GastroenterologiaJaponica Copyright 9 1978 by TheJapanese Societyof Gastroenterology

Vol. 13, No. 6 Printed inJapan

- - O r i g i n a l Article--

C L I N I C A L S I G N I F I C A N C E OF S E R U M B I L E ACID M E A S U R E M E N T I N L I V E R DISEASES T o m o f u m i M O R I T A * , M . D . , Yoshinori M A T S U Y A M A * , M. D., T a k a y o s h i F U J I M O T O * , M . D . , M a s a r u H I G U C H I * , M.D., T a d a s u TSUJII*, M . D . , a n d Yoichi M A T S U O K A * * , M.D. *The Third Department of Internal Medicine, Nara Medical University **Department o f Clinico-Laboratory Diagnostics, Nara Medical University

Summary To determine the clinical significance of serum bile acid measurements, changes in the serum bile acid composition in liver diseases and endogenous bile acid clearance due to test meal loads were investigated. In the case of changes in the serum bile acid composition, a characteristic pattern of a remarkable increase of chenodeoxycholic acid (CDCA) was found in fulminant hepatitis. In patients with acute hepatitis, increases in CDCA were somewhat greater than those of cholic acid (CA) and there was tendency for these changes to precede changes in other liver function tests. In cases of extrahepatic obstructive jaundice, the CA/CDCA ratio was a large value exceeding 1.0. In investigations of endogenous bile acid clearance, serum bile acid concentration two hours after the test meal load clearly reflected the hepatic disorder and it was useful in differentiating between active and inactive form in chronic hepatitis and compensation and decompensation in liver cirrhosis.

Key Words: serum bile acids, endogenous bile acid clearance, liverfunction Introduction Since the healthy liver efficiently ingests bile acids from the portal vein, the bile acid pool u n d e r n o r m a l conditions is present m a i n l y in the e n t e r o h e p a t i c circulation. However, in hepato-biliary diseases, the metabolic m e c h a n i s m of bile acids is d i s r u p t e d , the bile a c i d pool distribution is c h a n g e d a n d the bile acid c o n c e n t r a t i o n in the systemic circulating b l o o d , urine, skin, etc. increases. To d e t e r m i n e the m e t a b o l i c conditions of bile acids in p a t i e n t s with h e p a t o - b i l i a r y diseases, it is necessary to d e t e r m i n e the quantitative a n d ReceivedJune 15, 1978. Accepted August 14, 1978. Address requests for reprints to: Dr. Tomofumi Morita, M.D., 3rd Department of Internal Medicine, Nara Medical University, Shijo-machi, Kashiwara, Nara, 634 Japan.

test.

qualitative changes of bile acids in the blood, bile, small intestine contents, urine, feces, skin, etc. However, because o f the difficulties in collecting m a t e r i a l s a n d the m e a s u r i n g m e t h o d s , it is not easy to o b t a i n i n f o r m a t i o n from the all angles. Since the m a t e r i a l s were easy to o b t a i n a n d the bile acid c o m p o s i t i o n was c o m p a r a t i v e l y u n i f o r m , the authors m e a s u r e d the serum bile acids a n d investigated the significance of t h e m e a s u r e m e n t of serum bile acids in liver diseases to evaluate the status of the liver.

Methods T h e significance of serum bile acid m e a s u r e m e n t s in liver diseases was investigated from the following two standpoints:

492

T. M O R I T A

1) Pathological conditions of the liver and changes of serum bile acid composition. 2) Total serum bile acid concentrations before and after a test meal load in liver diseases. The first standpoint is concerned mainly with investigations of qualitative changes in the composition of bile acids in the blood, while the second standpoint involves observation of the endogenous bile acid clearance by means of the test meal load test to investigate its usefulness as a liver function test. 1. Pathological conditions of the liver and changes of serum bile acid composition The diseases examined were acute hepatitis, fulminant hepatitis, acute intrahepatic cholestasis and extrahepatic obstructive jaundice (carcinoma of the head of the pancreas). Blood samples were obtained from the antecubital veins before breakfast after an overnight fast, and the separated sera were kept at -20~ Serum bile acid composition were estimated by the procedure described by Sandberg et al. l) with some modification. Briefly, the procedure were as follows. A sample of serum (2-10 ml) was diluted 1 : 1 with distilled water and passed through a 5ml column of the anion exchanger Amberlyst A26. The absorbed bile acids were eluted with 150ml of 0.2 M (NH4)2CO~in 80% ethanol. The eluate was evaporated to dryness and then hydrolyzed in 5 ml of 1 N NaOH for 4 hours in an autoclave at a pressure of 1 Kg per cm 2 at 120~ After ether extraction of the acidfied hydrolysate, the bile acids were methyrated with diazomethane. Bile acid methyl esters were transferred to an aluminium oxide column with about 10ml of benzene-hexane (1:9, v/v) and washed with about 40ml of the same soluvent mixture. Lithocholic acid methyl ester was eluted with 70 ml of benzene and all the other bile acid methyl esters with 40 ml of methanol-acetone (1:9, v/v). The bile

ET AL.

