Levels
o f
Bile
I m m u n o r e a c t i v e
Acids
in
H e a l t h
a n d
G l y c i n e - c o n j u g a t e d Hepatobiliary
Disease LAURENCE M. DEMERS, P H . D . , AND GERSHON W. HEPNER,
M.D.
Departments of Pathology arid Medicine, The Milton S. Hershey Medical Center, The Pennsylvania University, Hershey, Pennsylvania 17033 ABSTRACT
study the dynamics of the enterohepatic circulation of cholate conjugates. 18 In further studies using this radioimmunoassay, it was found that levels of cholate conjugates were elevated in patients who had chronic active hepatitis when other liver function tests were normal 15 and in 12 Received April 26, 1976; received revised manu- patients with anicteric viral hepatitis. script June 9, 1976; accepted for publication June 9, We have developed a radioimmunoassay 1976. Supported in part by Grant AM 17303 from the for four glycine-conjugated bile acids— National Institute of Health and by a grant from the cholic, chenodeoxycholic, deoxycholic, and Pharmaceutical Manufacturers' Association Foundasulfolithocholic acids. T h e radioimmunotion. Address reprint requests to Dr. Demers: Depart- assay is highly specific for each of these ment of Pathology, M.S. Hershey Medical Center, four bile acids, whose levels were measured Pennsylvania Slate University, Hershey, Pennsylvania in sera from fasting healthy control sub17033.
SERUM BILE ACIDS are
known to
be
in-
creased in patients who have hepatobiliary diseise '• 2 ' 4_8 ' 10 ' 12_1S - 19 ' 22_25 - 28 ' 29 - 31-33 ' 37 A sensitive radioimmunoassay for conjugates of cholic acid was first described by Simmonds and associates33 and was used to
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Demers, Laurence M., and Hepner, Gershon W.: Levels of immunoreactive glycine-conjugated bile acids in health and hepatobiliary disease. Am J Clin Pathol 66: 831-839, 1976. A sensitive radioimmunoassay for cholylglycine, chenodeoxycholylglycine, deoxycholylglycine, and sulfolithocholylglycine was established using antibodies obtained from rabbits injected with albumin conjugates of these bile acids. Glycine-conjugated bile acid levels were measured in sera from 25 control subjects and 110 patients who had hepatic disease (alcoholic cirrhosis, hepatitis, cholestasis, and hepatic malignancy). Sulfolithocholylglycine was elevated in the sera of all 110 patients with hepatic disease. Cholylglycine was within normal range in only three. Chenodeoxycholylglycine was elevated in most sera of patients who had hepatitis, cholestasis, or hepatic malignancy. It was normal in most sera of patients who had alcoholic cirrhosis, suggesting that chenodeoxycholic acid may be subject to further biotransformations in these patients. Deoxycholylglycine was elevated in a minority of patients, none of whom had cholestasis. T h e data suggest that serum bile acids, particularly sulfolithocholylglycine, are a highly sensitive index for hepatic dysfunction. (Key words: Liver disease; Bile acids; Hepatobiliary disease; Liver function test; Radioimmunoassay.)
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DEMERS AND HEPNER
jects and patients who had portal cirrhosis, hepatitis, hepatic cancer, or cholestasis of extrahepatic or intrahepatic origin. Materials and Methods Radioimmunoassay of Bile Acids
T h e antiserum produced was highly specific, showing only minimal crossreactivity when tested against other structurally related bile acid compounds. There was less than 1% cross-reactivity between any two glycine-conjugated bile acids; there was 18% cross-reactivity between the glycine-conjugated bile acids and their taurine-conjugated analogs, and there was 1% cross-reactivity between the glycineconjugated bile acids and the free bile acid analogs. There was less than 1% cross-reactivity between sulfolithocholylglycine antibodies and the mono- and disulfate derivatives of chenodeoxycholylglycine and deoxycholylglycine, which were prepared by a modification of the method of Palmer and Bolt.26 Crossreactivity studies along with recovery and parallelism studies to test the reproducibility, accuracy, and precision of the bile acid radioimmunoassay have been performed. 9 Patients Studied.
The subjects studied included 25 controls without known hepatic disease; of T h e unlabeled bile acids were covalently these, six were patients who had carlinked with carbodiimide reagent (1-ethyl- cinomas without known metastatic involve3-[-3-dimethyl-aminopropyl]) carbodii- ment of the liver, as evidenced by normal mide hydrochloride (Sigma Chemical, St. serum biochemistry tests and liver scans, Louis, Mo.) to bovine serum albumin. six had duodenal ulcers, and 13 were Adding the tritiated bile acids to the normal volunteers. There were 37 patients original reaction mixture indicated that who had alcoholic cirrhosis, diagnosed by about 30% of the bile acids had become biopsy of the liver. In many patients the conjugated to bovine serum albumin. biopsies had been performed 12 months or Young adult female New Zealand rabbits more before the study, preventing correlawere immunized weekly for four weeks tion of radioimmunoassay data with acwith 3 0 - 4 0 /x.g of bile acid as the glycine tivity of hepatic disease. There were 14 paconjugate divided into four separate in- tients with acute hepatitis, 11 of whom had jections and injected subciitaneously as an acute viral hepatitis; two had infectious emulsion with Freund's complete adjuvant mononucleosis with hepatitis, and one had (1:1). T h e rabbits were bled after the fourth drug-induced hepatitis. There were 22 paweek and then at monthly intervals. Imme- tients with cholestasis not due to maligdiately after each bleeding a further nancy; among these were ten who had booster dose of albumin-conjugated bile stones in the common bile duct, three with sclerosing cholangitis, five with primary acid was administered.
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Three of the four glycine-conjugated bile acids, cholylglycine, chenodeoxycholylglycine, and deoxycholylglycine, were purchased from Maybridge Chemical Company, Tintagel, Cornwall, England. Sulfolithocholylglycine was a gift from Dr. Alan Hofmann. All other bile acids used for cross-reactivity studies were purchased from Maybridge Chemical Company. Radioactively labeled bile acids, tritiated in 2 - 4 position of the steroid nucleus, were purchased from New England Nuclear, Boston, Mass. Their specific activity was approximately 3.8 Ci/mmol. T h e bile acids were purified by thin-layer chromatography, running them on silicic acid plates with isoamyl acetate: propionic acid: n-propanol: water (40/30/40/ 10/v/v) as solvent, and were more than 98% pure before use.
A.J.C.P. —Vol. 66
November 1976
SERUM BILE ACIDS IN HEPATOBILIARY DISEASE
by centrifugation, decanted into liquid scintillation cocktail, and the radioactivity counted. Calculations were determined using Rodbard 27 statistics of logit plot analysis with weighted linear regression of the standard curve. Results were expressed as /i.mol glycine-conjugated bile acid per liter of serum (fimo\/\). During the course of the study, it was found that serum chenodeoxycholylglycine levels in sera of patients who had hepatic disease were often not as high as the levels of cholylglycine. A study was therefore performed to determine whether the ingestion of chenodeoxycholic acid caused an increase in immunoreactive chenodeoxycholylglycine. A single volunteer was given 1 g chenodeoxycholic acid by mouth and blood samples were taken for a 24-hour period so that serum chenodeoxycholylglycine, as well as the levels of cholylglycine and sulfolithocholylglycine, could be determined.
