- Email: [email protected]
Serum Bile Acids in Hepatobiliary Disease
1136
long-term emotional support and practical help, while their training is not always conducive to developing these skills. Many of the unmet needs of patients and relatives were regarded as due to inadequate assessment or to the diversity of agencies involved. It is recommended that greater efforts should be made to increase the independence and wellbeing of these patients, who are chronic but not long-stay. Since their medical supervision is often inadequate, community nurses might cover some of this gap, as well as ensuring regular medication. Some inpatient beds could be replaced by sheltered accommodation, probably saving money in the long run, while day care should be expanded and diversified. Each patient should have a counsellor with long-term responsibility, who could be a doctor, nurse, social worker or occupational and the counsellors should meet in a district therapist, rehabilitation committee, reviewing all cases regularly9. A social club with restaurant, perhaps run by volunteers, would be valuable both for the many patients who live alone and to relieve relatives. Questions of coordination and administration should receive much greater attention,’° since no mental health service now exists as such. The Camberwell workers say that the problems they have focused on in South London are universal in countries where large populations of psychiatric hospital residents have built up in the past, or are still doing so. Though emphasising that their data should not be generalised without allowance for local circumstances (something that has happened too much already) they offer these recommendations as a realistic starting-point for improving district services. This offer should be gratefully accepted, and ill-considered plans for closing mental hospitals put aside until a genuine alternative system of care exists outside them.
used routinely to assess liver function, none is ideal. Only some can be held to measure hepatocyte function (e.g., serum albumin and prothrombin time), and even they are rather insensitive.. Others such as serum transaminase levels, though sensitive, reflect hepatocyte damage rather than function. Serum bilirubin and alkaline phosphatase levels are good indicators of cholestasis but poor indicators of hepatocyte function. The serum gammaglutamyltranspeptidase is a more sensitive test of cholestasis and hepatocyte damage, but as a microsomal enzyme it is susceptible to induction by certain drugs. There is clearly room for better tests. tests
Of the many functions of the hepatobiliary system perhaps the most fundamental is the synthesis and excretion of bile. Royal College of Psychiatrists. Psychiatric Rehabilitation in the 1980s: Report Working Party. London: Royal College of Psychiatrists, 1980.
1959; 11: 109-34. Erlinger S. Hepatocyte bile secretion:
Current
views
and controversies.
Hepatology
1981; 1: 352-59. 5. Bjorkhem I, Danielsson H. Biosynthesis and metabolism of bile acids in man. In: Popper H, Schaffner F, eds. Progress in liver disease. New York: Grune and
Disease
9.
1. Thureborn E. Human hepatic bile. Composition changes due to altered enterohepatic circulation. Acta Chir Scand 1962; suppl 303: 1-63. 2. Holzbach RT, March M, Olszewski M, Holan K. Cholesterol solubility in bile. Evidence that supersaturated bile is frequent in healthy man. J Clin Invest 1973; 52: 1467-79. 3. Sperber I. Secretion of organic anions in the formation of urine and bile. Pharmacol Rev 4.
Serum Bile Acids in Hepatobiliary OF the blood
Bile acids (salts) are the predominant organic solute ofbile, 1,2 and indeed the main driving force of choleresis. 3,4 Almost all aspects of hepatobiliary function are involved in bile-acid metabolism.5,6 The hepatocytes synthesise bile acids by microsomal 7a-hydroxylation of cholesterol. In man two primary bile acids, cholic (trihydroxy) and chenodeoxycholic (dihydroxy) are produced in a ratio of about 2 to 1. These are then conjugated with glycine or taurine before active secretion into the canaliculi. Hydroxylation and conjugation render the basic steroid nucleus much more water soluble and endow it with detergent properties. Flow of bile into the duodenum depends on gallbladder function, on feeding, and on the time of day. 7,8 During fasting9 and sleep, most of the bile-acid pool is sequestered in the gallbladder, whereas after a meal gallbladder contraction releases the pool into the gut. Bile acids are then avidly reabsorbed, to a minor degree in the in the terminal ileum via a proximal small’gutlO but mainly carrier-mediated pathway."I The reabsorbed acids are efficiently extracted by the liver 12 from the portal vein to be recycled in the bile.13There are on average 6-8 complete cycles in a day with less than 10% of the bile-acid pool being lost in the faeces.7,8 Bacteria in the distal small bowel and large bowel deconjugatel4 and 7 a-dehydroxylatel5 cholic and chenodeoxylic to form deoxycholic and lithocholic acids, respectively. These secondary bile acids undergo enterohepatic circu1ationl6,17 too, but reabsorption is less efficient so they form a smaller proportion of the total bile acid pool. The liver also has the capacity to sulphate bile acids 18 but in health only lithocholate undergoes such esterification to any degree.16,17 This makes the potentially toxic lithocholate more polar and thus subject to renal excretion. Bile acids are detectable in peripheral blood at very low concentrations (of the order 10-6 mol/1) because hepatic extraction from the portal vein, though efficient, is incomplete. 12,13 The fractional extraction .rate varies
of a
10. Freeman HL, Cheadle AJ, Korer JR. A method for monitoring the treatment of schizophrenics in the community. Br J Psychiatry 1979; 134: 412-16.
