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Conjugated and Unconjugated Serum Bile Acid Levels in Patients with Hepatobiliary Diseases
Vol. 56, No.6
GASTRO ENTEROLOGY
Copyright
© 1969 by The Williams
Printed in U.S .A .
& Wilkins Co.
CONJUGATED AND UNCONJUGATED SERUM BILE ACID LEVELS IN PATIENTS WITH HEPATOBILIARY DISEASES ISAO MAKINO,
M . D.; SHOICHI NAKAGAWA,
M .D ., AND KEIMEI MASHIMO, M.D.
Second Department of M edicine , Hokkaido University School of Medicine, Sapporo , Japan
Total and unconjugated bile acid levels in serum of patients with various hepatobiliary diseases were determined by gas-liquid chromatography. In normal serum, ranges of total serum bile acid were found as follows : deoxycholic acid 0.22 ± 0.14, chenodeoxycholic acid 0.23 ± 0.14, cholic acid 0.14 ± 0.09, and total amount of bile acid 0.59 ± 0.31 J,lg per ml. The cholic to chenodeoxycholic acid ratio (C: CDC) was 0.61 ± 0.30. In hepatobiliary diseases, the level of to·· tal serum bile acid rose. The increase was remarkable in acute early hepatitis and in obstructive jaundice but moderate or slight in active portal cirrhosis and chronic hepatitis. Changes in the composition of serum bile acid were also observed. The C: CDC ratio was 1.01 ± 0.43 in portal cirrhosis and 2.74 ± 1.42 in obstructive jaundice. A correlation was observed between the total serum bile acid level and the serum bilirubin concentration in obstructive jaundice and portal cirrhosis. Unconjugated serum bile acid levels in healthy persons showed a range of 0.26 ± 0.20 J,lg per ml; and the unconjugated to total bile acid ratio (U: T) was 51.4 ± 22.0%. In active portal cirrhosis, the amount of unconjugated bile acid increased (2.54 ± 1.83 J,lg per ml). The value was significantly higher than that in normal, chronic hepatitis, and obstructive jaundice groups. Contrary to portal cirrhosis, the whole increase in serum bile acid in obstructive jaundice was occupied by conjugated bile acid and the U: T ratio was very low (0.2 ± 0.4 %). A possible cause for the rising of the total and unconjugated serum bile acid levels in hepatobiliary diseases is discussed. Although it is known that the total serum bile acid increases in patients with hepatobiliary diseases,l -s little work has been done to study the state of conjugation in these diseases. 2 . 5, 9 In 1965, Sandberg et al. 5 reported a method for the determination of serum bile acid with gas-liquid chromatography (GLC). This method has sufficient sensiReceived March 7, 1968. Accepted January 6, 1969. Address requests for reprints to: Dr. Isao Makino, Second Department of Medicine, Hokkaido University School of Medicine, West 5, North 14, Sapporo, Japan.
tivity and specificity to estimate the very low level of bile acid in serum. This report describes a study of the change of conjugated and unconjugated serum bile acid in patients with various hepatobiliary diseases.
Materials and Methods Reagents. Amberlyst A 26 anion exchanger resin was purchased from Japan Organo Company, Japan. Alumina was obtained from Merck AG., Darmstadt, Germany. Standard bile acids were obtained from Calbiochem Corporation, Los Angeles, California. Gas-liquid chromatography. A gas-liquid chromatograph, model GC-2C (Shimazu Man-
1033
1034
MAKINO ET AL.
ufacturing Company, Kyoto, Japan), with a hydrogen flame detector was used. The column was a commercial preparation (Shimazu Manufacturing Company), of 1.5% QF-1 using Chromosorb W (60 to 80 mesh) as support in a glass "U" tube 1.5 in length and 4 mm in diameter. The temperature of the column was· 225 C, and nitrogen as carrier gas was passed at a pressure of 2.5 kg per cm z at the inlet. Procedure. Serum bile acids were determined by the procedure described by Sandberg et al. 5 Briefly, the procedure was as follows. A sample of serum (3 to 5 m\) was diluted with the same volume of distilled water and passed through a 5-ml column of the anion exchanger Amberlyst A 26. The adsorbed bile acid was eluted with 150 ml of 0.2 M (NH.hCU a in 80S; ethanol. The eluate was evaporated to dryness and then hydrolyzed in 20 ml of 7.5% NaOH for 4 hr in an autoclave at a pressure of 1.0 kg per cm z • After ether extraction of the acidified hydrolysate, the bile acid was methylated by diazomethane, purified on an alumina column, and trifluoracetylated with trifluoracetic anhydride. The excess reagents were removed under the nitrogen stream. The residue was dissolved in carbon disulfide and an aliquot was used for GLC. To analyze the unconjugated bile acid, the hydrolysis step was omitted. The eluate from the ion exchange column was evaporated, the residue was dissolved in water, and after acidification the bile acid was directly extracted, methylated, and trifluoracetylated. The purity of the solvents was tested because the bile acid level in normal serum was very low. The same volume of each solvent used in this procedure was evaporated to dryness, treated with diazomethane and trifluoracetic anhydride, and dissolved in carbon disulfide. No peaks could be seen except for a small solvent front at the full sensitivity setting. For recovery, 14C-Iabeled cholic acid was added to normal (n = 2) and pathological (n = 4) sera. The entire procedure, excepting GLC, was carried out and the radioactivity in one aliquot of the sample was determined in a gas-flow counter. The recovery ranged from 62.0 to 78.3 % (70.0 ± 6.1). Quantitative determination. Standard bile acids (lithocholic, deoxycholic, chenodeoxycholic, and cholic) were treated with diazomethane and trifluoracetic anhydride and applied to GLC. Each derivative showed only one peak. Under these experimental conditions, complete separation of the bile acids mixture was evident. The relative retention times were
Vol. 56, No.6
0.67 for lithocholic acid, 1.00 (15.3 min) for deoxycholic acid, 1.20 for chenodeoxycholic acid, and 1.75 for cholic acid. The peaks rose linearly when the trifluoracetate of methyl bile acid was injected into GLC in amounts ranging from 0.1 to 0.5 Ilg. Conjugated deoxycholic, chenodeoxycholic, and cholic acids purified by thin-layer chromatography were treated with diazomethane and trifluoracetic anhydride without hydrolysis, and injected into GLC. No peak of unconjuga ted bile acid was detected. Subjects. Twenty healthy persons and 81 patients with hepatobiliary diseases were used in this study. The control subjects were persons without hepatobiliary diseases selected from the medical staff and nurses of our clinic. Of the group of hepatobiliary patients, 22 had acute hepatitis at early stage; 30 had active portal cirrhosis (flocculation and transaminase tests indicated abnormal values); 18 had obstructive jaundice; and 11 had active chronic hepatitis (transaminase tests showed abnormal values). All were treated at the Department of Medicine, Hokkaido University Hospital. Diagnoses were made on the basis of clinical symptoms, liver function test, liver biopsy, and in some cases with laparotomy. Blood samples were obtained from the antecubital veins in the morning before breakfast after overnight fasting; separated sera were kept frozen at -20 C.