Radioimmunoassay of Serum Bile Acids The radioimmunoassay procedures for the four individual bile acids were performed as described by Demers and Hepner. 9 The sensitivity of these radioimmunoassays is 10 picomoles, with the standard curve extended to 80 picomoles. Pure standards of cholylglycine, chenodeoxycholylglycine, deoxycholylglycine, and sulfolithocholylglycine were dissolved in .01 jumol/1 phosphate-buffered saline solution, pH 7.4. Standards and patient serum samples in dilutions ranging from 1:3 to 1:20, depending on serum bile acid concentrations, were allowed to incubate in the presence of specific bile acid antiserum and tritium-labeled bile acids for one hour at 42 C, followed by an overnight incubation at 4 C. Following the equilibrium reaction, bile acid-free normal rabbit serum was added to each tube and the antibody-bound and free bile acids separated using 60% ammonium sulfate. The free trace supernatant was obtained
Results T h e levels of serum cholylglycine, chenodeoxycholylglycine, deoxycholylglycine, and sulfolithocholylglycine in the sera of control subjects and patients with alcoholic cirrhosis, hepatitis, cholestasis and hepatic malignancy are shown in Figures 1-4. Cholylglycine levels were significantly elevated in most patients in all hepatobiliary disease groups. The increases were similar in all groups, extending to several hundred ju.mol/1, with the exception of a few patients in the malignancy group, where elevations that reached more than 1,000 /i.mol/1 were observed. Chenodeoxycholylglycine was increased predominantly in cholestasis, although the percentage increase was lower than that of cholylglycine. Slight elevations in chenodeoxycholylglycine were found in the sera of about half the patients who had hepatitis and hepatic malignancies, but only five of the 32 patients in the cirrhosis group. Deoxycholyl-
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biliary cirrhosis, two with drug-induced cholestasis, and two with traumatic strictures of the common bile-duct. There were 40 patients who had hepatic malignancies; one of these patients had a primary hepatoma, all the others had metastatic hepatic disease. Serum albumin, bilirubin, aspartate transaminase and alkaline phosphatase were measured in all patients, using a Technicon 12/60 AutoAnalyzer. Four of the patients who had hepatitis were studied when they were not jaundiced, and in two of these serum aspartate transaminase was less than twice the upper limit of normal. The other hepatitis patients were all jaundiced (bilirubin > [1.1 mg/100 ml], > [18.8 /xmol/1]), and had serum aspartate transaminase levels more than twice the upper limit of normal. All subjects fasted overnight before blood samples were drawn for these studies.
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DEMERS AND HEPNER
1000
100
10
Z
oX 5
1.0-
0.1 -
i 5?-
100
0.01 CONTROLS ' HEPATITIS MALIGNANCY CIRRHOSIS CHOLESTASIS
FIG. 1. Serum cholylglycine levels in control subjects, and patients with portal cirrhosis, hepatitis, cholestasis, and hepatic malignancy. In the normal population, the cholylglycine range was 0.1-0.85 /Ainol/1.
£ o O
x o
>X
o U J o o
•Oh
i
2 UJ
glycine was increased mostly in cirrhosis, with only about half of the patients in the remaining groups showing increases in this bile acid. Sulfolithocholylglycine was increased in all groups and in all sera of subjects with hepatobiliary disease. It is of interest that, although cholylglycine levels were more than 1,000 times higher than control levels in sera of some patients who had hepatic disease, serum chenodeoxycholylglycine levels were normal in many of these patients, and never reached the very high levels that were seen for cholylglycine.
oI 5 CC 0.1 U
•f -
0.01 CONTROLS HEPATITIS MALIGNANCY CIRRHOSIS CHOLESTASIS
FIG. 2. Serum chenodeoxycholylglycine levels in control subjects, and patients with portal cirrhosis, hepatitis, cholestasis, and hepatic malignancy. In the normal population, the chenodeoxycholylglycine range was 0.08-0.70 ju.mol/1.
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o
The serum levels of chenodeoxycholylglycine, cholylglycine, and sulfolithocholylglycine for 24 hours after ingestion of 1 g chenodeoxycholic acid by one volunteer are shown in Figure 5. Serum chenodeoxycholylglycine increased 1.5 hours after the ingestion of the chenodeoxycholic acid, and continued to increase for the next 1.5 hours, after which time a meal was taken. Thereafter, chenodeoxycholylglycine continued to increase, and remained elevated at a level more than six times higher than the fasting level until eight hours after the ingestion of the bile acid. Twenty-four hours later, the level of chenodeoxycholylglycine was 1.4 /imol/1, still significantly higher than the fasting level, 0.5 /imol/1.
November 1976
SERUM BILE ACIDS IN HEPATOBILIARY DISEASE
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T h e relationship between serum biochemistry and serum bile acids in patients who had cirrhosis or hepatic malignancies § i.o is shown in Table 1. Five of 37 patients who had cirrhosis and 9/40 patients with hepatic malignancies had elevated serum o $ bile acid as the only biochemical abi normality. In all of these patients, the 0.1 presence of hepatic disease was confirmed by hepatic biopsy. Serum cholylglycine correlated highly with serum bilirubin in patients with hepatic malignancies (r = 0.69, p < 0.01), but less markedly in 0.01 patients vvith cholestasis (r = 0.44, p < CONTROLS HEPATITIS MALIGNANCY 0.05). In patients who had cholestasis CIRRHOSIS CHOLESTASIS FIG. 3. Serum deoxycholyglycine levels in control serum CDG also correlated significantly subjects, and patients vvith portal cirrhosis, hepatitis, with serum bilirubin (r = 0.49, p < 0.05). cholestasis, and hepatic malignancy. In the normal There was no significant correlation bepopulation, the deoxycholylglycine range was 0.01 — tween serum bilirubin and the other serum 0.09 /xmol/1. bile acids in any of the other patient groups studied. The level of serum cholylglycine did not change significantly during the first three Discussion hours after the ingestion of chenodeoxyThe assay for the four glycine-conjucholic acid, but then rose in response to gated bile acids whose serum levels were ingestion of the meal. The level of cholylglycine was within normal limits five hours after the meal, and was back to the original 10 5 fasting level at the end of the study. Serum sulfolithocholylglycine r e m a i n e d un; changed for the first six hours of the study, and then began to increase, so that 24 hours I h { after the ingestion of chenodeoxycholic 1.0 • acid sulfolithocholylglycine serum was * r 0.4 /umol/1, twice the fasting level seen i during the first five hours of the study. T h e patient served as his own control in the sense that an observable increase in & 0.1 cholylglycine occurred in response to a meal in the face of a rising titer of chenodeoxycholylglycine. Like LaRusso and co-workers, 18 we observed parallel increases in cholylglycine and chenodeoxy1 i 0.01 T - f — HEPATITIS cholylglycine in response to a solid meal. CONTROLS ' ' MALIGNANCY CIRRHOSIS CHOLESTASIS In the patient given chenodeoxycholylglycine, only chenodeoxycholylglycine was FIG. 4. Serum sulfolithocholylglycine levels in observed to increase until a meal was control subjects, and patients with portal cirrhosis, hepatitis, cholestasis, and hepatic malignancy. In the given, which then provoked an increase in normal population the sulfolithocholylglycine group range was 0.04-0.20 /xmol/1. cholylglycine, as expected.