6.
Stratton, 1976. Bergstrom S, Danielsson H. Formation and metabolism of bile acids. In: Code CF, ed. Handbook of physiology. Washington D.C. American Physiology Society, 1968: 2391-407.
Wright R, Alberti KGMM, Karran S, Millward Sadler GH, biliary disease. London: W. B. Saunders, 1979: 233-54. 8. Javitt NB. Bile acids and hepatobiliary disease. In: Schiff L, ed. Diseases of the liver. Philadelphia: J. B Lippincott, 1981: 119-50. 9. von Bergmann K, Mok HYI, Grundy SM. Distribution of the bile acid pool in fasting man. Gastroenterology 1976; 71: A-41. 10. Angelin B, Bjorkhem I. Postprandial serum bile acids in healthy man. Evidence for differences in absorptive pattern between individual bile acids. Gut 1977; 18: 7. Heaton KW. Bile salts. In:
eds. Liver and
606-09. 11.
J Dietschy JM. Mechanisms for the intestinal absorption of bile acids. Lipid Res 1968;
9: 297-309. L, Simon FR. Identification and characterisation ofa bile acid receptor in isolated liver surface membrane. J Clin Invest 1976; 57: 496-508. 13. Lindblad L, Lundholm K, Schersten T. Bile acid concentrations in systemic and portal serum in presumably normal man and in cholestatic and cirrhotic conditions. Scand J Gastroenterol 1977; 12: 395-400. 14. Northfield TC, McColl I. Postprandial concentration of free and conjugated bile acids down the length of the normal human small intestine. Gut 1973; 14: 513-18. 15. Hepner GW, Hofmann AF, Thomas PJ. Metabolism of steroid and ammo acid moieties of conjugated bile acids in man. J Clin Invest 1972; 51: 1189-97. 16. Allan RN, Thistle JL, Hofmann AF. Lithocholate metabolism during chenotherapy for gall stone dissolution. Gut 1976; 17: 413-19. 17. Cowan AE, Korman MG, Hofmann AF, Coffin SB. Metabolism of lithocholate in healthy man. II. Enterohepatic circulation. Gastroenterology 1975; 69: 67-76 18. Palmer RH. Formation of bile acid sulphates: anew pathway of bile acid metabolism in humans. Proc Natl Acad Sci USA 1967; 58: 1047-50. 12. Accatino
1137
according to bile-acid structure; so, for instance, more cholate is extracted than chenodeoxycholate.19 Hence ratios of individual bile acids in peripheral blood differ from those in portal vein or bile.20 Blood levels of course depend on thestage of digestion, being lowest during fasting and highest at a variable point between one and three hours after food.21 Even fasting levels are variable because of intermittent gallbladder contraction associated with the interdigestive migrating motor complex.22 In health the main determinant of serum bile acid levels is the rate of intestinal reabsorption.23 The development of very sensitive assays has boosted the popularity of serum bile acid measurement as a test of liver function. Technical problems of measuring minor fluctuations of serum levels are now largely solved. The early enzymatic assays based on bacterial dehydrogenases24 were too insensitive even after later modifications.25,26 The position changed with the advent of radioimmunoassays and of gas-liquid chromatography2g with- mass spectrometry, 10 which are sensitive enough to detect the physiological postprandial rises. These assays, however, do not completely outclass the simpler enzymatic method-radioimmunoassay because it can only detect individual rather than total bile acids, and gas liquid chromatography/mass spectrometry because it requires special laboratory facilities. Lately a highly sensitive bioluminescent enzymatic method29 has been described and this may eventually prove the most convenient. There has been much argument as to whether fasting or postprandial serum bile acid measurement is the better determinant of abnormal liver function. Some30-35 but 19.