Results Total Serum Bile Acid Levels
Control subjects. In normal human serum, deoxycholic, chenodeoxycholic, and cholic acids are the major bile acids. The following ranges could be noticed: deoxycholic acid, 0.04 to 0.55 j.1.g per ml (0.22 ± 0.14); chenodeoxycholic acid, trace to 0.55 j.1.g per ml (0.23 ± 0.14); and cholic acid, 0.02 to 0.36 j.1.g per ml (0.14 ± 0.09). The total bile acid was 0.12 to 1.46 Ilg per ml (0.59 ± 0.31). The ratio of cholic acid to chenodeoxycholic acid (C: CDC) was 0.25 to 1.38 (0.61 ± 0.30) (tables 1 and 2). Acute hepatitis patients. In patients with acute hepatitis, the total serum bile acid level rose markedly at the early stage. In 1 it reached more than 160 j.1.g per ml, 100 times as high as the upper limit of normal values. In convalescence this bile acid level
June 1969 TABLE
1035
SERUM BILE ACID LEVELS 1. Total serum bile acid levels in normal subjects and in patients with liver diseases
.
Total serum bile acid level a
Liver function testb
No. of cases
-
DC
-
I
CDC
C
I
I
C:CDC
Total
I ratio
un1'ts
I"g/ml
Normal
20
Acute hepatitis (early) Chronic hepatitis (active) Portal cirrhosis (active) Obstruc t ive jaundice
22 11 30 18 1
0.22 ±0.14 1.20 ±1.15 0.77 ±0.69 0 .74 ±0.87 0.28 ±0 . 32
0.23 ±0.14 19.90 ±17.80 2.29 ±1.91 4.27 ±4.18 9.47 ±4.81
Kunkel
0.14 0 . 59 ±0 .09 ±0.31 26 . 81 47.98 ±25.83 ±37.78 1.91 4.97 ±2.09 ±4 .51 3.38 8.40 ±2.50 ±6.02 27.98 37.89 ±23.02 ±26.90
I T.Bilir.!
AI-Ph
--------- - -----
0.61 ±0.30 1.68 ±1.21 0.94 ±0.fi1 1.01 ±0.43 2.74 ±1.42
mg/ dl
I
SGOT
I SGPT
units
3- 10 0.3-1. 0 1 2 . 7- 10 5--40
3-35
296 8.9 12.0 I 15.1 ±4.5 ±8.4 ±9.7 ±146 5.6: 11.1 161 11.3 ±3.5 ±4.7 1 ±5 .9 ±98 1.8 18 .5 103 i3.5 ±5.1 ±2.7 I ±13 .3 ±95 8.4 16.3 1 41.fi 132 ±3.6 ±1D.0 ±23.1 ±83
220 ±128 168 ±118 53 ±46 97 ±50
- - -- - - - - - --
" DC, deoxycholic acid; CDC, chenodeoxycholic acid; C, cholic acid. b T.Bilir., total serum bilirubin concentratioll; AI-Ph , serum alkaline phosphatase (in King-Armstrong units); SGOT, serum glutamic oxalacetic transaminase; SGPT, serum glutamic pyruvic transaminase. 2. Statistical evaluation of total serum bile acid levels in normal subjects and in patients with liver diseases a
TABLE
I
Portal cirNormal rhasis
--------------------Total serum bile acid levels Portal cirrhosis .. Chronic hepatitis. Obstruction .. Acute hepatitis .... C:CDC ratios b Portal cirrhosis ... Chronic hepatitis ... Obstruction .. Acute hepatitis .....
*** *** *** ***
Chronic Obhepstrucatitis tion
--------
----
NS
100.0
r ~ 0.\ 1 p~0.02
~ 60.0
] 600 ';; 40.0
:=.
....
20.0
OL----------------------NS
*** ***
o
*** ***
NS
*** *** ***
e
NS
*** **
*** NS
*
a Data represent mean values plus or minus standard deviation. ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant.. b C, cholic acid; CDC, chenodeoxycholic acid.
soon dropped to the normal level. In these cases, chenodeoxycholic acid and cholic acid occupied a great part of the increased bile acids, while the increase of deoxycholic acid was not remarkable. The C: CDC ratio was variable in this disease. Chronic hepatitis patients. In patients with active chronic hepatitis, the increase
10.0 Tota I bili rubi n
20.0
(mgt dl)
30.0
FIG. 1. The correlation between total serum bile acid level and total serum bilirubin concentration in extrahepatic obstructive jaundice.
in bile acid was moderate or slight. The range was 0.90 to 18.25 Ilg per ml (4.97 ± 4.51). Portal cirrhosis patients. In patients with active portal cirrhosis, the total serum bile acid level ranged from 1.62 to 28.09 Ilg per ml (8.40 ± 6.02). The increase in chenodeoxycholic acid was more remarkable than that in other bile acids, and the C: CDC ratio was less than 1.5 (1.01 ± 0.43) in 26 of 30 patients. Obstructive jaundice patients. In obstructive jaundice the level of total bile acid was 9.07 to 109.49 Ilg per ml. (37.89 ± 26.90). A striking increase of cholic acid could be observed. The C: CDC ratio was
1036 TABLE
MAKINO ET AL.
Vol. 56, No.6
3. Unconjt.gated serum bile acid levels in normal subjects and in patients with hepatobilidry diseases
I Normal
No. of cases. ... 10 Total bile acid (lLg/ml) 0.50 ± 0 . 27 Unconjugate d bile acid ~g /ml) . ...... 0.26 ± 0.20 Un conjugated to total bile acid ratio (%) .. 51.4 ± 22 .0
Portal cirrhosis (active)
Hepatitis Obstruction Chronic (active)
Acute (early)
17 7.49 ± 5.53
11 4.97 ± 4.51
9 47.16 ± 27.69
11 48 .65 ± 26.85
2.54 ± 1.83
0.48 ± 0.30
1.52 ± 2. 29
0 . 14 ± 0.18
39.9 ± 24 . 9
15.6 ± 10.8
5.1 ± 7.4
0.2 ± 0 . 4
4. Statistical evaluation of unconjugated serum bile acid levels i n normal subjects and in patients with hepatobiliary diseases a
SERUM BILE lCIO
T A BLE
DI
5
10
(JJG 1M l) 15 20 0 .4 0.4
Portal Chronic ObNormal cirhepstrucrhasis atitis tion
0.5
-----------------1---- ---- ---- ---Unconjugated serum bile acid levels Portal cirrhosis ... . Chronic hepat itis ... Obstruction .... Acute hepatitis ..... U:T ratios b Portal cirrhosis .. . . Chronic hepatitis ... Obstruction Acute hepat itis.