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FIG. 5. Time courses of changes in serum cholylglycine (CG), chenodeoxycholylglycine (CDG), and sulfolithocholylglycine (SLCG) levels following oral ingestion of 1 g chenodeoxycholylglycine. A meal was taken three hours after the initial intake of chenodeoxycholylglycine.
determined in this study has not been previously described. Simmonds and associates33 first established radioimmunoassay for cholic acid conjugates; with this radioimmunoassay, both glycine and taurine conjugates of cholic acid have been determined in sera of healthy adults, patients with chronic active hepatic disease, 15 and patients with anicteric viral hepatitis. 12 In healthy adults, the reported ranges for fasting serum conjugates of cholic acid using the radioimmunoassay is between 0.1 and 1.0 /umol/1. Cholylglycine levels in our healthy adults were 0.1-0.85 )umol/l; since in healthy adults approximately 75% of bile acids are conjugated with glycine,11,34 the data from the two radioimmunoassays are in good accord. It is not clear why the cross-reactivities of the cholylglycine antibodies in our study differed from those reported by Simmonds and associates33; cross-reactivities may differ from animal to animal in other radioimmunoassays, and Hofmann has also found that cholylglycine antibodies from some animals cross-react poorly with cholyltaurine (Hofmann AF, personal communication). The levels of individual serum bile acids previously found using the method of gas chromatography reflect the total concen-
tration of individual non-sulfated bile acids, including glycine conjugates, taurine conjugates, and the free bile acids. It is expected, therefore, that the levels of glycine-conjugated bile acids we observed by radioimmunoassay would be lower than those previously found by gas chromatography, which also measures the serum taurine conjugates and free bile acids. In the largest study to date of serum bile acids in patients with a variety of hepatic diseases, Makino and co-workers, 23 using gas chromatography, found serum cholic acid levels considerably lower than that reported for cholylglycine in our study of patients with alcoholic cirrhosis, hepatitis and cholestasis. The discrepancy between the data from these two studies is particularly striking for the patients with alcoholic cirrhosis, whose mean serum cholate level reported by Makino and coworkers was 8.3 jamol/1, while in our study the mean serum cholylglycine level in patients with alcoholic cirrhosis was 57 /u,mol/l. On the other hand, serum chenodeoxycholylglycine a n d deoxycholylglycine in the cirrhotic patients in this study were substantially lower than corresponding levels in the cirrhotic patients studied by Makino and co-workers. 23 It is interesting that cholylglycine and sul-
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November 1976
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SERUM BILE ACIDS IN HEPATOBILIARY DISEASE
specific chenodeoxycholylglycine antibodies. It is possible that with hepatic disease, increased chenodeoxycholic acid in the serum is largely sulfated. 4,20,2I,35,3ti Although Stiehl and associates found only 4 - 5 % sulfation of serum bile acids in two cirrhotic patients they studied, 35 they found that in 17 cirrhotic patients more than 75% of the urinary dihydroxy bile acids were sulfated. Makino and co-workers 21 also found that more than 80% of serum bile acids were sulfated in patients with hepatobiliary disease. One possibility that might also be considered is that in patients who have hepatobiliary disease serum bile acids may undergo biotransformation to hyocholic acid 36 or become conjugated with unusual acids, such as glucuronic acid.3 There is also the possibility that with hepatic disease there may be an increase in unconjugated chenodeoxycholic acid or taurine-conjugated chenodeoxycholic acid, which does not cross-react significantly in the present radioimmunoassay with chenodeoxycholylglycine. T h e increased sulfolithocholylglycine found in all sera of patients with hepatic disease in the present study is of considerable interest. Sulfation of monohydroxy bile acid was first described by Palmer, who identified sulfate esters of lithocholyltaurine and lithocholylglycine in the bile. The range of sulfolithocholylglycine values for controls in our study was 0.04-0.20 /nmol/1, similar to the level of sulfolithocholic acid in healthy women reported by Back and associates. 4 T h e striking increases of sulfolithocholylglycine in all sera of patients with hepatic disease in
Table 1. Hepatic-disease Patients in Whom Elevated Serum Bile Acid was the Only Biochemical Abnormality* Total
Cholylglycine
Chenodeoxycholylglycine
Cirrhosis (37) Cancer (40) * Albumin, bilirubin. SCOT and alkaline phosphatase were normal in these patienl.s.
Deoxycholy! glycine
Sulfolithocholylglycine
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folithocholylglycine were similarly elevated in all hepatobiliary disease groups and not disproportionately higher in the cholestasis groups. This may be due to increased sulfation of bile acids in cholestasis or to differences in bile acid clearance by the liver. The observation that chenodeoxycholylglycine levels in patients with alcoholic cirrhosis were lower than those of cholylglycine differs from the observations of previous investigators 1,6,28,32,37 who, using gas-liquid chromatography, have found that chenodeoxycholic acid rises to higher levels in the sera of patients who have alcoholic cirrhosis than does cholic acid. Interestingly, while normal levels of chenodeoxycholylglycine were found in a large percentage of sera of our patients with alcoholic cirrhosis, the level of the other primary bile acid, cholylglycine, was normal in only two sera. On the other hand, chenodeoxycholylglycine was consistently elevated in the sera of patients who had hepatitis and cholestasis, although the levels were lower than those levels found by previous investigators measuring free chenodeoxycholic acid. T h e difference between the present data for immunoreactive serum chenodeoxycholylglycine and the data for chenodeoxycholic acid measured by gas chromatography in sera of patients with alcoholic cirrhosis suggests that in hepatic disease, especially alcoholic cirrhosis, when chenodeoxycholic acid accumulates in the systemic circulation it may undergo biotransformations 30 such as sulfation that render "chenodeoxycholylglycine" unreactive to the immunologically