Ahlberg J, Angelin B, Bjorghem I, Einarsson K. Individual bile acids in portal venous and systemic blood serum of fasting man. Gastroenterology 1977; 73: 1377-82. 20. Schalm SW, La Russo NF, Hofmann AF, Hofmann NE, Van Berge-Henegouwen GP, Korman MG. Diurnal serum levels of primary conjugated bile acids. Assessment by specific radioimmunoassays for conjugates of cholic and chenodeoxycholic acids. Gut 1978; 19: 1006-14. 21. Ponz De Leon M, Murphy GM, Dowling RH. Physiological factors influencing serum bile acid levels. Gut 1978; 19: 32-39. 22. Peeters TL, Van Trappen G, Janssens J. Bile acid output and the interdigestive migrating motor complex in normals and in cholecystectomy patients. Gastroenterology 1980; 79: 678-81. 23. La Russo NF, Hoffman NE, Korman MG, Hofmann AF, Cowen AE. Determinants of fasting and postprandial serum bile acid levels in healthy man. Dig Dis 1978; 23: 385-91. 24. Iwata Y, Yamasaki K. Enzymatic determination and thin layer chromatography of bile acids in blood. J Biochem 1964; 56: 424-31. 25. Osuga T, Mitamura K, Mashige F, Imai K. Evaluation of fluormetrically estimated serum bile acids in liver disease. Clin Chim Acta 1977; 75: 81-90. 26. Nicholas JC, Chaintreuil J, Descomps B, Crastes De Paulet A. Enzymatic microassay of serum bile acids: increased sensitivity with an enzyme amplification technique.
Analyt Biochem 1980; 103: 170-75. 27. Simmonds WJ, Korman MG, Go VLW, Hofmann A. Radioimmunoassay of conjugated cholyl bile acids in serum. Gastroenterology 1976; 65: 705-11. 28. Ross PE, Pennington CR, Bouchier IAD. Gas liquid chromatographic assay of serum bile acids. Analyt Biochem 1977; 80: 458-65. 29. Roda A, Kricke LJ, De Luca M. Bioluminescent measurement of bile acids using immobilised 7-hydroxysteroid dehydrogenase: application to serum bile acids. J Lipid Res (in press). 30. Kaplowitz N, Kok E, Javitt NB. Postprandial serum bile acid for the detection of hepatobiliary disease JAMA 1973; 225: 292-93. 31. Barnes S, Gallo GA, Trash DB, Morris JS. Diagnostic value of serum bile acid estimations in liver disease. J Clin Pathol 1975; 28: 506-09. 32. Fausa O, Gjone E. Serum bile acid concentrations in patients with liver disease. Scand J Gastroenterol 1976; 11: 537-43. 33. Thjodleifsson B, Barnes S, Chitranukroh A, Billing B, Sherlock S. Assessment of the plasma disappearance of cholyl-114 C-glycine as a test of hepatocellular disease. Gut 1977; 18: 697-702. 34. Jones MB, Wemstock S, Koretz RL, Lewin KJ, Higgins J, Gitnick GL. Clinical value of serum bile acid levels in chronic hepatitis. Dig Dis 1981; 26: 978-83. 35. Monroe PS, Baker AL, Schneider JF, Krager PS, Klein PD, Schoeller D. The aminopyrine breath test and serum bile acids reflect histologic severity in chronic hepatitis. Hepatology 1982; 2: 317-22. 36. Pennington CR, Ross PE, Bouchier IAD. Serum bile acids in the diagnosis of hepatobiliary disease. Cut 1977, 18: 903-08. 37. Davidson GP, Corey M, Morad-Hassel F, Sondheimer JM, Crozier D, Forstner GG. Immunoassay of serum conjugates of cholic acid in cystic fibrosis. J Clin Pathol 1980; 33: 390-94. 38. Tobiasson P, Boeryd B. Serum cholic and chenodeoxycholic acid conjugates and standard liver function tests in various morphological stages of alcoholic liver disease. Scand J Gastroenterol 1980; 15: 657-63.