:.:.:-:
*** NS NS NS NS
*** *** ***
** *** NS
** NS
NS
0-
~ ....
** *** ***
*** *
.:.;.;.;.;.;.
..... :-:.;.;.:-:-. :-:.... ;.:-:.:
***
Data represent mean values plus or minus standard devi ation. *** , P < 0.001; **, P < 0.01; *, P < 0.05; NS, not s ignificant. b Unco njugated to total bile acid ratio. a
greater than 1.5 (2.47 ± 1.42) in 17 of 18 patients. The total serum bilirubin concentration showed a close correlation to the serum bile acid levels in obstructive jaundice and portal cirrhosis patients. The coefficient of this correlation was 0.57 (P < 0.02) in obstructive jaundice (fig. 1) and 0.72 (P < 0.001) in portal cirrhosis. There was no correlation in acute hepatitis.
Unconjuated Serum Bile Acid Levels Control subjects. Unexpectedly, a relatively large amount of unconjugated serum bile acid was detected in normal serum. The values obtained from 10 healthy per-
16.5 18.9
0.9 0.9
16 .4
1. 4
83.9
0.9
13.1
FREE 61.8 TOTAl 168 % 60'. 5
0.6
....L 0 .9 CDC
36.4
2.2
1.3
1. 5
51.5
1.8
42.3
1.8
41.5
1 .0
11.9
2.0
0.5 BILIARY 6.5 I·········.,·, CON)
1.1
0 .6
1.0 0.9
_FREE
FIG. 2. Total and unconjugated serum bile acid levels in patients with active portal cirrhosis.
sons (including 4 women) ranged from 0.06 to 0.74 JIg per ml (0.26 ± 0.20) and the unconjugated to total bile acid ratio (U : T) was 51.4 ± 22.0% (tables 3 and 4). Portal cirrhosis patients. In 17 patients with active portal cirrhosis, the level of unconjugated bile acid ranged from 0.34 to 7.59 JIg per ml (2.54 ± 1.83). The unconjugated bile acid level of 14 of the 17 patients was greater than 1.0 JIg per ml and
June 1969
significantly higher than that of a normal group (P < 0.001), while the U: T ratio was 39.9 ± 24.9%, not statistically different from normal serum (fig . 2). In 2 cases of biliary cirrhosis, no increase in unconjugated bile acid was found (0.37 and 0.08 Ilg per ml) . The U: T ratios were 0.5 and 6.5%, respectively. Chronic hepatitis patients. In 11 patients with active chronic hepatitis, the range of
I
2
1037
SERUM BILE ACID LEVELS
SERUM BILE ACID 4 6
(VG/M L! 8 18
>-
;=
ga
""
SERUM BILE ACIP ( VG I ML) 20 40 60 80
.
20 0.9
l~~~~:r~~r:~~t~r::~~r~~r~::tt:~(~~~trlt:~~ 0:1]]]
7.0
6.5
1.1
kr~tt:rr~t~r:t:::~~rrr~~r~t:~~]
0
6.8
3.9
0.9
5~r~~~:~tt~~:r::~:~::~~:::~:~:::~::~~~:::~:i\1
0.2
4.0
11.0
0.2
~
rr:~:~~t~tt~~tt~r~r~::::}1
1.1
2.1
""
-...
~ttttttt~~~r::~:(tq
0.3
2 .0
~
{::::r::l:\.:l:· :::r::{t
,..=>
2i
_C_ O.2 CDC
>-
1.7
~
::0
>-
8 .4
'" ii
I
0.7
~
=>
C>
2 3 .2
§:!1
'"
0.6
"" ~
0 ""
'" ~
(.::::·:·:·:·:1 CONI
I 2 .6
1.3
21.8
0.4
34 .5
2,3
"" ""
0.1
2.0
FREE
~::::t:~:~:':::r::':~:::::1
Tom
rJ:::t·:·:tt::::1
%
C
0
CDC
0.1
2.6
LJ n. LJ 1:·:·:·:·:·:-:1 CONI
_FREE
3.0 1.9
r ::t:::::':::::J
~
FIG. 3. Total and unconjugated serum bile acid levels in patients with active chronic hepatitis.
TABLE
100
1.4
FREE 8.S TOTAL % 28.4
S
unconjugated bile acid levels was normal, 0.19 to 1.00 Ilg per ml (0.48 ± 0.30), i.e., under 1.0 Ilg per ml. The U: T ratio was 1.4 to 34.4% (15.6 ± 10.8), a significant difference from active portal cirrhosis (P < 0.01) was evident (fig. 3). Obstructive jaundice patients. The amount of unconjugated serum bile acid in 11 cases was trace to 0.62 Ilg per ml (0.14 ± 0.18), not significantly different from the normal group. However, unconjugated bile
_
o
1.6
o
1 .7
FREE
FIG. 4. Total and unconjugated serum bile acid levels in patients with obstructive jaundice.
5. S erum bile acid levels in portal blood obtained during operation" Total serum bile acid levels
Cases DC
I
CDC
I
C
I Total
Unconjugated serum bile acid levels /C:CDC
DC
",glml
No.1. (gastric cancer) ... Peripheral blood obtained simultaneously. . ' . No.2. (gastric cancer) ... Peripheral blood obtained simultaneously ... . ..
I
CDC
I
C
I Total
U:T
- -
%
",glml
0.06
0.99
1.84
2.89
1.83
0 .06
0 . 16
0.17
0.39
13. 5
0 . 12 0 .06
Trace 0 . 19
0 . 13 0.41
0 .25 0.66
2.16
0 .05
Trace
0.04
0.09
13.6
0 . 03
0 .01
0.04
0.08
4 .00
I
"DC, deoxycholic acid; CDC, chenodeoxycholic acid; C, cholic acid; U:T, unconjugated to total bile acid ratio.
