13. Greim, H., Trtilzsch. D., Czygan, P., Rudick. .I.. Hutterer, F., Schaffner, F.. and Popper, H. (1972) Gastroenteroloyy 63. 846-850. 14. DuPont. J.. Oh, S. Y., Odeen, L. A.. and Geller. S. (1974) Lipids 9, 294-296. 15. Greim, H.. Czygan, P., Schaffner. F., and Popper, H. (1973) Biochem. Med. 8, 280-286. 16. Nishikawa, Y., Yamashita, K., Ishibashi, M.. and Miyazaki, H. (1978) Chem. Phurm. Bull. 26, 2922-2923. H., Ishibashi, M.. Itoh, M.. and 17. Miyazaki. Nambara. T. ( 1977) Biomed. Mass Sprctrom. 4, 23-35.
ET AL 18. Axelson, M., and Sjovall. J. (197615. Chromnfogr. 126. 705-716. 19. Hamilton, J. G. (1963) Arch. Biochem. Bi0ph.v.v. 101, 7- 13. 20. Ganshirt. H., Koss. F. W., and Morianz. K. ( 1960) Arzeim. Forsch. IO. 943-947. 21. Makino, I.. and Sjovall, J. (1972) Anal. 1x11. 5, 341-349. 22. Palmer, Rrs. 23. Ali.
R. H., and 12, 671-679.
Bolt,
M.
G. (1971)
S. S.. and Javitt, N. B. (1970) Biochem. 48, 1054- 1057.
.I. Lipid Cnntrd.
24. Smith, A. G., Harland, W. A., and Brooks, W. (1977) J. Chromurogr. 142, 533-547. 25. Miyazaki, (1978)
H., Ishibashi. Biomed. Ma,ss.
26. Taguchi. G. (1972) Co., Tokyo.
J. C. J.
M., and Yamashita. Specrrom. 5, 469-476.
Statistical
Analysis,
Maruzen
K.
BIOCHEMISTRY
104, 75-86
Microanalysis
(1980)
of Bile Acid in Human Liver Tissue by Selected Ion Monitoring
JIRO YANAGISAWA,* MASAHIRO IT0H.t MASATAKA ISHIBASHI,t HIROSHI MIYAZAKI,~ AND FUMIO NAKAYAMA* *Department
of
Surgery
I. Kyushu
Fduoka 812. Japan; Nippon Ka,vnku
University iResearch Compurly.
Received
Fcircult~ Loborcltorirs 3-31 Shimo.
August
of
Mrdicirrr,
3-l-I
~)~Phnrt~~acc,frtical Kita-Xu. Tokw I/5,
,Mnedashi.
Higashi-Xu.
Diriviorf. Japcrn
23. 1979
A method of microquantitative determination of bile acid in 5-30 mg of human liver tissue was developed. Bile acids were converted to their ethyl ester dimethylethylsilyl ether derivatives and analyzed by capillary gas chromatography-selected ion monitoring, using [‘H,]lithocholic (LCA), L*H,]deoxycholic (DCA). L”H,]chenodeoxycholic (CDCA). rLHI]ursodeoxycholic (UDCA). and [*H,]cholic (CA) acids as internal standards. Precision and reproducibility of the present method were tested using surgically obtained liver specimens. The results were statistically analyzed according to one-way layout and the orthogonal polynomial equation. Bile acids except LCA were determined with 2.3 to 11.4% of the coefficient of variation. Recoveries of conjugated bile acids ranged from 72.2 to 96.05 with a mean of 84.3%. The amount of bile acids present in histologically normal liver specimens (n = 10) was found to be 29.56 ? 8.62 &g liver. The relative compositions (r?) of CDCA and CA were 38.8 + 8.9 and 41.1 + 11.0. respectively.
The metabolism of bile acid has aroused much interest because of the recent recognition of its importance in bile secretion and intestinal absorption as well as in various disease states such as cholelithiasis (1,2), Crohn’s disease (3.41, bacterial overgrowth (5,6), ileal resection (7), and liver disease (8). Bile is present in wellconfined body compartments forming the enterohepatic circulation (9), i.e., liver, bile, intestinal tract, and portal blood. These compartments are relatively inaccessible to sampling. Bile requires duodenal intubation Portal blood is usually inaccessible. Intestinal content can be sampled in the form of feces but the bile acid present is a spillover of bile acid from intestinal content further modified by intestinal flora so extensively that it does not give much information on the bile acid present in enterohepatic circulation. Bile acids present in systemic circulation can be analyzed by
sampling venous blood but they are also spillover of bile acid brought to the liver via portal blood and far from representing those in the enterohepatic circulation. The liver has been relatively inaccessible to sampling. Needle biopsy is increasingly used for histological diagnosis of liver disease. However, the small amount of liver tissue obtainable limits the utility of the specimen for chemical analysis of its constitutent bile acid. Since the reports of cellular and subcellular bile acids in the rat liver by Okishio and co-workers (10,l l), several papers (12- 15) describing the analysis of bile acid in the human and rat livers have been published. In these studies, however, bile acid was identified mainly by means of gas chromatography, which lacks sensitivity and specificity for microanalysis of bile acid in complexed biological mixtures. The analysis of bile acid can be improved by the use of gas chromatography-mass spectrom75
0003-2697/80/070075-
12$02.00:0
Copyright r 1980 by Academic Pres\. Inc. All rights of reproduction m any form reserved.