not a1l36-39 workers believe the postprandial level to be more discriminant. However, since the postprandial rise is governed largely by non-hepatic factors,4° fasting levels are probably more reproducible in the assessment of liver disease. The requirements of any new liver test are that it should surpass existing tests in simplicity, sensitivity, or
specificity. Serum bile acid measurement with good clearly not simple. Regarding sensitivity,
resolution is
most,30,31,35,37,38,41 but not all,32-34,36,39,42 recent studies indicate that, with an adequate assay, serum bile acid levels are superior to conventional liver function tests in detecting minor hepatobiliary dysfunction. As to specificity, if fasting or postprandial serum bile acid levels are raised, then a hepatobiliary defect is likely to be responsible (original reports of raised serum bile acid levels in stagnant loop syndrome4’ and hyperlipidaemia44 either remain unconfirmed or have been contradicted45). However, other serum bile acid abnormalities-for instance, absence of postprandial rise,46,47 or abnormal ratios of individual bile acids47 -may be associated with disorders of the alimentary limb of bile acid metabolism. Are serum bile acid measurements helpful in differentiating one type of liver disorder from another? It seems that total bile acids are not, whereas the ratios of individual acids or the degree of sulphation may separate cholestatic from hepatocellular lesions.48 These complex tests, however, may be no more informative than modern imaging techniques such as ultrasound. If serum bile acid levels are more sensitive than conventional liver tests (and the case is not uncontested), are they really useful? The answer would be yes only if detection of slight liver abnormalities aided diagnosis or prognosis, obviated a more invasive test, or caused a change in treatment. The condition best studied in this context is chronic hepatitis where differentiation between the persistent and aggressive forms can, by convention, be judged only by histological examination. Several groups 34,35,41,49 have claimed that serum bile acid levels are more closely
39.
Douglas JG, Beckett GJ, Nimmo IA, Finlayson NDC, Percy-Robb IW. Clinical value
of bile salt tests in anicteric liver disease. Gut 1981; 22: 141-48. 40. Gilmore IT, Hofmann AF. Altered drug metabolism and elevated serum bile acids in liver disease: a unified pharmacokinetic explanation. Gastroenterology 1980; 78: 177-79. 41. Matsui A, Psacharopoulos HT, Mowat AP, Portmann B, Murphy GM. Radioimmunoassay of serum glycocholic acid, standard laboratory tests of liver function and liver biopsy findings: comparative study of children with liver disease. J Clin Pathol 1982; 35: 1011-17. 42. Milton Mishler J, Barbosa L, Mihalko LJ, McCarter H. Serum bile acids and alanine aminotransferase concentrations. JAMA 1981; 246: 2340-44. 43. Lewis B, Panveliwalla D, Tabaqchali S, Wooton IDP. Serum bile acids in the stagnant loop syndrome. Lancet 1969; i: 219-20. 44. Pennington CR, Ross PE, Bateson MC, Bouchier IAD. Serum bile acids m patients with hyperlipidaemia. J Clin Pathol 1978; 31: 58-62. 45. Beckett GJ, Douglas-JG, Finlayson NDC, Percy-Robb IW. Are serum bile salt concentrations raised in hyperlipidaemia? Gut 1980; 21: 219-22 46. Suchy FS, Balistreri WF. Ileal dysfunction in Crohn’s disease assessed by the postprandial serum bile acid response. Gut 1981; 22: 948-52. 47. Aldini R, Roda A, Festi D, Mazzella G, Morselli AM, Sama C, Roda E, Scopmaro N, Barbara L. Diagnostic value of serum primary bile acids in detecting bile acid malabsorption. Gut 1982; 23: 829-34. 48. Bouchier IAD, Pennington CR. Serum bile acids in hepatobiliary disease. Gut 1978; 19: 492-96. 49. Korman MG, Hofmann AF, Summerskill WHJ. Assessment of activity in chronic active liver disease. N Engl J Med 1974; 290: 1339-1402.