1038
MAKINO ET AL.
acids could not be found in 5 cases in this group. Therefore, the increased bile acid values in obstructive jaundice patients were almost all the result of increased levels of conjugated bile acid; the U: T ratio was 0 to 1.1% (0.2 ± 0.4) (fig. 4). Acute hepatitis patients. Un conjugated serum bile acid levels in the 9 patients were variable. They ranged from 0.17 to 7.75 JLg per ml; the U: T ratio was 0.2 to 24.2% (5.1 ± 7.4).
Vol. 56, No.6
less than 1.0 in liver cirrhosis patients, and he regarded this index as a prognostic and diagnostic sign. Our result confirmed that the difference in the TBA: DBA ratio was not due to an increase or decrease in deoxycholic acid of the secondary bile acid. The C: CDC ratio was less than 1.5 in 26 of 30 patients with portal cirrhosis and more than 1.5 in 17 of 18 patients with obstructive jaundice. The C: CDC ratio in the normal group did not significantly differ from that of the patients with portal cirSerum Bile Acid Levels in Portal Blood rhosis. The portal blood obtained during operaThe rise of the serum bile acid level tion contained high levels of conjugated and some changes of bile acid observed in bile acid. The U: T ratio was 13.5 and hepatobiliary patients could be caused by 13.6%. The data showed that conjugated one or more of the following factors: 1. A fiow of bile acid from the bile into bile acid was reabsorbed more easily from intestine than was unconjugated bile acid blood as a result of the destruction of he(table 5). patic acini and intrahepatic biliary ·ducts. Osborn et al. 6 observed that the level of Discussion serum bile acid correlated with the serum The normal values of total serum bile total bilirubin concentration rather than acid determined in this study are similar to the concentration of serum glutamic to those reported by Sandberg et al. 5 oxalacetic transaminase or serum glutamic Iwata and Yamazaki!O described a higher pyruvic transaminase in obstructive jaunnormal bile acid level (2.20 ± 0.54 JLg per dice. This obseryation was confirmed in ml) obtained by their enzymatic method. these studies. Th\Is it seems that this is But their estimation might include sub- one of the important reasons for the ascent stances other than bile acid because they of the serum bile acid level in obstructive jaundice. used a crude enzyme preparation. 2. Quantitative and qualitative change In the hepatobiliary diseases the total serum bile acid level was increased. The in the synthesis of bile acid in the liver. elevations were the greatest in the early The change in the C: CDC ratio in hepatostage of acute hepatitis and in obstructive biliary diseases seems to be due to the jaundice. Slight or moderate elevation oc- changed metabolic pathway of bile acid. 3. A fiow of the reabsorbed bile acid into curred in portal cirrhosis and in chronic hepatitis patients. However, the total bile the blood of systemic circulation as the acid levels in each group overlap each result of a decreased removal by the liver. There have been few reports on unconanother markedly, so that the diagnostic value of the estimation of the total bile jugated bile acid levels in serum. Rudman acid level is questionable. The results are and Kendall, 2 who made an experiment similar to the values reported by Osborn with reverse phase chromatography, and et al.,6 Rudman and KendalV and Sandberg et al.,5 using gas-liquid chroCarey.! matography, reported the presence of unThe C: CDC ratio of bile acid was sig- conjugated bile acid in the sera of a small nificantly different in hepatobiliary dis- number of healthy persons and hepatoeases, especially in obstructive jaundice biliary patients, but no pathological and and portal cirrhosis. Carei reported that diagnostic significances have been disthe ratio of trihydroxycholanic to dihy- cussed. In healthy persons the unconjugated bile droxycholanic acid (TBA: DBA) was over 1.0 in obstructive jaundice patients and acid occupied approximately one-half of
June 1969
SERUM BILE ACID LEVELS
1039
the total bile acid in serum, but the ab- seems to be useful for the differential diagsolute amount of unconjugated bile acid nosis of portal cirrhosis, chronic hepatitis, was small. In obstructive jaundice pa- and obstructive jaundice. tients the conjugated bile acid increased REFERENCES markedly, but the unconjugated bile acid 1. Carey, J. B., Jr. 1958. The serum trihydroxychanged little, so that the U: T ratio was dihydroxy bile acid ratio in liver and biliary very low. On the contrary, in active cirtract disease. J. Clin. Invest. 37: 1494-1503. rhosis patients, the amount of unconju2. Rudman, D., and F. E. Kendall. 1957. Bile acid gated bile acid increased significantly and content of human serum. 1. Serum bile acids the total bile acid rose slightly or moderin patients with hepatic disease. J. Clin. In-' ately, so that the U: T ratio was signifivest. 36: 530- 537. cantly high. In some cases, the unconju3. Carey, J. B., Jr. 1961. Bile acids in the serum of gated bile acid occupied 80% of the total jaundiced patients. Gastroenterology 41: 285bile acid. In chronic hepatitis patients no 287. 4. Sherlock, S., and V. Walshe. 1948. Blood cholates increase in unconjugated bile acid was obin normal subjects and in liver disease. Clin. served. Sci. 6: 223-234. The amount of unconjugated bile acid 5. Sandberg, D. H., J . Sjovall, K. Sjovall, and D. A. was less than l. 0 f.Lg per ml in all 11 paTurner. 1965. Measurement of human serum tients with active chronic hepatitis (0.48 bile acids by gas-liquid chromatography. J. ± 0.30 f.Lg per ml) but over l.0 f.Lg per Lipid Res. 6: 182- 192. ml in 14 of 17 patients with active portal 6. Osobrn, E. C., I. D. P. Wootton, L. C. DeSilva, cirrhosis (2.54 ± 1.83 f.Lg per m!). The and S. Sherlock. 1959. Serum bile acid levels level of unconjugated serum bile acid inin liver disease. Lancet 2: 1049-1053. creased significantly in portal cirrhosis. 7. Carey, J. B. , Jr . 1956. Chenodeoxycholic acid in human blood serum. Science 123: 892. These data suggest that the conjugation 8. Sjovall, K., and J. Sjovall. 1966. Serum bile acid of bile acid with taurine or glycine was level in pregnancy with pruritus. Bile acids deficient owing to the reduction in the acand Steroid 158. Clin. Chim. Acta 13: 207-211. tivity of the conjugating enzyme system 9. Blum, M., and N . Spritz. 1966. The metabolism in portal cirrhosis. However, the degree of of intravenously injected isotopic cholic acid serum unconjugated bile acid level did not in Laennec's cirrhosis. J. Clin. Invest. 45: 187always correlate with the degree of clinical 193. symptoms. Investigators are now discussing 10. Iwata, T., and K. Yamazaki. 1964. Enzymatic the cases with a high level of serum undetermination and thin-layer chromatography conjugated bile acid. of bile acids in blood. J. Biochem. (Tokyo) 56: 424-431. Measurement of serum bile acid levels
GASTRO ENTEROLOGY
Copyright
© 1969 by The Williams
Printed in U.S .A .