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Methodik sowie Ergebnisse bei Patienten mit akuter Hepatitis. Z Gastroenterol 10:349-358, 1972 11. Garbutt JT, Lack L, Tyor MP: Physiological basis of alterations in the relative conjugation of bile acids with glycine and taurine. Am ] Clin Nutr 24:218-228, 1971 12. Hofmann AF, Korman MG, Krugman S: Sensitivity of serum bile acid assay for detection of liver damage in viral hepatitis Type B. Prospective study in five patients. Am J Dig Dis 19:908-911, 1974 13. Javitt NB, Morrissey KP, Siegel E, etal: Cholestastic syndromes in infancy: Diagnostic value of serum bile acid pattern and cholestryamine administration. Pediatr Res 7:119-125, 1973 14. Kaplowitz N, Kok E, Javitt NB: Postprandial serum bile acid for the detection of hepatobiliary disease. JAMA 225:292-293, 1973 15. Korman MG, Hofmann AF, Summerskill WHJ: Assessment of activity of chronic active liver disease: Serum bile acids compared with conventional tests and histology. N Engl J Med 290:1399-1402, 1974 16. Korman MG, LaRusso NF, Hoffman NE, et al: Intravenous bile acid tolerance test in health. N Engl J Med 292:1205-1209, 1975 17. LaRusso NF, Hoffman NE, Hofmann AF, et al: Validity and sensitivity of an intravenous bile acid tolerance test in patients with liver disease. N Engl J Med 292:1209-1214, 1975 18. LaRusso NF, Korman MG, Hoffman NE, et al: Dynamics of the enterohepatic circulation of Acknowledgment. Technical assistance was probile acids. Postprandial serum concentrations vided by Ms. Kathy Smith and Ms. Naomi Cole. of conjugates of cholic acid in health, cholecystectomized patients, and patients with bile acid malabsorption. N Engl J Med 291:689References 692, 1974 1. Back P, Spaczynski K, Gerok W: Bile-salt glu- 19. Maclntyre I, Wootton IDP: Clinical biochemistry. curonides in urine. Hoppe-Seyler's Z Physiol I. Bile acids in blood. Annu Rev Biochem 29: Chem 355:749-752, 1974 635-641, 1960 2. Back P: Identification and quantitative deter- 20. Makino I, Shinozaki K, Nakagawa S, et al: mination of urinary bile acids excreted in Measurement of sulfated and non-sulfated cholestasis. Clin Chim Acta 44:199-207, 1973 bile acids in human serum and urine. J Lipid 3. Back P, Spaczynski K, Gerok W: Bile-salt gluRes 15:132-138, 1974 curonides in urine. Hoppe-Seyler's Z Physiol 21. Makino I, Hashimoto H, Shinozaki K, et al: Chem 355:749-752, 1974 Sulfated and non-sulfated bile acids in urine, 4. Back P, Sjovall J, Sjovall K: Monohydroxy bile serum, and bile of patients with hepatobiliary acids in plasma in intrahepatic cholestasis of disease. Gastroenterology 68:545-553, 1975 pregnancy. Identification by computerized 22. Makino I, Nakagawa S, Shinozaki K, et al: gas chromatography —mass spectrometry. Sulfated and non-sulfated bile acid in human Med Biol 52:31-38, 1974 serum. Lipids 7:750-752, 1972 5. Blomstrand R: Gas-liquid chromatography of 23. Makino I, Nakagawa S, Mashimo K: Conjugated human bile acids. Proc Soc Exp Biol Med 107: and unconjugated serum bile acid levels in 126-128, 1960 patients with hepatobiliary disease. Gastro6. Carey JB Jr: The serum trihydroxy-dihydroxy enterology 56:1033-1039, 1969 bile acid ratio in liver and biliary tract disease. 24. Neale G, Lewis B, Weaver V, et al: Serum bile J Clin Invest 37:1494-1503, 1958 acids in liver disease. Gut 12:145-152, 1971 7. Carey JB Jr: Bile acid in serum of jaundice pa- 25. Osborn EC, Wootton IDP, Da Silva LC, et al: tients. Gastroenterology 41:285-287, 1961 Serum bile acids in liver disease. Lancet 2: 8. Carey JB Jr: Lithocholic acid in human blood 1049-1053, 1959 serum. Science 150:620-622. 1965 26. Palmer RH, Bolt MG: Bile acid sulfates. I. 9. Demers LM, Hepner GW: Radioimmunoassay of Synthesis of lithocholic acid sulfates and their serum bile acids. Clin Chem 245:602-606, identification in human bile. ] Lipid Res 1976 12:671-679, 1971 10. Erb W, Schreiber J. Walczak M: Gaschromatische 27. Rodbard D: Statistical quality control and Untersuchunge der Serumgallensauren: routine data processing for radioimmunoassays
this study suggest that this bile acid is less well cleared by the liver than glycine conjugates of dihydroxy or trihydroxy bile acids. This poor clearance may also explain the presence of this secondary bile acid in the sera of patients who have cholestasis. In these patients interruption of the enterohepatic circulation might be expected to cause a decrease in the production of chenodeoxycholic acid bacterial metabolites. T h e potential usefulness of measuring serum sulfolithocholylglycine to screen patients with suspected hepatobiliary disease is thus clearly considerable. Since no patient who had hepatobiliary disease had normal serum sulfolithocholylglycine, measurement of this serum bile acid would appear to be a more convenient hepatobiliary function test than the intravenous bile acid tolerance test recently proposed by Hofmann and colleagues. 16-17
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30.
31. 32.
SERUM BILE ACIDS IN HEPATOBILIARY DISEASE
33. Simnionds WJ, Kornian MG, Go VWL, el al: Radioimmunoassay of conjugated cholyl bile acids in serum. Gastroenterology 65:705-711, 1973 34. Sjovall J: Bile acids in man under normal and pathological conditions. Bile acids and steroids 73. Clin Chem Acta 5:33-41, 1960 35. Stiehl A, Earnest DL, Admirand WH: Sulfation and renal excretion of bile salts in patients with cirrhosis of the liver. Gastroenterology 68: 534-544, 1975 36. Van Berge Henegouwen GP, Brandt K-H. Eyssen H, et al: Variations in serum and urinary bile acid patterns in patients with cholestasis (abstr). Gastroenterology 68:1005, 1975 37. Van Berge Henegouwen GP, Ruben A, Brandt K-H: Quantitative analysis of bile acids in serum and bile, using gas-liquid chromatography. Clin Chim Acta 54:249-261. 1974
News and Notices Clinical Cytopathology for Pathologists—Postgraduate Course The Eighteenth Postgraduate Institute for Pathologists in Clinical Cytopathology is to be given at The Johns Hopkins University School of Medicine and The Johns Hopkins Hospital, Baltimore, Maryland, April 11-22, 1977. The full two-week program is designed for Pathologists who are Certified (or qualified) by the American Board of Pathology, (PA), or their international equivalents. It will provide an intensive refresher in all aspects of the field of Clinical Cytopathology, with time devoted to newer technics, special problems, and recent applications. Topics will be covered in lectures, explored in small informal conferences, and discussed over the microscope with the Faculty. Self-instructional material will be available to augment at individual pace. A loan set of slides with text will be sent to each participant for home-study during March and April before the Institute. Credit hours 120 in A MA Category 1. Application is to be made before February 28, 1977. For details, write: John K. Frost, M.D., 610 Pathology Building, The Johns Hopkins Hospital, Baltimore, Maryland 21205, U. S. A. The entire course is given in English.
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and immunoradiometric assays. Clin Chem 20: 1255-1270, 1974 Roovers J, Evrard E, Vanderhaege H: An improved method for measuring human blood bile acids. Clin Chim Acta 19:449-457, 1968 Rudman D, Kendall RE: Bile acid content of human serum. I. Serum bile acids in patients with hepatic disease. J Clin Invest 36:530-537, 1957 Salen G, Tint GS, Eliav B, et al.: Increased formation of ursodeoxycholic acid in patients treated with chenodeoxycholic acid. J Clin Invest 53:612-621, 1974 Sandberg DH: Bile acid concentrations in serum during infancy and childhood. Pediatr Res 4: 262-267, 1970 Sandberg DH, Sjovall J, Sjovall K, et al: Measurement of human serum bile acids by gasliquid chromatography. J Lipid Res 6:182192, 1965
839
o f
Bile
I m m u n o r e a c t i v e
Acids
in
H e a l t h
a n d
G l y c i n e - c o n j u g a t e d Hepatobiliary
Disease LAURENCE M. DEMERS, P H . D . , AND GERSHON W. HEPNER,
M.D.