1138
linked to changes in the histological appearance of chronic hepatitis than are the results of conventional tests. In none of these reports, however, was the correlation between serum bile acid levels and histological findings so close that liver biopsy could be deemed unnecessary. Rather than a marker of disease, serum bile acid levels may be of more value in ruling it out; thus, where Gilbert’s syndrome is suspected because of lone unconjugated hyperbilirubinaemia, the finding of normal serum bile acid levels would make that diagnosis more certain. 50 In screening for liver disease serum bile acid levels are perhaps more sensitive than any other individual blood test, but probably not more so than the standard combination of liver tests offered by most laboratories. The assay systems for serum bile acid measurement do not lend themselves to auto-analysis. In view of the questionable clinical relevance of an extra, albeit more sensitive and specific, liver function test, measurement of serum bile-acid levels is likely to remain a research tool, or at least be restricted to centres with a special interest.
MORE ABOUT HUMAN ONCOGENES
ONCOGENES-specific segments of DNA which confer the properties of malignancy’ -have now been isolated from several human cancer cell lines. There is fierce competition to be the first to determine a precise mechanism for human cancer, and progress has been remarkable. Two separate groups have now found the difference between a human
bladder-cancer oncogene and its corresponding normal gene.2,3 The result is amazingly simple: the swapping (transversion) of a base pair from guanosine-cytosine to adenine-thymine at a defined position in the gene. Over the past two years several groups have isolated pieces of human DNA with oncogenic potential by means of the transfection assay. Here pieces of DNA are precipitated with calcium phosphate and mixed with an indicator cell line such as NIH 3T3, a non-transformed mouse fibroblast. Oncogenic DNA changes the growth pattern of the cells from a confluent monolayer into clumps of piled up, transformed fibroblasts. These cells now grow as tumours in mice whereas the NIH 3T3 cells do not. The beauty of this technique is that the oncogenic DNA can come from any species including man. By cloning of genes in suitable plasmid or bacteriophage vectors, unlimited quantities of defined pieces of DNA can be produced. The combination of gene cloning and the transfection assay provides a powerful tool by which human cancer genes can be isolated and analysed. In the newly reported experiments from the United States, DNA was prepared from two human bladder-carcinoma lines, EJ and T24. Restriction enzymes were used to JM, Berk PD, Hofmann AF, Martin JF, Wolkoff AW, Scharschmidt BF. fasting-state levels of serum cholyl conjugated bile acids in Gilbert’s syndrome: an aid to the diagnosis Hepatology 1982; 2: 340-43.
50. Vierling
Normal
1. Editorial. Human oncogenes Lancet 1982; ii: 195-96. 2. Tobin CT, Bradley SM, Borgmann CI, Weinberg RA, Papageorge AG, Scolnick EM, Dhor R, Lowry DR, Chang EG. Mechanism of activation ofa human oncogene. Nature 1982; 300: 143-49. 3. Reddy EP, Reynolds RK, Santos E, Barbacid M A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogenes Nature 1982; 300: 149-52. 4. Weinberg RA. Use of transfection to analyse genetic information and malignant transformation Biochem Biophys Act 1981, 651: 25-35.