& Wilkins Co.
CONJUGATED AND UNCONJUGATED SERUM BILE ACID LEVELS IN PATIENTS WITH HEPATOBILIARY DISEASES ISAO MAKINO,
M . D.; SHOICHI NAKAGAWA,
M .D ., AND KEIMEI MASHIMO, M.D.
Second Department of M edicine , Hokkaido University School of Medicine, Sapporo , Japan
Total and unconjugated bile acid levels in serum of patients with various hepatobiliary diseases were determined by gas-liquid chromatography. In normal serum, ranges of total serum bile acid were found as follows : deoxycholic acid 0.22 ± 0.14, chenodeoxycholic acid 0.23 ± 0.14, cholic acid 0.14 ± 0.09, and total amount of bile acid 0.59 ± 0.31 J,lg per ml. The cholic to chenodeoxycholic acid ratio (C: CDC) was 0.61 ± 0.30. In hepatobiliary diseases, the level of to·· tal serum bile acid rose. The increase was remarkable in acute early hepatitis and in obstructive jaundice but moderate or slight in active portal cirrhosis and chronic hepatitis. Changes in the composition of serum bile acid were also observed. The C: CDC ratio was 1.01 ± 0.43 in portal cirrhosis and 2.74 ± 1.42 in obstructive jaundice. A correlation was observed between the total serum bile acid level and the serum bilirubin concentration in obstructive jaundice and portal cirrhosis. Unconjugated serum bile acid levels in healthy persons showed a range of 0.26 ± 0.20 J,lg per ml; and the unconjugated to total bile acid ratio (U: T) was 51.4 ± 22.0%. In active portal cirrhosis, the amount of unconjugated bile acid increased (2.54 ± 1.83 J,lg per ml). The value was significantly higher than that in normal, chronic hepatitis, and obstructive jaundice groups. Contrary to portal cirrhosis, the whole increase in serum bile acid in obstructive jaundice was occupied by conjugated bile acid and the U: T ratio was very low (0.2 ± 0.4 %). A possible cause for the rising of the total and unconjugated serum bile acid levels in hepatobiliary diseases is discussed. Although it is known that the total serum bile acid increases in patients with hepatobiliary diseases,l -s little work has been done to study the state of conjugation in these diseases. 2 . 5, 9 In 1965, Sandberg et al. 5 reported a method for the determination of serum bile acid with gas-liquid chromatography (GLC). This method has sufficient sensiReceived March 7, 1968. Accepted January 6, 1969. Address requests for reprints to: Dr. Isao Makino, Second Department of Medicine, Hokkaido University School of Medicine, West 5, North 14, Sapporo, Japan.
tivity and specificity to estimate the very low level of bile acid in serum. This report describes a study of the change of conjugated and unconjugated serum bile acid in patients with various hepatobiliary diseases.
Materials and Methods Reagents. Amberlyst A 26 anion exchanger resin was purchased from Japan Organo Company, Japan. Alumina was obtained from Merck AG., Darmstadt, Germany. Standard bile acids were obtained from Calbiochem Corporation, Los Angeles, California. Gas-liquid chromatography. A gas-liquid chromatograph, model GC-2C (Shimazu Man-
1033
1034
MAKINO ET AL.
ufacturing Company, Kyoto, Japan), with a hydrogen flame detector was used. The column was a commercial preparation (Shimazu Manufacturing Company), of 1.5% QF-1 using Chromosorb W (60 to 80 mesh) as support in a glass "U" tube 1.5 in length and 4 mm in diameter. The temperature of the column was· 225 C, and nitrogen as carrier gas was passed at a pressure of 2.5 kg per cm z at the inlet. Procedure. Serum bile acids were determined by the procedure described by Sandberg et al. 5 Briefly, the procedure was as follows. A sample of serum (3 to 5 m\) was diluted with the same volume of distilled water and passed through a 5-ml column of the anion exchanger Amberlyst A 26. The adsorbed bile acid was eluted with 150 ml of 0.2 M (NH.hCU a in 80S; ethanol. The eluate was evaporated to dryness and then hydrolyzed in 20 ml of 7.5% NaOH for 4 hr in an autoclave at a pressure of 1.0 kg per cm z • After ether extraction of the acidified hydrolysate, the bile acid was methylated by diazomethane, purified on an alumina column, and trifluoracetylated with trifluoracetic anhydride. The excess reagents were removed under the nitrogen stream. The residue was dissolved in carbon disulfide and an aliquot was used for GLC. To analyze the unconjugated bile acid, the hydrolysis step was omitted. The eluate from the ion exchange column was evaporated, the residue was dissolved in water, and after acidification the bile acid was directly extracted, methylated, and trifluoracetylated. The purity of the solvents was tested because the bile acid level in normal serum was very low. The same volume of each solvent used in this procedure was evaporated to dryness, treated with diazomethane and trifluoracetic anhydride, and dissolved in carbon disulfide. No peaks could be seen except for a small solvent front at the full sensitivity setting. For recovery, 14C-Iabeled cholic acid was added to normal (n = 2) and pathological (n = 4) sera. The entire procedure, excepting GLC, was carried out and the radioactivity in one aliquot of the sample was determined in a gas-flow counter. The recovery ranged from 62.0 to 78.3 % (70.0 ± 6.1). Quantitative determination. Standard bile acids (lithocholic, deoxycholic, chenodeoxycholic, and cholic) were treated with diazomethane and trifluoracetic anhydride and applied to GLC. Each derivative showed only one peak. Under these experimental conditions, complete separation of the bile acids mixture was evident. The relative retention times were
Vol. 56, No.6
0.67 for lithocholic acid, 1.00 (15.3 min) for deoxycholic acid, 1.20 for chenodeoxycholic acid, and 1.75 for cholic acid. The peaks rose linearly when the trifluoracetate of methyl bile acid was injected into GLC in amounts ranging from 0.1 to 0.5 Ilg. Conjugated deoxycholic, chenodeoxycholic, and cholic acids purified by thin-layer chromatography were treated with diazomethane and trifluoracetic anhydride without hydrolysis, and injected into GLC. No peak of unconjuga ted bile acid was detected. Subjects. Twenty healthy persons and 81 patients with hepatobiliary diseases were used in this study. The control subjects were persons without hepatobiliary diseases selected from the medical staff and nurses of our clinic. Of the group of hepatobiliary patients, 22 had acute hepatitis at early stage; 30 had active portal cirrhosis (flocculation and transaminase tests indicated abnormal values); 18 had obstructive jaundice; and 11 had active chronic hepatitis (transaminase tests showed abnormal values). All were treated at the Department of Medicine, Hokkaido University Hospital. Diagnoses were made on the basis of clinical symptoms, liver function test, liver biopsy, and in some cases with laparotomy. Blood samples were obtained from the antecubital veins in the morning before breakfast after overnight fasting; separated sera were kept frozen at -20 C.