Vol. 13, No. 6

acids were converted to their methyl ester trifluoroacetates and dissolved in carbone disulfide and an aliquot was injected into the gas-liquid chromatography (GLC). A Shimazu GC4BMPF hydrogen flame ionizarion detector was used for GLC. The column was a 2% OV210 glass column 3ram in diameter. The column temperature was 230~ and the detector temperature 258~ Nitrogen was used as the carrier gas, the flow rate was 60ml/min. and the sensitivity 10~ range 0.04 V. 2. Total serum bile acid concentrations before and after a test meal in various status of liver The diseases examined were acute hepatitis, chronic hepatitis and liver cirrhosis. In all cases, the diagnosis had been confirmed by liver biopsy with Laparoscopy. Chronic hepatitis was divided into the active and inactive forms. The histological definition was as follows: active form showed enlargement of portal area with fibrosis and inflammatory cells infiltration, piecemeal necrosis due to destruction of limiting plate in mesenchymal region and focal necrosis and remarkable mobilization of Kupffer's cell in parenchymal region. While inactive form showed no destruction of limiting plate and minimal change of parenchymal region. Liver cirrhosis was divided into compensated and decompensated types. For measurement of the bile acids, blood samples were obtained in the morning after an overnight fasting and one, two and three hours after the test meal load and the measurements were performed b y simple and sensitive assay2) using 3a-hydroxysteroid dehydrogenase (3a-HSD), NAD, diaphorase and Resazurin. The test meal consisted of 200 g of milk, one egg, 5 g of sugar and a small amount of vanilla essence, mixed by shaking and given as a liquid meal. Other liver function tests were performed at the same

D e c e m b e r 1978

Serum Bile Acid in Liver Diseases

t i m e as the bile acid measurements and the correlation between the serum bile acid concentrations and other liver function tests was investigated.

Results 1.

Pathological conditions of the liver and changes of serum bile acid composition 1) Healthy controls

T h e total serum bile acid concentration measured by GLC was 0 . 6 0 - 0 . 2 6 / ~ g / m l (n=6),

lithocholic acid deoxycholic acid

(LCA) (DCA)

chenodeoxycholic

acid

0.02--0.02Mg/ml, 0.10+0.04#g/ml,

(CDCA)

0.30-+0.09

# g / m l and cholic acid (CA) 0.14-+0.11 Mg/ml. These were extremely small amounts in all cases.

Ym, 60

50

40

~

493

2) Acute hepatitis The course of a typical case is shown in Fig. 1. In the florid stage of acute hepatitis, the total bile acid concentration reached m o r e than 100 times that in healthy controls and

CDCA and CA increased and these two acids accounted for the great majority of the total bile acids. However, the CDCA increase was somewhat greater and the C A / C D C A ratio was less than 1.0. There were also slight increases in

LCA and DCA. As was indicated by the liver function tests, with the i m p r o v e m e n t of liver

condition the bile acids decreased and they returned to normal after about three months. In this case, the changes in bile acids tended to

precede those in bilirubin and GPT. 3) Fulminant hepatitis

Bile acids

\ 40

~

45

~

Bile acids

Totalb,~eac,d

Total bde acld

. . . . . L CA -----D CA . . . . CDCA --~--CA

\

---,-

L CA

'd 30

~ t \ " l

m---- C A

30

\

20

15'

\, i\

10-

i

:::.51

i

l,Dec. IO,6ec.

8,Is,.

31,Jan.

24,Feb.

24,May Bihrubm

Bilirubin

AI-p (~It/dl) (K.A) GPT 30,

1420 9 ,400. 15. 100 80 6O 1o . 40 2O

i

~

,

Liver

~

10

i

&Nov. I6,N~.

~'~ ~

function

GPT

tests

1220! 1200-

2-'~

. . . . . . . . . . . . ~ i i i i 9,Nov. 18,Nov. 3,0ec. 10,Dec.

~,~,_ i &Jan.

i 31Jan.

BIIIrubu~ i 24,Feb.