fragment the DNA, and the smallest piece with oncogenic potential in a transfection assay was immortalised by gene cloning. This turned out to be a sequence of 6600 nucleotide base pairs. The sequence showed remarkable homology to an RNA tumour virus oncogene (v-Ha-ras) which causes in rats.s The oncogene codes for a 21 000 dalton protein, p21. The cloned human oncogene was then compared with the corresponding DNA sequence isolated from normal bladder epithelium. By breaking and recombination of these two DNA sequences the site of the difference was identified and the DNA sequence determined. A single base-pair transversion was noted at position 35 in the functional p21 gene. Such a transversion causes the twelth aminoacid of p2l to be changed from glycine to valine. Several key questions are outstanding. How does p21 result in the alteration of a cell’s growth-control apparatus to cause malignant change? How universal is the mechanism involved? Perhaps most important of all is, how can this discovery be harnessed to reverse the changes of malignancy in patients? Biopsy material from patients with common solid tumours often contains DNA sequences related to the oncogenes of RNA tumour viruses, suggesting that these intriguing results are not artifacts of cell lines but provide a promising new avenue of research in clinical oncology. There are vast sums of money available to fund clinical cancer research but a dearth of new ideas. The time has come to group young clinicians able and willing to grasp the impact of modern molecular biology with energetic scientists in order to bring these developments to the bedside of the cancer sarcomas
patient as soon as possible. TOPICAL DILEMMAS IN ACNE TREATMENT THE mainstay in management of moderate or severe acne is long-term, low-dose antibiotic therapy with either erythromycin or one of the original tetracyclines such as tetracycline or oxytetracycline. Most patients so treated will respond well, but for maximum benefit the treatment must be continued for at least six months.6 In patients who do not respond the dose of the original antibiotic can be increased from the usual 0 - 5 g daily to 1 or even 2 g daily in severe cases, or the oral antibiotic can be changed to co-trimoxazole, minocycline, or clindamycin. Oral clindamycin is particularly effective in acne but when taken orally it occasionally causes antibiotic-associated colitis. What about the possibility of treating acne, and in particular minor degrees of acne, with topical antibiotics? Topical preparations of tetracycline, clindamycin, and erythromycin are all available in the United States and have been approved by the Food and Drugs Administration;but many dermatologists believe that topical antibiotics are generally less effective than conventional oral treatment.’ Three papers by Cunliffe and his co-workers 8-10 raise important points about topical application of antibiotics in 5. Prodha LF, Tobin CJ, Shih C, Weinberg RA. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus rat gene. Nature 1982; 297: 474-78. 6. Cunliffe WJ, Clayden AD, Gould D, Simpson MB. Acne vulgaris—its aetiology and treatment. A review. Clin Exp Dermatol 1981, 6: 461-69. 7. Stoughton RB. Topical antibiotics for acne vulgaris. Current uses. Arch Dermatol 1979; 115: 486-89. 8. Eady EA, Holland KT, Cunliffe WJ. The use of antibiotics in acne therapy oral or topical administration? J Antimicrob Chemother 1982; 10: 89-115. 9. Eady EA, Holland KT, Cunliffe WJ. Topical antibiotics in acne therapy.J Am Acad Dermatol 1981; 5: 455-56. 10. Eady EA, Holland KT, Cunliffe WJ. Should topical antibiotics be used for the treatment of acne vulgaris? Br J Dermatol 1982; 107: 235-46
long-term emotional support and practical help, while their training is not always conducive to developing these skills. Many of the unmet needs of patients and relatives were regarded as due to inadequate assessment or to the diversity of agencies involved. It is recommended that greater efforts should be made to increase the independence and wellbeing of these patients, who are chronic but not long-stay. Since their medical supervision is often inadequate, community nurses might cover some of this gap, as well as ensuring regular medication. Some inpatient beds could be replaced by sheltered accommodation, probably saving money in the long run, while day care should be expanded and diversified. Each patient should have a counsellor with long-term responsibility, who could be a doctor, nurse, social worker or occupational and the counsellors should meet in a district therapist, rehabilitation committee, reviewing all cases regularly9. A social club with restaurant, perhaps run by volunteers, would be valuable both for the many patients who live alone and to relieve relatives. Questions of coordination and administration should receive much greater attention,’° since no mental health service now exists as such. The Camberwell workers say that the problems they have focused on in South London are universal in countries where large populations of psychiatric hospital residents have built up in the past, or are still doing so. Though emphasising that their data should not be generalised without allowance for local circumstances (something that has happened too much already) they offer these recommendations as a realistic starting-point for improving district services. This offer should be gratefully accepted, and ill-considered plans for closing mental hospitals put aside until a genuine alternative system of care exists outside them.
used routinely to assess liver function, none is ideal. Only some can be held to measure hepatocyte function (e.g., serum albumin and prothrombin time), and even they are rather insensitive.. Others such as serum transaminase levels, though sensitive, reflect hepatocyte damage rather than function. Serum bilirubin and alkaline phosphatase levels are good indicators of cholestasis but poor indicators of hepatocyte function. The serum gammaglutamyltranspeptidase is a more sensitive test of cholestasis and hepatocyte damage, but as a microsomal enzyme it is susceptible to induction by certain drugs. There is clearly room for better tests. tests
Of the many functions of the hepatobiliary system perhaps the most fundamental is the synthesis and excretion of bile. Royal College of Psychiatrists. Psychiatric Rehabilitation in the 1980s: Report Working Party. London: Royal College of Psychiatrists, 1980.