Results Total Serum Bile Acid Levels
Control subjects. In normal human serum, deoxycholic, chenodeoxycholic, and cholic acids are the major bile acids. The following ranges could be noticed: deoxycholic acid, 0.04 to 0.55 j.1.g per ml (0.22 ± 0.14); chenodeoxycholic acid, trace to 0.55 j.1.g per ml (0.23 ± 0.14); and cholic acid, 0.02 to 0.36 j.1.g per ml (0.14 ± 0.09). The total bile acid was 0.12 to 1.46 Ilg per ml (0.59 ± 0.31). The ratio of cholic acid to chenodeoxycholic acid (C: CDC) was 0.25 to 1.38 (0.61 ± 0.30) (tables 1 and 2). Acute hepatitis patients. In patients with acute hepatitis, the total serum bile acid level rose markedly at the early stage. In 1 it reached more than 160 j.1.g per ml, 100 times as high as the upper limit of normal values. In convalescence this bile acid level
June 1969 TABLE
1035
SERUM BILE ACID LEVELS 1. Total serum bile acid levels in normal subjects and in patients with liver diseases
.
Total serum bile acid level a
Liver function testb
No. of cases
-
DC
-
I
CDC
C
I
I
C:CDC
Total
I ratio
un1'ts
I"g/ml
Normal
20
Acute hepatitis (early) Chronic hepatitis (active) Portal cirrhosis (active) Obstruc t ive jaundice
22 11 30 18 1
0.22 ±0.14 1.20 ±1.15 0.77 ±0.69 0 .74 ±0.87 0.28 ±0 . 32
0.23 ±0.14 19.90 ±17.80 2.29 ±1.91 4.27 ±4.18 9.47 ±4.81
Kunkel
0.14 0 . 59 ±0 .09 ±0.31 26 . 81 47.98 ±25.83 ±37.78 1.91 4.97 ±2.09 ±4 .51 3.38 8.40 ±2.50 ±6.02 27.98 37.89 ±23.02 ±26.90
I T.Bilir.!
AI-Ph
--------- - -----
0.61 ±0.30 1.68 ±1.21 0.94 ±0.fi1 1.01 ±0.43 2.74 ±1.42
mg/ dl
I
SGOT
I SGPT
units
3- 10 0.3-1. 0 1 2 . 7- 10 5--40
3-35
296 8.9 12.0 I 15.1 ±4.5 ±8.4 ±9.7 ±146 5.6: 11.1 161 11.3 ±3.5 ±4.7 1 ±5 .9 ±98 1.8 18 .5 103 i3.5 ±5.1 ±2.7 I ±13 .3 ±95 8.4 16.3 1 41.fi 132 ±3.6 ±1D.0 ±23.1 ±83
220 ±128 168 ±118 53 ±46 97 ±50
- - -- - - - - - --
" DC, deoxycholic acid; CDC, chenodeoxycholic acid; C, cholic acid. b T.Bilir., total serum bilirubin concentratioll; AI-Ph , serum alkaline phosphatase (in King-Armstrong units); SGOT, serum glutamic oxalacetic transaminase; SGPT, serum glutamic pyruvic transaminase. 2. Statistical evaluation of total serum bile acid levels in normal subjects and in patients with liver diseases a
TABLE
I
Portal cirNormal rhasis
--------------------Total serum bile acid levels Portal cirrhosis .. Chronic hepatitis. Obstruction .. Acute hepatitis .... C:CDC ratios b Portal cirrhosis ... Chronic hepatitis ... Obstruction .. Acute hepatitis .....
*** *** *** ***
Chronic Obhepstrucatitis tion
--------
----
NS
100.0
r ~ 0.\ 1 p~0.02
~ 60.0
] 600 ';; 40.0
:=.
....
20.0
OL----------------------NS
*** ***
o
*** ***
NS
*** *** ***
e
NS
*** **
*** NS
*
a Data represent mean values plus or minus standard deviation. ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant.. b C, cholic acid; CDC, chenodeoxycholic acid.
soon dropped to the normal level. In these cases, chenodeoxycholic acid and cholic acid occupied a great part of the increased bile acids, while the increase of deoxycholic acid was not remarkable. The C: CDC ratio was variable in this disease. Chronic hepatitis patients. In patients with active chronic hepatitis, the increase
10.0 Tota I bili rubi n
20.0
(mgt dl)
30.0
FIG. 1. The correlation between total serum bile acid level and total serum bilirubin concentration in extrahepatic obstructive jaundice.
in bile acid was moderate or slight. The range was 0.90 to 18.25 Ilg per ml (4.97 ± 4.51). Portal cirrhosis patients. In patients with active portal cirrhosis, the total serum bile acid level ranged from 1.62 to 28.09 Ilg per ml (8.40 ± 6.02). The increase in chenodeoxycholic acid was more remarkable than that in other bile acids, and the C: CDC ratio was less than 1.5 (1.01 ± 0.43) in 26 of 30 patients. Obstructive jaundice patients. In obstructive jaundice the level of total bile acid was 9.07 to 109.49 Ilg per ml. (37.89 ± 26.90). A striking increase of cholic acid could be observed. The C: CDC ratio was
1036 TABLE
MAKINO ET AL.
Vol. 56, No.6
3. Unconjt.gated serum bile acid levels in normal subjects and in patients with hepatobilidry diseases
I Normal
No. of cases. ... 10 Total bile acid (lLg/ml) 0.50 ± 0 . 27 Unconjugate d bile acid ~g /ml) . ...... 0.26 ± 0.20 Un conjugated to total bile acid ratio (%) .. 51.4 ± 22 .0
Portal cirrhosis (active)
Hepatitis Obstruction Chronic (active)
Acute (early)
17 7.49 ± 5.53
11 4.97 ± 4.51
9 47.16 ± 27.69
11 48 .65 ± 26.85
2.54 ± 1.83
0.48 ± 0.30
1.52 ± 2. 29
0 . 14 ± 0.18
39.9 ± 24 . 9
15.6 ± 10.8
5.1 ± 7.4
0.2 ± 0 . 4
4. Statistical evaluation of unconjugated serum bile acid levels i n normal subjects and in patients with hepatobiliary diseases a
SERUM BILE lCIO
T A BLE
DI
5
10
(JJG 1M l) 15 20 0 .4 0.4
Portal Chronic ObNormal cirhepstrucrhasis atitis tion
0.5
-----------------1---- ---- ---- ---Unconjugated serum bile acid levels Portal cirrhosis ... . Chronic hepat itis ... Obstruction .... Acute hepatitis ..... U:T ratios b Portal cirrhosis .. . . Chronic hepatitis ... Obstruction Acute hepat itis.