76
YANAGISAWA
e&y-selected ion monitoring (gc-MS-SIM)’ with a wall-coated open tubular (WCOT) glass capillary column (16). The present study deals with the development of an analytical method for trace amounts of bile acids in biopsy specimens of human livers. MATERIALS
AND
METHODS
Lithocholic (LCA), deoxycholic (DCA), chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), and cholic (CA) acids and sodium salts of their taurine-conjugated bile acids and glycine-conjugated CDCA were purchased from Tokyo Kasei Kogyo Company, Tokyo, Japan, Applied Science Laboratories, Inc., State College, Pennsylvania, and Sigma Chemical Company Ltd., St. Louis, Missouri. Taurolithocholic acid3-sulfate (TLCAS) and glycolithocholic acid-3-sulfate (GLCAS) were kindly supplied by Dr. G. A. D. Haslewood and lithocholic acid-3-sulfate (LCAS) by Dr. R. H. Palmer. [24-‘“C]Cholic, glyco[G-“H]cholic, and [24-14C]taurocholic acids were obtained from New England Nuclear Corporation, Boston, Massachusetts. Their purities were checked by gas chromatography and thin-layer chromatography (see below). Solvents were of analytical grade and used without further purification. Pyridine was redistilled before use over P,O,. Trimethylsilyl imidazole (TMSI) was purchased from Tokyo Kasei Kogyo Company.Dimethylethylsilyl imidazole (DMESI) was prepared in our laboratory by the ’ Abbreviations used: gc-MS-SIM. gas chromatography-mass spectrometry-selected ion moniwall-coated open tubular (glass toring: WCOT, capillary column): LCA, lithocholic acid; DCA. deoxycholic acid; CDCA, chenodeoxycholic acid; UDCA. ursodeoxycholic acid: CA, cholic acid: TLCAS. taurolithocholic acid-3-sulfate; GLCAS, glycolithocholic acid-3-sulfate; LCAS. lithocholic acid-3-sulfate: TMSI. trimethylsilyl imidarole; DMESI. dimethylethylsilyl imidazole: tic. thinlayer chromatography; LSC. liquid scintillation counting; IS, internal standard.
ET AL.
method described previously (17). [6,6,7,7‘H,]LCA, [6,6,7,7,8-“H,]DCA, [l I,1 1, 12, I2-?H,]CDCA, and [ 11,l 1, 12P-2H,,]CA were prepared in our laboratory (H. Miyazaki, M. Ishibashi, and M. Itoh, in preparation) and ]11,11,12.12-ZH,]UDCA was kindly supplied by Tokyo Tanabe Company, Tokyo, Japan. Amberlite XAD-2 resin was obtained from Rohm Haas Company, Philadelphia, Pennsylvania, and Sephadex LH20 (25-100 pm) from Pharmacia Fine Chemicals AB. Uppsala, Sweden. The former was washed prior to use according to the method of Axelson and Sjovall (18). Kieselgel 60 (70-230 mesh) was purchased from Merck, Darmstadt, West Germany. Thin-luyrr c.hrornatograpk~ (tic). Thinlayer chromatography was carried out using precoated plates with kieselgel 60 (Merck). Solvent systems used were isopropyl etherisooctane-acetic acid, 10:5:5 (v/v/v) (19). for unconjugated bile acids and n-butanolacetic acid-water, 10: 1: I (v/v/v) (20), for conjugated bile acids. Gas chromutogruphy (gci. A Shimazu GC-7AG gas chromatograph equipped with a flame ionization detector was employed. The column was 2.0 m x 2.5mm glass with 1.5%’ OV-101 (Ohio Valley Specialty Chemical Co.) on Gas-Chrom Q, 80-100 mesh (Applied Science Laboratories, Inc.). The temperature of the column oven was maintained at 275°C. The flow rate of the carrier gas (nitrogen) was 40 ml min’. The temperature of the injection port and detector were 300°C. Gus cllronlcrtoRrrtp~l~-tncrss rtry -.sclrctrd iota monitoritlK.