1959; 11: 109-34. Erlinger S. Hepatocyte bile secretion:
Current
views
and controversies.
Hepatology
1981; 1: 352-59. 5. Bjorkhem I, Danielsson H. Biosynthesis and metabolism of bile acids in man. In: Popper H, Schaffner F, eds. Progress in liver disease. New York: Grune and
Disease
9.
1. Thureborn E. Human hepatic bile. Composition changes due to altered enterohepatic circulation. Acta Chir Scand 1962; suppl 303: 1-63. 2. Holzbach RT, March M, Olszewski M, Holan K. Cholesterol solubility in bile. Evidence that supersaturated bile is frequent in healthy man. J Clin Invest 1973; 52: 1467-79. 3. Sperber I. Secretion of organic anions in the formation of urine and bile. Pharmacol Rev 4.
Serum Bile Acids in Hepatobiliary OF the blood
Bile acids (salts) are the predominant organic solute ofbile, 1,2 and indeed the main driving force of choleresis. 3,4 Almost all aspects of hepatobiliary function are involved in bile-acid metabolism.5,6 The hepatocytes synthesise bile acids by microsomal 7a-hydroxylation of cholesterol. In man two primary bile acids, cholic (trihydroxy) and chenodeoxycholic (dihydroxy) are produced in a ratio of about 2 to 1. These are then conjugated with glycine or taurine before active secretion into the canaliculi. Hydroxylation and conjugation render the basic steroid nucleus much more water soluble and endow it with detergent properties. Flow of bile into the duodenum depends on gallbladder function, on feeding, and on the time of day. 7,8 During fasting9 and sleep, most of the bile-acid pool is sequestered in the gallbladder, whereas after a meal gallbladder contraction releases the pool into the gut. Bile acids are then avidly reabsorbed, to a minor degree in the in the terminal ileum via a proximal small’gutlO but mainly carrier-mediated pathway."I The reabsorbed acids are efficiently extracted by the liver 12 from the portal vein to be recycled in the bile.13There are on average 6-8 complete cycles in a day with less than 10% of the bile-acid pool being lost in the faeces.7,8 Bacteria in the distal small bowel and large bowel deconjugatel4 and 7 a-dehydroxylatel5 cholic and chenodeoxylic to form deoxycholic and lithocholic acids, respectively. These secondary bile acids undergo enterohepatic circu1ationl6,17 too, but reabsorption is less efficient so they form a smaller proportion of the total bile acid pool. The liver also has the capacity to sulphate bile acids 18 but in health only lithocholate undergoes such esterification to any degree.16,17 This makes the potentially toxic lithocholate more polar and thus subject to renal excretion. Bile acids are detectable in peripheral blood at very low concentrations (of the order 10-6 mol/1) because hepatic extraction from the portal vein, though efficient, is incomplete. 12,13 The fractional extraction .rate varies
of a
10. Freeman HL, Cheadle AJ, Korer JR. A method for monitoring the treatment of schizophrenics in the community. Br J Psychiatry 1979; 134: 412-16.
6.
Stratton, 1976. Bergstrom S, Danielsson H. Formation and metabolism of bile acids. In: Code CF, ed. Handbook of physiology. Washington D.C. American Physiology Society, 1968: 2391-407.
Wright R, Alberti KGMM, Karran S, Millward Sadler GH, biliary disease. London: W. B. Saunders, 1979: 233-54. 8. Javitt NB. Bile acids and hepatobiliary disease. In: Schiff L, ed. Diseases of the liver. Philadelphia: J. B Lippincott, 1981: 119-50. 9. von Bergmann K, Mok HYI, Grundy SM. Distribution of the bile acid pool in fasting man. Gastroenterology 1976; 71: A-41. 10. Angelin B, Bjorkhem I. Postprandial serum bile acids in healthy man. Evidence for differences in absorptive pattern between individual bile acids. Gut 1977; 18: 7. Heaton KW. Bile salts. In:
eds. Liver and
606-09. 11.