:.:.:-:
*** NS NS NS NS
*** *** ***
** *** NS
** NS
NS
0-
~ ....
** *** ***
*** *
.:.;.;.;.;.;.
..... :-:.;.;.:-:-. :-:.... ;.:-:.:
***
Data represent mean values plus or minus standard devi ation. *** , P < 0.001; **, P < 0.01; *, P < 0.05; NS, not s ignificant. b Unco njugated to total bile acid ratio. a
greater than 1.5 (2.47 ± 1.42) in 17 of 18 patients. The total serum bilirubin concentration showed a close correlation to the serum bile acid levels in obstructive jaundice and portal cirrhosis patients. The coefficient of this correlation was 0.57 (P < 0.02) in obstructive jaundice (fig. 1) and 0.72 (P < 0.001) in portal cirrhosis. There was no correlation in acute hepatitis.
Unconjuated Serum Bile Acid Levels Control subjects. Unexpectedly, a relatively large amount of unconjugated serum bile acid was detected in normal serum. The values obtained from 10 healthy per-
16.5 18.9
0.9 0.9
16 .4
1. 4
83.9
0.9
13.1
FREE 61.8 TOTAl 168 % 60'. 5
0.6
....L 0 .9 CDC
36.4
2.2
1.3
1. 5
51.5
1.8
42.3
1.8
41.5
1 .0
11.9
2.0
0.5 BILIARY 6.5 I·········.,·, CON)
1.1
0 .6
1.0 0.9
_FREE
FIG. 2. Total and unconjugated serum bile acid levels in patients with active portal cirrhosis.
sons (including 4 women) ranged from 0.06 to 0.74 JIg per ml (0.26 ± 0.20) and the unconjugated to total bile acid ratio (U : T) was 51.4 ± 22.0% (tables 3 and 4). Portal cirrhosis patients. In 17 patients with active portal cirrhosis, the level of unconjugated bile acid ranged from 0.34 to 7.59 JIg per ml (2.54 ± 1.83). The unconjugated bile acid level of 14 of the 17 patients was greater than 1.0 JIg per ml and
June 1969
significantly higher than that of a normal group (P < 0.001), while the U: T ratio was 39.9 ± 24.9%, not statistically different from normal serum (fig . 2). In 2 cases of biliary cirrhosis, no increase in unconjugated bile acid was found (0.37 and 0.08 Ilg per ml) . The U: T ratios were 0.5 and 6.5%, respectively. Chronic hepatitis patients. In 11 patients with active chronic hepatitis, the range of
I
2
1037
SERUM BILE ACID LEVELS
SERUM BILE ACID 4 6
(VG/M L! 8 18
>-
;=
ga
""
SERUM BILE ACIP ( VG I ML) 20 40 60 80
.
20 0.9
l~~~~:r~~r:~~t~r::~~r~~r~::tt:~(~~~trlt:~~ 0:1]]]
7.0
6.5
1.1
kr~tt:rr~t~r:t:::~~rrr~~r~t:~~]
0
6.8
3.9
0.9
5~r~~~:~tt~~:r::~:~::~~:::~:~:::~::~~~:::~:i\1
0.2
4.0
11.0
0.2
~
rr:~:~~t~tt~~tt~r~r~::::}1
1.1
2.1
""
-...
~ttttttt~~~r::~:(tq
0.3
2 .0
~
{::::r::l:\.:l:· :::r::{t
,..=>
2i
_C_ O.2 CDC
>-
1.7
~
::0
>-
8 .4
'" ii
I
0.7
~
=>
C>
2 3 .2
§:!1
'"
0.6
"" ~
0 ""
'" ~
(.::::·:·:·:·:1 CONI
I 2 .6
1.3
21.8
0.4
34 .5
2,3
"" ""
0.1
2.0
FREE
~::::t:~:~:':::r::':~:::::1
Tom
rJ:::t·:·:tt::::1
%
C
0
CDC
0.1
2.6
LJ n. LJ 1:·:·:·:·:·:-:1 CONI
_FREE
3.0 1.9
r ::t:::::':::::J
~
FIG. 3. Total and unconjugated serum bile acid levels in patients with active chronic hepatitis.
TABLE
100
1.4
FREE 8.S TOTAL % 28.4
S
unconjugated bile acid levels was normal, 0.19 to 1.00 Ilg per ml (0.48 ± 0.30), i.e., under 1.0 Ilg per ml. The U: T ratio was 1.4 to 34.4% (15.6 ± 10.8), a significant difference from active portal cirrhosis (P < 0.01) was evident (fig. 3). Obstructive jaundice patients. The amount of unconjugated serum bile acid in 11 cases was trace to 0.62 Ilg per ml (0.14 ± 0.18), not significantly different from the normal group. However, unconjugated bile
_
o
1.6
o
1 .7
FREE
FIG. 4. Total and unconjugated serum bile acid levels in patients with obstructive jaundice.
5. S erum bile acid levels in portal blood obtained during operation" Total serum bile acid levels
Cases DC
I
CDC
I
C
I Total
Unconjugated serum bile acid levels /C:CDC
DC
",glml
No.1. (gastric cancer) ... Peripheral blood obtained simultaneously. . ' . No.2. (gastric cancer) ... Peripheral blood obtained simultaneously ... . ..
I
CDC
I
C
I Total
U:T
- -
%
",glml
0.06
0.99
1.84
2.89
1.83
0 .06
0 . 16
0.17
0.39
13. 5
0 . 12 0 .06
Trace 0 . 19
0 . 13 0.41
0 .25 0.66
2.16
0 .05
Trace
0.04
0.09
13.6
0 . 03
0 .01
0.04
0.08
4 .00
I
"DC, deoxycholic acid; CDC, chenodeoxycholic acid; C, cholic acid; U:T, unconjugated to total bile acid ratio.
1038
MAKINO ET AL.
acids could not be found in 5 cases in this group. Therefore, the increased bile acid values in obstructive jaundice patients were almost all the result of increased levels of conjugated bile acid; the U: T ratio was 0 to 1.1% (0.2 ± 0.4) (fig. 4). Acute hepatitis patients. Un conjugated serum bile acid levels in the 9 patients were variable. They ranged from 0.17 to 7.75 JLg per ml; the U: T ratio was 0.2 to 24.2% (5.1 ± 7.4).