spcctrotw
An LKB2091 gc-MS system equipped with a data processing system (Shimazu GCMSPAC90) and Van den Berg’s solventless injector was employed. The column used was WCOT, 25 m x 0.35 mm i.d., coated with SE-30 (LKB-Producktor, Stockholm, Sweden). The operating conditions were injection port temperature 285”C, column oven temperature 270°C. separator temperature
MICROANALYSIS
OF BILE
ACID TABLE
MASS
SPECTROMETRIC
DATA
OF THE DMES
ETHER
IN
HUMAN
M,
Lithocholic acid Deoxycholic acid Chenodeoxycholic acid Ursodeoxycholic acid Cholic acid
490 592 592 592 694
IMI:
[M-IS]
2.1 0.1 0.1 0.2 0.1
5.9 6.3 0.1 5.8 5.3
27o”C, ionization source temperature 290°C. flow rate of helium carrier gas I .5 ml min-‘. ionization energy 22.5 eV, acceleration voltage 3.5 kV, and trap current 100 PA. SIM was carried out using a multiple ion detector. Liyuid scintillation counting (LSC). The accuracy and reproducibility of the sample preparation procedure was checked by counting the radioactivity of the sample before and after the procedure in the scintillation cocktail (Aquasol 2, NEN). LSC was performed with a Packard liquid scintillation counter, model 3380, equipped with AAA. Liver specimens. Surgical biopsies were performed during laparotomy at the Department of Surgery 1, Kyusyu University Faculty of Medicine, for histological examination. A part of the liver tissue was used in the present investigation. Cleunup procedure. Five to 30 mg of liver tissue was rinsed with ice-cold saline, briefly dried on a filter paper, and weighed. After the addition of adequate amounts of L2H4]LCA, I’H,]DCA, [‘HJCDCA, [‘HJUDCA, and [“H,,]CA as internal standards (IS). the tissue was solubilized with 1 ml of 5%’ aqueous sodium hydroxide solution at 70-8O”C, diluted with 9 ml of distilled water, and passed through a column (8 x 120 mm) of the XAD-2 resin (21). After washing with water until neutral, the bile salts were eluted with 15 ml of ethanol. After evaporation under reduced pressure, the residue was subjected to solvolysis
77
TISSUE
I DERIVATIVES Relative
Compound
LIVER
OF BILL intensity
ACID
Other 386 443 459 488 561
ESTERS
(%)
[M-29]’ 100.0 100.0 6.8 100.0 100.0
ETHYL
(57) (3) (76) (19) (67)
ions
257 (30) 384 (8, 385 (100) 459 (15) 383 (33)
215 255 255 383 253
(53) (60) (20) (24) (26)
according to the method described by Palmer and Bolt (22). After solvolysis the residue was dissolved in 5% aqueous sodium hydroxide solution. Hydrolysis of glycine and taurine conjugates was performed in a sealed glass tube at 120°C for 7 h. After acidification to pH 1 with concentrated hydrochloric acid, the bile acids were extracted three times with IO-ml volumes of ethyl acetate. The combined ethyl acetate extract was evaporated to dryness and the residue was derivatized to ethyl ester-DMES ether as described below. Deri\,utizcction. To each authentic bile acid or hydrolyzed samples, 0.5 ml of 5% (w/v) hydrogen chloride ethanol solution was added and allowed to stand for 60 min at room temperature. After evaporation under a stream of nitrogen, the residue was transferred onto a silica gel column (6 x 50 mm, silica gel 60) with a small volume of n-hexane-ethyl acetate, 9:l (v/v), mixture and washed with 10 ml of the solvent (23). The bile acid ethyl ester derivatives were eluted with 10 ml of ethermethanol, 9: 1 (v/v), mixture. After evaporation to dryness under reduced pressure, the residue was treated with 5’0 ~1 of DMESI and allowed to stand for 15 min. Excess silylating reagent was removed on a Sephadex LH-20 column (6 x 60 mm) prepared in the solvent system, n-hexane-chloroform-methanol, 10: 10: 1 (v/v/v), mixture (24). The DMES ether derivatives of bile acid ethyl esters were recovered in the first
78
YANAGISAWA
ET AL.
TABLE DEUTERIUM
CONTENTS
2
OF DEUTERIUM-LABELED
BILE
ACIDS
‘H contents Deuterium-labeled
bile acid
Measured
L’H,]Lithocholic acid I’HJDeoxycholic acid [‘H,]Chenodeoxycholic acid (‘H,JUrsodeoxycholic acid 1’HJCholic acid “Measured
as the methyl
ion”
(M-TMSOH]+ [M-2 x TMSOH]: [M-2 x TMSOH]? [M-2 x TMSOH]+ IM-2 x TMSOH]’ ester-TMS
ether
‘H”
‘H,
‘H,
‘H,,
YH,
-
-
16.0 -
71.0
12.9
-
6.1
0.1 -
0.4 2.2
38.6 79.5 77.0
55.3
0.3
0.5
1.4 3.0 24.4
16.6 19.4 73.4
RESULTS AND DISCUSSION Gas Chromatography -Mass Spectrometry
Gas chromatographic separation of bile acids on a glass capillary column was found to be superior to that on a packed column, even though various liquid phases and derivatives were available for the latter. The dimethylethylsilyl (DMES) ether derivatives of bile acid ethyl esters gave, as reported earlier (16), sharp, symmetrical peaks and eluted in regular order according to the number of hydroxyl groups. Furthermore, excellent resolution can be obtained using a glass capillary column coated with SE-30.