Fig. l . Changes of serum bile acid composition and liver function tests in a 40-year-old patient with acute hepatitis.

20

2/,May

A(KA) l p (.~/dl)

Liver

l,Jun.

.... 3JLIn

function

6.

30 4' ~

""~'--.,,

,0oi'20' 80 60 4OI

.......

/

/"

ll,Jun.

21,Jan.

tests

"~,,

0"2' ~ ~j~ ~,~,/.i

\, ~.

GPT

10. 2'

11 I

11' |

11

.

.

kBlllrub,n

Exchang blood transfusion

.

24,May

.

27,May

1,lun.

.

3,lun.

~, 11Jun.

, 21,Jan.

Fig. 9. Changes of serum bile acid composition a n d liver function tests in a 53-year-old patient with f u l m i n a n t hepatitis.

494

T. M O R I T A

Fig. 2 shows the changes in total serum bile acid concentration and in the composition in a case of fulminant hepatitis who was saved by several exhange blood transfusions. The total bile acids before the start of the transfusions was about 80 times greater than that of the normal controls and about 80% of this was CDCA. Only about 9% was CA, which made the C A / C D C A ratio very low. There were also slight increases in LCA and DCA. There were r a p i d decreases in both total bile acids and CDCA as the exchange blood transfusions increased in number. There were further decreases when the patient was revived, but there was a reverse tendency for CA to increase and the gap between the CDCA and CA values tended to decrease. However, the C A / C D C A ratio remained at less than 1.0 two months after it was confirmed that the patient had been saved. Eight months after the outbreak of the disease, there was a slight GPT abnormality (80 u), but it was found that the total bile acid concentration and composition were basically normal. 4) A c u t e i n t r a h e p a t i c cholestasis Fig. 3 shows a case of acute viral intrahepatic cholestasis. In the florid stage, the total bile acid value increased to about 30 times the normal value. The individual bile acids all increased but the CDCA, CA and LCA increases were noteworthy. In the early stages, the CDCA value was higher than that of CA and the CA/CDCA ratio was less than 1.0. Two months and four months after the outbreak, total bile acid values and the values for each bile acids rapidly decreased in parallel with the improvements of liver function tests. Thereafter, abnormal values of bile acids continued in parallel with abnormal alkaline phosphatase (A1-P). One year and a half after the outbreak, the values h a d nearly returned to normal. The same bile acid composition pattern was

ET AL.

Voh 13, N o . 6

ml ~ 2o

Bileacids , Total btle acid

\

-----

k C A

~-~

D C A

i,.m

C D C A

i

C A

i

~

II 5,Sep. 19,~a~. 8ilirubin AI-p ( ~ / d l ) (K,A.) GPT 40, I00- O"

60-

5,May

25,0ct.

Liver function tests AI-p

B- 30. 6-

40-

17,~.

20.

4-

20- 2-

k

\

10,

5,~p. 19,~1L

17,~t.

5,May

25,~t.

Fig. 3. C h a n g e s o f s e r u m bile acid c o m p o s i t i o n a n d liver f u n c t i o n tests in a 3 3 - y e a r - o l d p a t i e n t w i t h int r a h e p a t i c cholestasis.

also seen in a case of drug-induced acute intrahepatic cholestasis. There was a return to normal as the disease improved in parallel with bilirubin changes. 5) Extrahepatic obstructive jaundice Fig. 4 shows the course of a case with carcinoma in the head of pancreas which obstructed bile duct. In this case T-tube was implanted. Before implantation of the T-tube, the total serum bile acid value was about 30 times the normal value and in the composition CA and CDCA increased. The CA value increased more than the CDCA value and the C A / C D C A ratio was greater than 1.0. After the T-tube was implanted, there were sharp decreases in the total bile acids and CA and

December 1978

1) 18

495

Serum Bile Acid ~n Liver Diseases

Healthy controls

T h e fasting bile acid c o n c e n t r a t i o n in the

Bile acids

16

k

14

k

.....L C A

k

-----D C A

Total bile acid

healthy controls was 6 . 2 + 2 . 7 , u m o l e / 1 (n=17). T h e value one hour after the test meal was

12

1 0 . 6 + 2 . 9 p m o l e / 1 . After two hours, it was 9,2+l.2pmole/1 a n d after three hours,

10

5.5_+2.dpmole/l.

' "

Clinical significance of serum bile acid measurement in liver diseases.

GastroenterologiaJaponica Copyright 9 1978 by TheJapanese Societyof Gastroenterology Vol. 13, No. 6 Printed inJapan - - O r i g i n a l Article-- C...
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