Vol. 56, No.6
less than 1.0 in liver cirrhosis patients, and he regarded this index as a prognostic and diagnostic sign. Our result confirmed that the difference in the TBA: DBA ratio was not due to an increase or decrease in deoxycholic acid of the secondary bile acid. The C: CDC ratio was less than 1.5 in 26 of 30 patients with portal cirrhosis and more than 1.5 in 17 of 18 patients with obstructive jaundice. The C: CDC ratio in the normal group did not significantly differ from that of the patients with portal cirSerum Bile Acid Levels in Portal Blood rhosis. The portal blood obtained during operaThe rise of the serum bile acid level tion contained high levels of conjugated and some changes of bile acid observed in bile acid. The U: T ratio was 13.5 and hepatobiliary patients could be caused by 13.6%. The data showed that conjugated one or more of the following factors: 1. A fiow of bile acid from the bile into bile acid was reabsorbed more easily from intestine than was unconjugated bile acid blood as a result of the destruction of he(table 5). patic acini and intrahepatic biliary ·ducts. Osborn et al. 6 observed that the level of Discussion serum bile acid correlated with the serum The normal values of total serum bile total bilirubin concentration rather than acid determined in this study are similar to the concentration of serum glutamic to those reported by Sandberg et al. 5 oxalacetic transaminase or serum glutamic Iwata and Yamazaki!O described a higher pyruvic transaminase in obstructive jaunnormal bile acid level (2.20 ± 0.54 JLg per dice. This obseryation was confirmed in ml) obtained by their enzymatic method. these studies. Th\Is it seems that this is But their estimation might include sub- one of the important reasons for the ascent stances other than bile acid because they of the serum bile acid level in obstructive jaundice. used a crude enzyme preparation. 2. Quantitative and qualitative change In the hepatobiliary diseases the total serum bile acid level was increased. The in the synthesis of bile acid in the liver. elevations were the greatest in the early The change in the C: CDC ratio in hepatostage of acute hepatitis and in obstructive biliary diseases seems to be due to the jaundice. Slight or moderate elevation oc- changed metabolic pathway of bile acid. 3. A fiow of the reabsorbed bile acid into curred in portal cirrhosis and in chronic hepatitis patients. However, the total bile the blood of systemic circulation as the acid levels in each group overlap each result of a decreased removal by the liver. There have been few reports on unconanother markedly, so that the diagnostic value of the estimation of the total bile jugated bile acid levels in serum. Rudman acid level is questionable. The results are and Kendall, 2 who made an experiment similar to the values reported by Osborn with reverse phase chromatography, and et al.,6 Rudman and KendalV and Sandberg et al.,5 using gas-liquid chroCarey.! matography, reported the presence of unThe C: CDC ratio of bile acid was sig- conjugated bile acid in the sera of a small nificantly different in hepatobiliary dis- number of healthy persons and hepatoeases, especially in obstructive jaundice biliary patients, but no pathological and and portal cirrhosis. Carei reported that diagnostic significances have been disthe ratio of trihydroxycholanic to dihy- cussed. In healthy persons the unconjugated bile droxycholanic acid (TBA: DBA) was over 1.0 in obstructive jaundice patients and acid occupied approximately one-half of
June 1969
SERUM BILE ACID LEVELS
1039
the total bile acid in serum, but the ab- seems to be useful for the differential diagsolute amount of unconjugated bile acid nosis of portal cirrhosis, chronic hepatitis, was small. In obstructive jaundice pa- and obstructive jaundice. tients the conjugated bile acid increased REFERENCES markedly, but the unconjugated bile acid 1. Carey, J. B., Jr. 1958. The serum trihydroxychanged little, so that the U: T ratio was dihydroxy bile acid ratio in liver and biliary very low. On the contrary, in active cirtract disease. J. Clin. Invest. 37: 1494-1503. rhosis patients, the amount of unconju2. Rudman, D., and F. E. Kendall. 1957. Bile acid gated bile acid increased significantly and content of human serum. 1. Serum bile acids the total bile acid rose slightly or moderin patients with hepatic disease. J. Clin. In-' ately, so that the U: T ratio was signifivest. 36: 530- 537. cantly high. In some cases, the unconju3. Carey, J. B., Jr. 1961. Bile acids in the serum of gated bile acid occupied 80% of the total jaundiced patients. Gastroenterology 41: 285bile acid. In chronic hepatitis patients no 287. 4. Sherlock, S., and V. Walshe. 1948. Blood cholates increase in unconjugated bile acid was obin normal subjects and in liver disease. Clin. served. Sci. 6: 223-234. The amount of unconjugated bile acid 5. Sandberg, D. H., J . Sjovall, K. Sjovall, and D. A. was less than l. 0 f.Lg per ml in all 11 paTurner. 1965. Measurement of human serum tients with active chronic hepatitis (0.48 bile acids by gas-liquid chromatography. J. ± 0.30 f.Lg per ml) but over l.0 f.Lg per Lipid Res. 6: 182- 192. ml in 14 of 17 patients with active portal 6. Osobrn, E. C., I. D. P. Wootton, L. C. DeSilva, cirrhosis (2.54 ± 1.83 f.Lg per m!). The and S. Sherlock. 1959. Serum bile acid levels level of unconjugated serum bile acid inin liver disease. Lancet 2: 1049-1053. creased significantly in portal cirrhosis. 7. Carey, J. B. , Jr . 1956. Chenodeoxycholic acid in human blood serum. Science 123: 892. These data suggest that the conjugation 8. Sjovall, K., and J. Sjovall. 1966. Serum bile acid of bile acid with taurine or glycine was level in pregnancy with pruritus. Bile acids deficient owing to the reduction in the acand Steroid 158. Clin. Chim. Acta 13: 207-211. tivity of the conjugating enzyme system 9. Blum, M., and N . Spritz. 1966. The metabolism in portal cirrhosis. However, the degree of of intravenously injected isotopic cholic acid serum unconjugated bile acid level did not in Laennec's cirrhosis. J. Clin. Invest. 45: 187always correlate with the degree of clinical 193. symptoms. Investigators are now discussing 10. Iwata, T., and K. Yamazaki. 1964. Enzymatic the cases with a high level of serum undetermination and thin-layer chromatography conjugated bile acid. of bile acids in blood. J. Biochem. (Tokyo) 56: 424-431. Measurement of serum bile acid levels