Departments of Pathology arid Medicine, The Milton S. Hershey Medical Center, The Pennsylvania University, Hershey, Pennsylvania 17033 ABSTRACT
study the dynamics of the enterohepatic circulation of cholate conjugates. 18 In further studies using this radioimmunoassay, it was found that levels of cholate conjugates were elevated in patients who had chronic active hepatitis when other liver function tests were normal 15 and in 12 Received April 26, 1976; received revised manu- patients with anicteric viral hepatitis. script June 9, 1976; accepted for publication June 9, We have developed a radioimmunoassay 1976. Supported in part by Grant AM 17303 from the for four glycine-conjugated bile acids— National Institute of Health and by a grant from the cholic, chenodeoxycholic, deoxycholic, and Pharmaceutical Manufacturers' Association Foundasulfolithocholic acids. T h e radioimmunotion. Address reprint requests to Dr. Demers: Depart- assay is highly specific for each of these ment of Pathology, M.S. Hershey Medical Center, four bile acids, whose levels were measured Pennsylvania Slate University, Hershey, Pennsylvania in sera from fasting healthy control sub17033.
SERUM BILE ACIDS are
known to
be
in-
creased in patients who have hepatobiliary diseise '• 2 ' 4_8 ' 10 ' 12_1S - 19 ' 22_25 - 28 ' 29 - 31-33 ' 37 A sensitive radioimmunoassay for conjugates of cholic acid was first described by Simmonds and associates33 and was used to
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Demers, Laurence M., and Hepner, Gershon W.: Levels of immunoreactive glycine-conjugated bile acids in health and hepatobiliary disease. Am J Clin Pathol 66: 831-839, 1976. A sensitive radioimmunoassay for cholylglycine, chenodeoxycholylglycine, deoxycholylglycine, and sulfolithocholylglycine was established using antibodies obtained from rabbits injected with albumin conjugates of these bile acids. Glycine-conjugated bile acid levels were measured in sera from 25 control subjects and 110 patients who had hepatic disease (alcoholic cirrhosis, hepatitis, cholestasis, and hepatic malignancy). Sulfolithocholylglycine was elevated in the sera of all 110 patients with hepatic disease. Cholylglycine was within normal range in only three. Chenodeoxycholylglycine was elevated in most sera of patients who had hepatitis, cholestasis, or hepatic malignancy. It was normal in most sera of patients who had alcoholic cirrhosis, suggesting that chenodeoxycholic acid may be subject to further biotransformations in these patients. Deoxycholylglycine was elevated in a minority of patients, none of whom had cholestasis. T h e data suggest that serum bile acids, particularly sulfolithocholylglycine, are a highly sensitive index for hepatic dysfunction. (Key words: Liver disease; Bile acids; Hepatobiliary disease; Liver function test; Radioimmunoassay.)
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jects and patients who had portal cirrhosis, hepatitis, hepatic cancer, or cholestasis of extrahepatic or intrahepatic origin. Materials and Methods Radioimmunoassay of Bile Acids
T h e antiserum produced was highly specific, showing only minimal crossreactivity when tested against other structurally related bile acid compounds. There was less than 1% cross-reactivity between any two glycine-conjugated bile acids; there was 18% cross-reactivity between the glycine-conjugated bile acids and their taurine-conjugated analogs, and there was 1% cross-reactivity between the glycineconjugated bile acids and the free bile acid analogs. There was less than 1% cross-reactivity between sulfolithocholylglycine antibodies and the mono- and disulfate derivatives of chenodeoxycholylglycine and deoxycholylglycine, which were prepared by a modification of the method of Palmer and Bolt.26 Crossreactivity studies along with recovery and parallelism studies to test the reproducibility, accuracy, and precision of the bile acid radioimmunoassay have been performed. 9 Patients Studied.
The subjects studied included 25 controls without known hepatic disease; of T h e unlabeled bile acids were covalently these, six were patients who had carlinked with carbodiimide reagent (1-ethyl- cinomas without known metastatic involve3-[-3-dimethyl-aminopropyl]) carbodii- ment of the liver, as evidenced by normal mide hydrochloride (Sigma Chemical, St. serum biochemistry tests and liver scans, Louis, Mo.) to bovine serum albumin. six had duodenal ulcers, and 13 were Adding the tritiated bile acids to the normal volunteers. There were 37 patients original reaction mixture indicated that who had alcoholic cirrhosis, diagnosed by about 30% of the bile acids had become biopsy of the liver. In many patients the conjugated to bovine serum albumin. biopsies had been performed 12 months or Young adult female New Zealand rabbits more before the study, preventing correlawere immunized weekly for four weeks tion of radioimmunoassay data with acwith 3 0 - 4 0 /x.g of bile acid as the glycine tivity of hepatic disease. There were 14 paconjugate divided into four separate in- tients with acute hepatitis, 11 of whom had jections and injected subciitaneously as an acute viral hepatitis; two had infectious emulsion with Freund's complete adjuvant mononucleosis with hepatitis, and one had (1:1). T h e rabbits were bled after the fourth drug-induced hepatitis. There were 22 paweek and then at monthly intervals. Imme- tients with cholestasis not due to maligdiately after each bleeding a further nancy; among these were ten who had booster dose of albumin-conjugated bile stones in the common bile duct, three with sclerosing cholangitis, five with primary acid was administered.
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Three of the four glycine-conjugated bile acids, cholylglycine, chenodeoxycholylglycine, and deoxycholylglycine, were purchased from Maybridge Chemical Company, Tintagel, Cornwall, England. Sulfolithocholylglycine was a gift from Dr. Alan Hofmann. All other bile acids used for cross-reactivity studies were purchased from Maybridge Chemical Company. Radioactively labeled bile acids, tritiated in 2 - 4 position of the steroid nucleus, were purchased from New England Nuclear, Boston, Mass. Their specific activity was approximately 3.8 Ci/mmol. T h e bile acids were purified by thin-layer chromatography, running them on silicic acid plates with isoamyl acetate: propionic acid: n-propanol: water (40/30/40/ 10/v/v) as solvent, and were more than 98% pure before use.
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by centrifugation, decanted into liquid scintillation cocktail, and the radioactivity counted. Calculations were determined using Rodbard 27 statistics of logit plot analysis with weighted linear regression of the standard curve. Results were expressed as /i.mol glycine-conjugated bile acid per liter of serum (fimo\/\). During the course of the study, it was found that serum chenodeoxycholylglycine levels in sera of patients who had hepatic disease were often not as high as the levels of cholylglycine. A study was therefore performed to determine whether the ingestion of chenodeoxycholic acid caused an increase in immunoreactive chenodeoxycholylglycine. A single volunteer was given 1 g chenodeoxycholic acid by mouth and blood samples were taken for a 24-hour period so that serum chenodeoxycholylglycine, as well as the levels of cholylglycine and sulfolithocholylglycine, could be determined.