IN AMBERLITE
COLUMN
1.7 -
LIVER
-
Propriety of Deuterium-Lahelrd Compounds as Internal Standards
When [‘HJCDCA was used as an internal standard for the determination of bile acids in human liver, the analytical errors for UDCA and LCA were found to be large in comparison to those for CDCA and CA. This is probably due to an error
3
OF RADIOISOTOPE-LABEI.ED TO SOLUBILIZED HUMAN
XAD-2
‘H,
The mass spectrometric data of the DMES ether derivatives of bile acid ethyl esters are listed in Table 1. All bile acids except CDCA showed the characteristic fragment ions of [M-29]+ formed by the elimination of the ethyl group at the silicon atom, while the CDCA derivative yielded the [M-dimethylethylsilanol-29]+ ion as a prominent peak (25). The appearance of a prominent ion at the high mass region seems to be of great advantage in avoiding interference when biological materials are to be thus enhancing reliability in analyzed, quantitation.
TABLE RECOVERY ACID ADDED
-
‘H,
derivatives
2.5 ml of effluent. Solvent was evaporated to dryness under reduced pressure. The residue was redissolved in 5% (v/v) pyridine-n-hexane solution and injected to gc-MS.
TABLE
(%)
CHOLIC TISSUE
CHROMATOGRAPHY
Bile acid
Recovery (%) i SD (n = 3)
124-“C]Cholic acid [24-‘“ClTaurocholic acid Glyco[G-JH]cholic acid
95.6 2 1.2 96.5 ? 0.5 94.0 -e 2.9
COMPLETENESS LABELED
4
OF HYDROLYSIS OF RADIOISOTOPECONJUGATED CHOLIC ACID ADDED
Completeness of hydrolysis (%) i SD (n = 4)
Bile acid [24-‘“ClTaurocholic GlycolG-“Hlcholic
acid acid
94.0 IT 3.5 87.6 i- 7.0
MICROANALYSIS
0.01
0.03
OF BILE
ACID
IN HUMAN
LIVER
79
TISSUE
0.05
O.liJ
I 0
I 1.0
I ?.O
I
LTR '
(.n-"'rJ)
40
I 0
I 1 0
1 2.0
1 4.0
DCA
(
I
I 0.5
1 LOCAL 1 .I!
b
0.25
I 0
0.25
I 0
Cl.?5
I
0 20
,j)
1 UDCA(.lO-' 1.0
q)
I
1 CP 1.0
q)
I 0.5 of
.lil-"
31
I 05
Fmounts
1O-9
blip
( 10-B
ac>di
FIG. I _ Calibration curves of bile acids. The weight ratio of a component relative to the corresponding deuterated internal standard is plotted on the abscissa and the peak height ratio of the component to the internal standard is plotted on the ordinate. (0) LCA (,,I/; 4611465): (r‘,) DCA (I>I/: 5631568): (A) CDCA (/n/z 4591463); (B) UDCA (rni; 5631567); (0) CA (r,r/,l 6651668).
in SIM as well as the difference in recovery of individual bile acids during the cleanup procedure. Thus, instead of a single IS, the five deuterium-labeled analogs corresponding to each bile acid were prepared and used as IS. The deuterium contents in these compounds were measured as methyl ester-TMS ether derivatives and are summarized in Table 2. No loss of deuterium atoms in these compounds was found to occur during alkaline hydrolysis, confirming that the deuterated bile acids can be added before the sample preparation.
The recovery of “H- or 13C-labeled bile acid added to the solubilized liver tissue corresponding to about 50 mg of fresh liver tissue was studied in the Amberlite XAD-2 coiumn chromatographic extraction procedure. Triplicate runs with 0.1 FCi of ‘%or 0.5 $Zi of “H-labeled bile acid were made. The radioactivity was measured with the liquid scintillation counter before and after Amberlite XAD-2 column chromatography. As shown in Table 3, taurine- and glycineconjugated and unconjugated CA were quantitatively recovered. Radioisotope-
80
YANAGISAWA
ET AL (e)
[‘I 567J
568
I\
..,
j
-'
I
1
I
I
0
5
10
15
RETENTION
TlME(min)
FIG. 2. Selected ion recording of an authentic mixture of the DMES ether derivatives of LCA (a), DCA (b), CDCA (c), UDCA (d). and CA (e) ethyl esters. Monitoring ions were selected as follows: m/z 4611465 for LCA/]‘H,]LCA, 5631568 for DCA/]‘H,]DCA, 4591463 for CDCA/]“H1]CDCA, 5631567 for UDCA/]?H,]UDCA. and 6651668 for CA/(‘H:,]CA. Monitoring ions were changed at the positions indicated by the asterisks.
labeled compounds added to human liver were subjected to alkaline hydrolysis. After neutralization, an aliquot was applied to a
silica gel G plate (0.25 mm thick, Merck) and developed with isopropyl ether-isooctane-acetic acid, 10:5:5 (v/v/v). The
1
,
I
I
0
5
10
15
RETENTION FIG. 3. Selected ion recording from human liver tissue (Patient
TiME(mio)
of the DMES ether 1. Table 9): (a) LCA,
derivatives (b) DCA,
of bile acid ethyl esters in extract (c) CDCA, (d) UDCA. and (e) CA.