Radioimmunoassay of Serum Bile Acids The radioimmunoassay procedures for the four individual bile acids were performed as described by Demers and Hepner. 9 The sensitivity of these radioimmunoassays is 10 picomoles, with the standard curve extended to 80 picomoles. Pure standards of cholylglycine, chenodeoxycholylglycine, deoxycholylglycine, and sulfolithocholylglycine were dissolved in .01 jumol/1 phosphate-buffered saline solution, pH 7.4. Standards and patient serum samples in dilutions ranging from 1:3 to 1:20, depending on serum bile acid concentrations, were allowed to incubate in the presence of specific bile acid antiserum and tritium-labeled bile acids for one hour at 42 C, followed by an overnight incubation at 4 C. Following the equilibrium reaction, bile acid-free normal rabbit serum was added to each tube and the antibody-bound and free bile acids separated using 60% ammonium sulfate. The free trace supernatant was obtained
Results T h e levels of serum cholylglycine, chenodeoxycholylglycine, deoxycholylglycine, and sulfolithocholylglycine in the sera of control subjects and patients with alcoholic cirrhosis, hepatitis, cholestasis and hepatic malignancy are shown in Figures 1-4. Cholylglycine levels were significantly elevated in most patients in all hepatobiliary disease groups. The increases were similar in all groups, extending to several hundred ju.mol/1, with the exception of a few patients in the malignancy group, where elevations that reached more than 1,000 /i.mol/1 were observed. Chenodeoxycholylglycine was increased predominantly in cholestasis, although the percentage increase was lower than that of cholylglycine. Slight elevations in chenodeoxycholylglycine were found in the sera of about half the patients who had hepatitis and hepatic malignancies, but only five of the 32 patients in the cirrhosis group. Deoxycholyl-
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biliary cirrhosis, two with drug-induced cholestasis, and two with traumatic strictures of the common bile-duct. There were 40 patients who had hepatic malignancies; one of these patients had a primary hepatoma, all the others had metastatic hepatic disease. Serum albumin, bilirubin, aspartate transaminase and alkaline phosphatase were measured in all patients, using a Technicon 12/60 AutoAnalyzer. Four of the patients who had hepatitis were studied when they were not jaundiced, and in two of these serum aspartate transaminase was less than twice the upper limit of normal. The other hepatitis patients were all jaundiced (bilirubin > [1.1 mg/100 ml], > [18.8 /xmol/1]), and had serum aspartate transaminase levels more than twice the upper limit of normal. All subjects fasted overnight before blood samples were drawn for these studies.
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1000
100
10
Z
oX 5
1.0-
0.1 -
i 5?-
100
0.01 CONTROLS ' HEPATITIS MALIGNANCY CIRRHOSIS CHOLESTASIS
FIG. 1. Serum cholylglycine levels in control subjects, and patients with portal cirrhosis, hepatitis, cholestasis, and hepatic malignancy. In the normal population, the cholylglycine range was 0.1-0.85 /Ainol/1.
£ o O
x o
>X
o U J o o
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i
2 UJ
glycine was increased mostly in cirrhosis, with only about half of the patients in the remaining groups showing increases in this bile acid. Sulfolithocholylglycine was increased in all groups and in all sera of subjects with hepatobiliary disease. It is of interest that, although cholylglycine levels were more than 1,000 times higher than control levels in sera of some patients who had hepatic disease, serum chenodeoxycholylglycine levels were normal in many of these patients, and never reached the very high levels that were seen for cholylglycine.
oI 5 CC 0.1 U
•f -
0.01 CONTROLS HEPATITIS MALIGNANCY CIRRHOSIS CHOLESTASIS
FIG. 2. Serum chenodeoxycholylglycine levels in control subjects, and patients with portal cirrhosis, hepatitis, cholestasis, and hepatic malignancy. In the normal population, the chenodeoxycholylglycine range was 0.08-0.70 ju.mol/1.
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o
The serum levels of chenodeoxycholylglycine, cholylglycine, and sulfolithocholylglycine for 24 hours after ingestion of 1 g chenodeoxycholic acid by one volunteer are shown in Figure 5. Serum chenodeoxycholylglycine increased 1.5 hours after the ingestion of the chenodeoxycholic acid, and continued to increase for the next 1.5 hours, after which time a meal was taken. Thereafter, chenodeoxycholylglycine continued to increase, and remained elevated at a level more than six times higher than the fasting level until eight hours after the ingestion of the bile acid. Twenty-four hours later, the level of chenodeoxycholylglycine was 1.4 /imol/1, still significantly higher than the fasting level, 0.5 /imol/1.
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T h e relationship between serum biochemistry and serum bile acids in patients who had cirrhosis or hepatic malignancies § i.o is shown in Table 1. Five of 37 patients who had cirrhosis and 9/40 patients with hepatic malignancies had elevated serum o $ bile acid as the only biochemical abi normality. In all of these patients, the 0.1 presence of hepatic disease was confirmed by hepatic biopsy. Serum cholylglycine correlated highly with serum bilirubin in patients with hepatic malignancies (r = 0.69, p < 0.01), but less markedly in 0.01 patients vvith cholestasis (r = 0.44, p < CONTROLS HEPATITIS MALIGNANCY 0.05). In patients who had cholestasis CIRRHOSIS CHOLESTASIS FIG. 3. Serum deoxycholyglycine levels in control serum CDG also correlated significantly subjects, and patients vvith portal cirrhosis, hepatitis, with serum bilirubin (r = 0.49, p < 0.05). cholestasis, and hepatic malignancy. In the normal There was no significant correlation bepopulation, the deoxycholylglycine range was 0.01 — tween serum bilirubin and the other serum 0.09 /xmol/1. bile acids in any of the other patient groups studied. The level of serum cholylglycine did not change significantly during the first three Discussion hours after the ingestion of chenodeoxyThe assay for the four glycine-conjucholic acid, but then rose in response to gated bile acids whose serum levels were ingestion of the meal. The level of cholylglycine was within normal limits five hours after the meal, and was back to the original 10 5 fasting level at the end of the study. Serum sulfolithocholylglycine r e m a i n e d un; changed for the first six hours of the study, and then began to increase, so that 24 hours I h { after the ingestion of chenodeoxycholic 1.0 • acid sulfolithocholylglycine serum was * r 0.4 /umol/1, twice the fasting level seen i during the first five hours of the study. T h e patient served as his own control in the sense that an observable increase in & 0.1 cholylglycine occurred in response to a meal in the face of a rising titer of chenodeoxycholylglycine. Like LaRusso and co-workers, 18 we observed parallel increases in cholylglycine and chenodeoxy1 i 0.01 T - f — HEPATITIS cholylglycine in response to a solid meal. CONTROLS ' ' MALIGNANCY CIRRHOSIS CHOLESTASIS In the patient given chenodeoxycholylglycine, only chenodeoxycholylglycine was FIG. 4. Serum sulfolithocholylglycine levels in observed to increase until a meal was control subjects, and patients with portal cirrhosis, hepatitis, cholestasis, and hepatic malignancy. In the given, which then provoked an increase in normal population the sulfolithocholylglycine group range was 0.04-0.20 /xmol/1. cholylglycine, as expected.
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FIG. 5. Time courses of changes in serum cholylglycine (CG), chenodeoxycholylglycine (CDG), and sulfolithocholylglycine (SLCG) levels following oral ingestion of 1 g chenodeoxycholylglycine. A meal was taken three hours after the initial intake of chenodeoxycholylglycine.
determined in this study has not been previously described. Simmonds and associates33 first established radioimmunoassay for cholic acid conjugates; with this radioimmunoassay, both glycine and taurine conjugates of cholic acid have been determined in sera of healthy adults, patients with chronic active hepatic disease, 15 and patients with anicteric viral hepatitis. 12 In healthy adults, the reported ranges for fasting serum conjugates of cholic acid using the radioimmunoassay is between 0.1 and 1.0 /umol/1. Cholylglycine levels in our healthy adults were 0.1-0.85 )umol/l; since in healthy adults approximately 75% of bile acids are conjugated with glycine,11,34 the data from the two radioimmunoassays are in good accord. It is not clear why the cross-reactivities of the cholylglycine antibodies in our study differed from those reported by Simmonds and associates33; cross-reactivities may differ from animal to animal in other radioimmunoassays, and Hofmann has also found that cholylglycine antibodies from some animals cross-react poorly with cholyltaurine (Hofmann AF, personal communication). The levels of individual serum bile acids previously found using the method of gas chromatography reflect the total concen-
tration of individual non-sulfated bile acids, including glycine conjugates, taurine conjugates, and the free bile acids. It is expected, therefore, that the levels of glycine-conjugated bile acids we observed by radioimmunoassay would be lower than those previously found by gas chromatography, which also measures the serum taurine conjugates and free bile acids. In the largest study to date of serum bile acids in patients with a variety of hepatic diseases, Makino and co-workers, 23 using gas chromatography, found serum cholic acid levels considerably lower than that reported for cholylglycine in our study of patients with alcoholic cirrhosis, hepatitis and cholestasis. The discrepancy between the data from these two studies is particularly striking for the patients with alcoholic cirrhosis, whose mean serum cholate level reported by Makino and coworkers was 8.3 jamol/1, while in our study the mean serum cholylglycine level in patients with alcoholic cirrhosis was 57 /u,mol/l. On the other hand, serum chenodeoxycholylglycine a n d deoxycholylglycine in the cirrhotic patients in this study were substantially lower than corresponding levels in the cirrhotic patients studied by Makino and co-workers. 23 It is interesting that cholylglycine and sul-
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specific chenodeoxycholylglycine antibodies. It is possible that with hepatic disease, increased chenodeoxycholic acid in the serum is largely sulfated. 4,20,2I,35,3ti Although Stiehl and associates found only 4 - 5 % sulfation of serum bile acids in two cirrhotic patients they studied, 35 they found that in 17 cirrhotic patients more than 75% of the urinary dihydroxy bile acids were sulfated. Makino and co-workers 21 also found that more than 80% of serum bile acids were sulfated in patients with hepatobiliary disease. One possibility that might also be considered is that in patients who have hepatobiliary disease serum bile acids may undergo biotransformation to hyocholic acid 36 or become conjugated with unusual acids, such as glucuronic acid.3 There is also the possibility that with hepatic disease there may be an increase in unconjugated chenodeoxycholic acid or taurine-conjugated chenodeoxycholic acid, which does not cross-react significantly in the present radioimmunoassay with chenodeoxycholylglycine. T h e increased sulfolithocholylglycine found in all sera of patients with hepatic disease in the present study is of considerable interest. Sulfation of monohydroxy bile acid was first described by Palmer, who identified sulfate esters of lithocholyltaurine and lithocholylglycine in the bile. The range of sulfolithocholylglycine values for controls in our study was 0.04-0.20 /nmol/1, similar to the level of sulfolithocholic acid in healthy women reported by Back and associates. 4 T h e striking increases of sulfolithocholylglycine in all sera of patients with hepatic disease in
Table 1. Hepatic-disease Patients in Whom Elevated Serum Bile Acid was the Only Biochemical Abnormality* Total
Cholylglycine
Chenodeoxycholylglycine
Cirrhosis (37) Cancer (40) * Albumin, bilirubin. SCOT and alkaline phosphatase were normal in these patienl.s.
Deoxycholy! glycine
Sulfolithocholylglycine
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folithocholylglycine were similarly elevated in all hepatobiliary disease groups and not disproportionately higher in the cholestasis groups. This may be due to increased sulfation of bile acids in cholestasis or to differences in bile acid clearance by the liver. The observation that chenodeoxycholylglycine levels in patients with alcoholic cirrhosis were lower than those of cholylglycine differs from the observations of previous investigators 1,6,28,32,37 who, using gas-liquid chromatography, have found that chenodeoxycholic acid rises to higher levels in the sera of patients who have alcoholic cirrhosis than does cholic acid. Interestingly, while normal levels of chenodeoxycholylglycine were found in a large percentage of sera of our patients with alcoholic cirrhosis, the level of the other primary bile acid, cholylglycine, was normal in only two sera. On the other hand, chenodeoxycholylglycine was consistently elevated in the sera of patients who had hepatitis and cholestasis, although the levels were lower than those levels found by previous investigators measuring free chenodeoxycholic acid. T h e difference between the present data for immunoreactive serum chenodeoxycholylglycine and the data for chenodeoxycholic acid measured by gas chromatography in sera of patients with alcoholic cirrhosis suggests that in hepatic disease, especially alcoholic cirrhosis, when chenodeoxycholic acid accumulates in the systemic circulation it may undergo biotransformations 30 such as sulfation that render "chenodeoxycholylglycine" unreactive to the immunologically