MICROANALYSIS
OF
BILE
ACID
completeness of hydrolysis was calculated from the radioactivity of unconjugated bile acid. As shown in Table 4, hydrolysis was found to be fairly complete, i.e., up to 87%. Consequently, these results suggested that these deuterium-labeled unconjugated bile acids could be used as convenient IS for the determination of conjugated bile acids in the present procedure, as the degree of hydrolysis could be compensated. Since only a minute amount of nonlabeled component was found to be present in the deuterated bile acids used, i.e., 0.3% these compounds may be added to approximately 10 times the bile acids of interest as IS and carriers. TABLE REYRODUCIBILITY:
5
CHENODEOXYCHOLIC
LEVELS
IN HLIMAN I. ANAL
LIVER
YTITAI.
ACID
TISSUE
DATA
Found
Sample
t&g
A B C D E
7.54 7.81 7.51 7.63 7.37 II.
liver) 7.36 7.68 7.81 7.48 7.52
ANALYSIS
7.41 7.34 7.15 7.21 7.62 or
Mean
2 SD
7.44 7.61 7.69 7.44 7.50
2 O.OY it 0.24 ? 0.16 ? 0.21 -c_ 0.13
VARIANCE
Source
L’
Sample preparation Error (selected ion monitoring)
0. II306
“’4
0.037015
0.30774
10
0.030774
Total
0.45580
14
10, 0.05)
= 3.478
P(4. S: residual ,f‘: number
Fu 1.203
sum of squares of degrees of freedom,
.f, : .fm?,l<.*wpam~,,m .f; : .f,,r,,, V: unbiased variance F,,: observed value following F distribution variance ratio ( Vsam,~, PrPPalatlWli V,,,,,, ) F(j’, J;,(Y): Density function of F distribution withf’, and fi degrees of freedom
IN HUMAN
LIVER
TABLE C~EFFICIENTOF
VARIATION
AND
SELECTED
Bile acid Lithocholic acid Deoxycholic acid Chenodeoxycholic acid Ursodeoxycholic acid Cholic acid ” Coefficient ’ Coefficient * Statistically
Calihrtrtion
81
TISSUE
of variation of variation significant
6 IN SAMPLE
ION
PREPARATION
MONITORING
CV;,” (‘V )
CV,.” (7)
12.1” I.5 0.6
7.4 9.3 2.3 Il.4 3.9
1.3 during sample preparation. for selected ion monitoring. at 5% level.
Clirr-1,s
Figure 1 shows the calibration curves for five kinds of bile acids. The weight ratio of a component relative to the corresponding IS was plotted on the abscissa and the peak height ratio of the component to the IS was plotted on the ordinate. There were good linearities in the range of 20-400 pg for LCA, 0.05-l ng for UDCA, 0.2-4 ng for DCA, and OS- 10 ng for CDCA and CA. Figure 2 illustrates the representative selected ion recording of an authentic bile acid mixture. The four channels in the multiple ion detector were initially focused on ml: 4611465 for LCA and 5631568 for DCA. The monitored ions were changed to ml,: 4591463 for CDCA, 5631567 for UDCA, and 6651668 for CA. The gain in the amplifier for the recording of each endogenous compound was adjusted to IO-fold that for IS. Figure 3 shows the typical selected ion recording obtained by analyzing the liver extract. The peaks of LCA. DCA, CDCA, UDCA, and CA in the selected ion recording corresponded to approximately 0.04. 1.4, 7.0. 0.4. and 10.0 ng. respectively. Statistic& Analysis c?f‘Accuracy and Precision qf‘thr Present Method
The accuracy and precision of the present method as applied to the determination of
82
YANAGISAWA
ET AL.
TABLE RECOVERY
7
OF CONJUGATED
BILE
ACID
ADDED"
Sample
IX,, + ml Bile
acid"
(n = 0. I. 2. 3)
Amount (f&g
added liver)
Amount found t&g liver)
Recovery I%) + SD
0 0
0.165 0.133
0.171 0.149
0.163 0.150
0.089 0.089
0.250 0.218
0.220 0.201
0.244 0.227
80.5
k 20.2
Estimated value (,?,,I k confidence limit (95%) (&g/g liver)
0 178 -c 0.024
TLCA E Flx,,,
+ ?ri, I
0.178 0.178
0.321 0.315
0.314 0.327
0.314 0.314
91.3
i
3.0
G "IX,,,.