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Methodik sowie Ergebnisse bei Patienten mit akuter Hepatitis. Z Gastroenterol 10:349-358, 1972 11. Garbutt JT, Lack L, Tyor MP: Physiological basis of alterations in the relative conjugation of bile acids with glycine and taurine. Am ] Clin Nutr 24:218-228, 1971 12. Hofmann AF, Korman MG, Krugman S: Sensitivity of serum bile acid assay for detection of liver damage in viral hepatitis Type B. Prospective study in five patients. Am J Dig Dis 19:908-911, 1974 13. Javitt NB, Morrissey KP, Siegel E, etal: Cholestastic syndromes in infancy: Diagnostic value of serum bile acid pattern and cholestryamine administration. Pediatr Res 7:119-125, 1973 14. Kaplowitz N, Kok E, Javitt NB: Postprandial serum bile acid for the detection of hepatobiliary disease. JAMA 225:292-293, 1973 15. Korman MG, Hofmann AF, Summerskill WHJ: Assessment of activity of chronic active liver disease: Serum bile acids compared with conventional tests and histology. N Engl J Med 290:1399-1402, 1974 16. Korman MG, LaRusso NF, Hoffman NE, et al: Intravenous bile acid tolerance test in health. N Engl J Med 292:1205-1209, 1975 17. LaRusso NF, Hoffman NE, Hofmann AF, et al: Validity and sensitivity of an intravenous bile acid tolerance test in patients with liver disease. N Engl J Med 292:1209-1214, 1975 18. LaRusso NF, Korman MG, Hoffman NE, et al: Dynamics of the enterohepatic circulation of Acknowledgment. Technical assistance was probile acids. Postprandial serum concentrations vided by Ms. Kathy Smith and Ms. Naomi Cole. of conjugates of cholic acid in health, cholecystectomized patients, and patients with bile acid malabsorption. N Engl J Med 291:689References 692, 1974 1. Back P, Spaczynski K, Gerok W: Bile-salt glu- 19. Maclntyre I, Wootton IDP: Clinical biochemistry. curonides in urine. Hoppe-Seyler's Z Physiol I. Bile acids in blood. Annu Rev Biochem 29: Chem 355:749-752, 1974 635-641, 1960 2. Back P: Identification and quantitative deter- 20. Makino I, Shinozaki K, Nakagawa S, et al: mination of urinary bile acids excreted in Measurement of sulfated and non-sulfated cholestasis. Clin Chim Acta 44:199-207, 1973 bile acids in human serum and urine. J Lipid 3. Back P, Spaczynski K, Gerok W: Bile-salt gluRes 15:132-138, 1974 curonides in urine. Hoppe-Seyler's Z Physiol 21. Makino I, Hashimoto H, Shinozaki K, et al: Chem 355:749-752, 1974 Sulfated and non-sulfated bile acids in urine, 4. Back P, Sjovall J, Sjovall K: Monohydroxy bile serum, and bile of patients with hepatobiliary acids in plasma in intrahepatic cholestasis of disease. Gastroenterology 68:545-553, 1975 pregnancy. Identification by computerized 22. Makino I, Nakagawa S, Shinozaki K, et al: gas chromatography —mass spectrometry. Sulfated and non-sulfated bile acid in human Med Biol 52:31-38, 1974 serum. Lipids 7:750-752, 1972 5. Blomstrand R: Gas-liquid chromatography of 23. Makino I, Nakagawa S, Mashimo K: Conjugated human bile acids. Proc Soc Exp Biol Med 107: and unconjugated serum bile acid levels in 126-128, 1960 patients with hepatobiliary disease. Gastro6. Carey JB Jr: The serum trihydroxy-dihydroxy enterology 56:1033-1039, 1969 bile acid ratio in liver and biliary tract disease. 24. Neale G, Lewis B, Weaver V, et al: Serum bile J Clin Invest 37:1494-1503, 1958 acids in liver disease. Gut 12:145-152, 1971 7. Carey JB Jr: Bile acid in serum of jaundice pa- 25. Osborn EC, Wootton IDP, Da Silva LC, et al: tients. Gastroenterology 41:285-287, 1961 Serum bile acids in liver disease. Lancet 2: 8. Carey JB Jr: Lithocholic acid in human blood 1049-1053, 1959 serum. Science 150:620-622. 1965 26. Palmer RH, Bolt MG: Bile acid sulfates. I. 9. Demers LM, Hepner GW: Radioimmunoassay of Synthesis of lithocholic acid sulfates and their serum bile acids. Clin Chem 245:602-606, identification in human bile. ] Lipid Res 1976 12:671-679, 1971 10. Erb W, Schreiber J. Walczak M: Gaschromatische 27. Rodbard D: Statistical quality control and Untersuchunge der Serumgallensauren: routine data processing for radioimmunoassays
this study suggest that this bile acid is less well cleared by the liver than glycine conjugates of dihydroxy or trihydroxy bile acids. This poor clearance may also explain the presence of this secondary bile acid in the sera of patients who have cholestasis. In these patients interruption of the enterohepatic circulation might be expected to cause a decrease in the production of chenodeoxycholic acid bacterial metabolites. T h e potential usefulness of measuring serum sulfolithocholylglycine to screen patients with suspected hepatobiliary disease is thus clearly considerable. Since no patient who had hepatobiliary disease had normal serum sulfolithocholylglycine, measurement of this serum bile acid would appear to be a more convenient hepatobiliary function test than the intravenous bile acid tolerance test recently proposed by Hofmann and colleagues. 16-17
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30.
31. 32.
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33. Simnionds WJ, Kornian MG, Go VWL, el al: Radioimmunoassay of conjugated cholyl bile acids in serum. Gastroenterology 65:705-711, 1973 34. Sjovall J: Bile acids in man under normal and pathological conditions. Bile acids and steroids 73. Clin Chem Acta 5:33-41, 1960 35. Stiehl A, Earnest DL, Admirand WH: Sulfation and renal excretion of bile salts in patients with cirrhosis of the liver. Gastroenterology 68: 534-544, 1975 36. Van Berge Henegouwen GP, Brandt K-H. Eyssen H, et al: Variations in serum and urinary bile acid patterns in patients with cholestasis (abstr). Gastroenterology 68:1005, 1975 37. Van Berge Henegouwen GP, Ruben A, Brandt K-H: Quantitative analysis of bile acids in serum and bile, using gas-liquid chromatography. Clin Chim Acta 54:249-261. 1974
News and Notices Clinical Cytopathology for Pathologists—Postgraduate Course The Eighteenth Postgraduate Institute for Pathologists in Clinical Cytopathology is to be given at The Johns Hopkins University School of Medicine and The Johns Hopkins Hospital, Baltimore, Maryland, April 11-22, 1977. The full two-week program is designed for Pathologists who are Certified (or qualified) by the American Board of Pathology, (PA), or their international equivalents. It will provide an intensive refresher in all aspects of the field of Clinical Cytopathology, with time devoted to newer technics, special problems, and recent applications. Topics will be covered in lectures, explored in small informal conferences, and discussed over the microscope with the Faculty. Self-instructional material will be available to augment at individual pace. A loan set of slides with text will be sent to each participant for home-study during March and April before the Institute. Credit hours 120 in A MA Category 1. Application is to be made before February 28, 1977. For details, write: John K. Frost, M.D., 610 Pathology Building, The Johns Hopkins Hospital, Baltimore, Maryland 21205, U. S. A. The entire course is given in English.
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and immunoradiometric assays. Clin Chem 20: 1255-1270, 1974 Roovers J, Evrard E, Vanderhaege H: An improved method for measuring human blood bile acids. Clin Chim Acta 19:449-457, 1968 Rudman D, Kendall RE: Bile acid content of human serum. I. Serum bile acids in patients with hepatic disease. J Clin Invest 36:530-537, 1957 Salen G, Tint GS, Eliav B, et al.: Increased formation of ursodeoxycholic acid in patients treated with chenodeoxycholic acid. J Clin Invest 53:612-621, 1974 Sandberg DH: Bile acid concentrations in serum during infancy and childhood. Pediatr Res 4: 262-267, 1970 Sandberg DH, Sjovall J, Sjovall K, et al: Measurement of human serum bile acids by gasliquid chromatography. J Lipid Res 6:182192, 1965
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