+ 30, I
0.267 0.267
0.361 0.384
0.409 0.372
0.394 0 377
85.3
t
6.3
0 0
1.56 I.62
1.78 1.98
1.72 1.78
0.52 0.52
2.02 2.02
2.14 1.05
2.40 2.15
76. I ? 28. I
1.03 1.03
2.66 2.66
2.67 2.64
2.55 2.63
86.9
ic
4.3
1.55 1.55
2.97 3.23
3.26 3.31
3.20 3.37
96.0
t
8.9
0 0
7.54 7.37
7.36 7.52
7.41 7.62
3.02 3.02
10.31 9.82
10.29 10.09
9.99 9.67
84.7
e
8.5
6.04 6.04
12.84 II.76
Il.50 II.85
12.43 12.46
77.3
e
8.5
9.06 9.06
15.22 IS.60
14.60 15.05
14.59 15.08
834+
A Blx,,,,) C Dw<,,,
+ (1111
IX,,,
= 0.1ss*
I.76
lO.l84P]
_t 0.26
TDCA
$x,,,,
+ 3a,,J
IX,,,,
= 1.74*
9.06
(7.06,**]
k 0.80
TCDCA E FlXm"
C DLL,
+ 2N< II)
+ (I,,)
IX,,
(8.86$'*j
4.2
0 0
0.158 0.156
0.166 0.171
0.144 0.165
0. I I I 0. I I I
0.260 0.241
0.253 0.250
0.249 0.258
82.8
t
6.2
0.222 0.222
0.387 0.354
0.353 0.335
0.370 0.345
88.9
t
8.4
0.333 0 333
0.471 0.507
0.475 0.453
0.469 0.481
94.9
t
5.3
0 0
x.42 8.93
8.76 8.48
8.42 8.99
0.162
t 0.019
TUDCA
c,(X,,, + ui 1
3.20 3 20
10.83 10.84
10.79 II.20
10.97 II.24
72 2 t
6.2
E Flx#,,
6.40 6.40
14.54 13.50
13.96 13.08
14.09 13.39
79 6 f
8.3
9.60 9.60
16.90 17.52
16.69 17.50
16.89 17.15
87.9
3.6
IX,,,
= 0.160*
9.69
(0.190)**1
5 0.92
TCA + ?U,)
2
IX<,,
= 8.67*
(10.28)'*]
MICROANALYSIS
OF
BILE TABLE
ACID
IN HUMAN
LIVER
7 (Conrinrccd)
Sample Bile
acid”
IX, + ml (II = 0. 1. 2. 3)
Amount C&g
added liver)
U 0
Amount found I @gig liver)
Recovery I’-‘:) + SD
7.54 7.37
7.36 7.52
7.41 7.62
5.10 5.10
11.60 11.2
II.88 IO.90
11.52 11.82
78.8
t
7.3
1,)
10.70 10.20
17.04 16.93
16.13 16.22
16.08 17.0x
89.5
2
4.5
+ 3ri’, 1,)
IS.30 15.30
20.02 19.03
20. I3 19.91
21.03 3l.05
82.1
r
4.1
A B lx’w,) I J IX,,,,,
+
K L IX,,,.,,
+
O’rn)
83
TISSUE
Estimated 2 confidence C&g
8.91
value limit liver)
(,?,I 195~6)
-c 0.x.5
GCDCA
M , N (X,,,,,
k’,
IX,..,>
= 7.47s
(8 86)*“]
jq = x4.3*** fl All amounts were calculated as their free form. b Abbreviations used: TLCA, taurolithocholic acid: TDCA. taurodeoxycholic acid; TCDCA, taurochenodeoxycholic TUDCA, tauroursodeoxycholic acid; TCA. taurocholic acid: GCDCA. glycochenodeoxycholic acid. * The mean ofthe total amounts ofindividual bile acid in original liver tissue without correction. ** The mean corrected by dividing x,, by I?. *** The mean ofrecoveries of individual bile acids added.
acid:
bile acids in human liver tissue were investiand significantly high error during sample gated. The liver specimen used was re- preparation in LCA may be due to the moved from the patient suffering from minute amount present in comparison to hepatolithiasis. The tissue seemed to be that of other bile acids and interference normal in appearance as well as in histology. from adjacent peaks which could not be The solubilized tissue was divided into 17 removed adequately by the present cleanaliquots equivalent to 25 mg each of fresh up procedure. For the recovery experiment, to three liver tissue. After the addition of IS, five samples were subjected to cleanup pro- groups of two samples each, known amounts cedure. The others were allotted to six of taurine-conjugated bile acids were added, groups of two each for the recovery experiand to the other three groups of two samples ment. SIM was carried out in triplicate each, glycochenodeoxycholic acid was for each sample. added. Table 7 shows analytical data, reReproducibilities were investigated by coveries of conjugated bile acids added, analyzing five samples in triplicate by SIM. estimated values of each endogenous bile The result was analyzed according to one- acid, and 95% confidence limits. The reby dividing the way layout (26), where the analytical errors covery was calculated were divided into two sources of sample amount recovered by that of bile acid added. preparation and measurement of SIM. The estimated values and their confidence Table 5 shows the analytical data and the limits were obtained as an index of precision analysis of variance in CDCA. For other according to the orthogonal polynomial bile acids, the same statistical analyses were equation (26). The relatively low recoveries done and the results are summarized in observed were thought to be attributable Table 6. Almost all of the variance in the to the incomplete hydrolysis of conjugated analysis of bile acids except LCA was bile acids in addition to the losses during thought to be attributable to the measurethe present cleanup procedure. As shown ment of SIM, because the errors during in this table, the recoveries for DCA and sample preparation were negligible. The UDCA improved with increases of the relatively large coefficient of variation (CV) amount added, indicating a small sample
84
YANAGISAWA
ET AL.
TABLE RECOVERY
Bile Lithocholic
acid
OF BILL
Amount C&g
Sample
acid-3-sulfate
Taurolithocholic acid-3-sulfate
Glycolithocholic acid-3-sulfate
ACID
8
SULFAI
added liver)
I E ADDED”
Amount (pig/g
found liver)
Recovery
(%)
+ SD
A B
I.526 1.526
1.328 i 0.038 (N : 3) 1.354 -+ 0.007 (?I = 3)
87.9 r
A B
2.126 2.126
1.580 + 0.026 1.496 + 0.006
(n = 3) (n = 3)
72.4 f 2.3 (86.1
k 2.7)*
A B
2.016 2.016
1.672 -t 0.035 (n = 3) 1.574 -t 0.026 (n = 3)
80.5 5 3.0 (95.9
+ 3.6)*
” All amounts were calculated as their free form. * Value corrected by the correction factor of 0.84.
size is partly responsible for low recovery. Considerably narrow ranges of confidence limits were suggestive of satisfactory precision of the present method even for bile acid in minute amounts. If a large amount of sample is used and analyzed by SIM, the accuracy and precision should be greatly improved. The recovery of all bile acids added ranged from 72.2 to 96.0% with a mean of 84.3%. The statistical analysis by one-way layout indicated no significant differences among individual bile acids. When the mean of original samples was
8. (see Table
B11.t Acius
IN HISTOI
OCKALLY
1.9
7)
divided by that of recoveries, 0.84, the value obtained coincided closely with the estimated value. These results seem to verify the adequacy of 0.84 as a correction factor. Since the value obtained by the present method seems to be underestimated as shown in Table 7, the amounts of bile acids in the human liver tissue should be corrected by dividing the value by the factor of 0.84. Thus. the present method permits analysis of each bile acid in minute amounts, i.e., 5-30 mg of liver tissue is satisfactory for the purpose.
TABLE
9 PROVEF~
NORMAL
L1w.R
‘I’lSSut
Bile acid\ (w’yg IlverY’
Patlent NO.
Age
Sex
I
16 4x 44 67 51 55 47 79 70 62
IM F M F M F F M M
3 4 s 6 7 x 9 IO
E CONJLIGA
Clinical dlagnocl, Ga,tnc Gartnc Gastric Gastric Gastric Gastric Gastric Gastric Gastric Gastric Mean
” Figures m parsntkes
LCA
ulcer ulcer ulcer cancer cancer cancer cancer cancer cancer cancer + SD
reprewnf
percentage
DCA
0 OS (0 2) I.22 (3.91 0.0s (0 2) 0.33 (0.9) 0 50 (1.X) 0.28 (1.2) 0.47 , 1.0) 0.15 (0.X1 0.69 12.3) 04?11 II
I.60 (7.51 7.35 123 51 0.83 (4.01 ? 24ll4.1) 3.18 ill.41 2.33 ,10.3) 9.54 (?O.Kl 3.3x 11X.0) 7.62 (23 I) 5.43 (13 71
0.42 t 0.35 (1.3 1 II)
4 65 z 2.x7 (14.8 t 6 9)
ofeach
CDCA X.64 X.1’) 9.89 15.78 12.81
140.6) (26 I) 147.7) (42.5) (46.0)
5 21 (23.1) 20.05 8 05 13.76 Ii.‘)8
(43.71 (42.X) (45.21 00.2)
Il.44 t 4.37 (3R.R + X.91
Me aud m total bde acid\ of liver.
LJDCA
CA
049 (2.31 0.57 (1 81 ?.16(104) 2.81 (7.61 0 57 (2.0) 0.88 I3 9) 1.08 12.4) 0.45 (2 4) 1.12 13.7) LOX I2 7)
IO 4’) 14.00 7 79 12.93 10.79 13.88 I4 71 6 76 7.22 20.76
(49.3) (44.7) I37 6) (34.91 13X.7) (61.51 (32 I, 136.0) (23.71 (52.31
1.12 IO.7X (3 9 e 2.X)
I I .Y3 f 4.27 (41.1 + 11.0,
Total 2, 27 31 33 20.72 37 w 27.X 22.58 45.85 1x.7’, 3u.41 79 70 2’3.56 4 X.62
MICROANALYSIS
OF BILE
ACID
Bile Acid Sulfate Conjugate As it was difficult to obtain the radioisotope-labeled bile acid sulfate conjugate, the recovery experiment for investigating the loss of the sulfate during the extraction procedure was carried out using the known amounts of TLCAS, GLCAS, or LCAS added to liver extract equivalent to 30 mg of fresh tissue. Prior to addition, the level of endogenous LCA in the liver tissue was determined by SIM. It was found to be 17.0 rig/g liver, which was negligible as compared to the amounts of bile acids added. Table 8 shows amounts of bile acid sulfate conjugates added and their recoveries. When the recoveries of TLCAS and GLCAS were corrected with the factor of 0.84. they were 86.2 and 95.855, respectively. Some losses found in Table 8 may have occurred during the Amberlite XAD-2 column chromatographic extraction and solvolysis. Preliminary experiments showed the amounts of bile acid sulfate conjugates present in histologically normal liver tissue were relatively low in comparison to the total amount of bile acids. Therefore, no correction was made for bile acid sulfate conjugate. The investigation on the sulfated bile acid present in human liver will be published elsewhere.
Table 9 summarizes the amounts of bile acids present in livers of 10 patients, confirmed to have no hepatobiliary abnormality by clinical and histological examinations. The mean and standard deviation of the total bile acids in normal liver tissue was 29.59 2 8.62 pgig liver. The relative compositions (5%) of primary bile acids, CDCA and CA, agreed well among the patients and were 38.8 2 8.9 and 41.1 ? 11.0, respectively. However, those of secondary bile acids differed considerably. The levels of CDCA and CA were comparable to those reported by Greim et 111. (13.15). Though the values obtained were the sum of bile
IN HUMAN
LIVER
TISSUE
85
acids present in the hepatocyte, serum contained in the specimen, and bile present in the bile canaliculi, the results would aid diagnosis df the disease state present. If a larger analytical error is allowed or the quantitation of only primary bile acids is required, it is possible to use smaller sample sizes, probably as little as I mg of liver tissue, i.e.. well within the range of needle biopsy specimens. The present method seems to open up a wider area of application such as profiling the bile acid composition of liver tissue and studing bile acid metabolism in various liver diseases where needle biopsy is indicated. ACKNOWLEDGMENTS The authors are indebted to Dr. W. Tanaka. Research Laboratories of Nippon Kayaku Company, for his encouragement throughout this work. to Dr. G. A. D. Haslewood. Biochemistry and Chemistry Department, Guy’s Hospital Medical School. London. Great Britain, for providing samples of sulfate esters of taurolithocholic and glycolithocholic acids. to Dr. R. H. Palmer. College of Physicians and Surgeons of Columbia University. Department of Medicine, New York. for sulfate ester of lithocholic acid. and to Research Laboratories of Tokyo Tanabe Company for deuterated ursodeoxycholic acid.
REFERENCES 1. Admirand. W. H.. and Small, D. M. (1968) .I. C/in. Iu1,csr. 47, 1043- 1052. 2. Vlahcevic. Z. R.. Bell. C. C.. Jr., Buhac. 1.. Farrar. J. T.. and Swell. L. (19701 G;trrtr.oc~rrftvdo~?. 59, 165- 173. 3. Meihoff. W. E.. and Kern. F.. Jr. (196X) .1. Cl;,,. In\~esr. 37, 26 I-267. 4. Hofmann. A. F. (1967) (;tl\tro~lt/(,~.~)l~,t?. 52, 751-7.57. 3. Donaldson, R. M.. Jr. t 1970) AC/I.LIII. Irlfcru. \Ic,c!. 16, 191-:I?. 6. Gracey. M. (1971) C;rrr 12. 403-410. 7. Hofmann, A. F. (1972, .4rc,/r. /rz/rrrr. .%lcjtl. 130, 597-60.5. 8. Makino. I.. Nakagawa, S.. and Mashimo. K. (1969) (;‘(~sfro(,nt(.r~/~h’?’ 56, lO33- 1039. 9. Heaton. K. W. (1972) Bile Salts in Health and Disease. pp. SX-81. Churchill Livingstone. Edinburgh/London. IO. Okishio. T., and Nair, P. P. i 1966) Bicrc$rt,,ni.t!r\ 5. 3663-3668.
86
YANAGISAWA
11. Okishio, T.. Nair, P. P.. and Gordon, M. (1967) Biochem. J. 102, 654-659. 12. Greim, H., Triilzsch, D., Roboz, J., Dressler. K., Czygan, P., Hutterer, F.. Schaffner, F., and Popper, H. (1972) Gastroenrerolog~ 63, 837-845. 13. Greim, H., Trtilzsch. D., Czygan, P., Rudick. .I.. Hutterer, F., Schaffner, F.. and Popper, H. (1972) Gastroenteroloyy 63. 846-850. 14. DuPont. J.. Oh, S. Y., Odeen, L. A.. and Geller. S. (1974) Lipids 9, 294-296. 15. Greim, H.. Czygan, P., Schaffner. F., and Popper, H. (1973) Biochem. Med. 8, 280-286. 16. Nishikawa, Y., Yamashita, K., Ishibashi, M.. and Miyazaki, H. (1978) Chem. Phurm. Bull. 26, 2922-2923. H., Ishibashi, M.. Itoh, M.. and 17. Miyazaki. Nambara. T. ( 1977) Biomed. Mass Sprctrom. 4, 23-35.
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R. H., and 12, 671-679.
Bolt,
M.
G. (1971)
S. S.. and Javitt, N. B. (1970) Biochem. 48, 1054- 1057.
.I. Lipid Cnntrd.
24. Smith, A. G., Harland, W. A., and Brooks, W. (1977) J. Chromurogr. 142, 533-547. 25. Miyazaki, (1978)
H., Ishibashi. Biomed. Ma,ss.
26. Taguchi. G. (1972) Co., Tokyo.
J. C. J.
M., and Yamashita. Specrrom. 5, 469-476.
Statistical
Analysis,
Maruzen
K.