J Dietschy JM. Mechanisms for the intestinal absorption of bile acids. Lipid Res 1968;
9: 297-309. L, Simon FR. Identification and characterisation ofa bile acid receptor in isolated liver surface membrane. J Clin Invest 1976; 57: 496-508. 13. Lindblad L, Lundholm K, Schersten T. Bile acid concentrations in systemic and portal serum in presumably normal man and in cholestatic and cirrhotic conditions. Scand J Gastroenterol 1977; 12: 395-400. 14. Northfield TC, McColl I. Postprandial concentration of free and conjugated bile acids down the length of the normal human small intestine. Gut 1973; 14: 513-18. 15. Hepner GW, Hofmann AF, Thomas PJ. Metabolism of steroid and ammo acid moieties of conjugated bile acids in man. J Clin Invest 1972; 51: 1189-97. 16. Allan RN, Thistle JL, Hofmann AF. Lithocholate metabolism during chenotherapy for gall stone dissolution. Gut 1976; 17: 413-19. 17. Cowan AE, Korman MG, Hofmann AF, Coffin SB. Metabolism of lithocholate in healthy man. II. Enterohepatic circulation. Gastroenterology 1975; 69: 67-76 18. Palmer RH. Formation of bile acid sulphates: anew pathway of bile acid metabolism in humans. Proc Natl Acad Sci USA 1967; 58: 1047-50. 12. Accatino
1137
according to bile-acid structure; so, for instance, more cholate is extracted than chenodeoxycholate.19 Hence ratios of individual bile acids in peripheral blood differ from those in portal vein or bile.20 Blood levels of course depend on thestage of digestion, being lowest during fasting and highest at a variable point between one and three hours after food.21 Even fasting levels are variable because of intermittent gallbladder contraction associated with the interdigestive migrating motor complex.22 In health the main determinant of serum bile acid levels is the rate of intestinal reabsorption.23 The development of very sensitive assays has boosted the popularity of serum bile acid measurement as a test of liver function. Technical problems of measuring minor fluctuations of serum levels are now largely solved. The early enzymatic assays based on bacterial dehydrogenases24 were too insensitive even after later modifications.25,26 The position changed with the advent of radioimmunoassays and of gas-liquid chromatography2g with- mass spectrometry, 10 which are sensitive enough to detect the physiological postprandial rises. These assays, however, do not completely outclass the simpler enzymatic method-radioimmunoassay because it can only detect individual rather than total bile acids, and gas liquid chromatography/mass spectrometry because it requires special laboratory facilities. Lately a highly sensitive bioluminescent enzymatic method29 has been described and this may eventually prove the most convenient. There has been much argument as to whether fasting or postprandial serum bile acid measurement is the better determinant of abnormal liver function. Some30-35 but 19.
Ahlberg J, Angelin B, Bjorghem I, Einarsson K. Individual bile acids in portal venous and systemic blood serum of fasting man. Gastroenterology 1977; 73: 1377-82. 20. Schalm SW, La Russo NF, Hofmann AF, Hofmann NE, Van Berge-Henegouwen GP, Korman MG. Diurnal serum levels of primary conjugated bile acids. Assessment by specific radioimmunoassays for conjugates of cholic and chenodeoxycholic acids. Gut 1978; 19: 1006-14. 21. Ponz De Leon M, Murphy GM, Dowling RH. Physiological factors influencing serum bile acid levels. Gut 1978; 19: 32-39. 22. Peeters TL, Van Trappen G, Janssens J. Bile acid output and the interdigestive migrating motor complex in normals and in cholecystectomy patients. Gastroenterology 1980; 79: 678-81. 23. La Russo NF, Hoffman NE, Korman MG, Hofmann AF, Cowen AE. Determinants of fasting and postprandial serum bile acid levels in healthy man. Dig Dis 1978; 23: 385-91. 24. Iwata Y, Yamasaki K. Enzymatic determination and thin layer chromatography of bile acids in blood. J Biochem 1964; 56: 424-31. 25. Osuga T, Mitamura K, Mashige F, Imai K. Evaluation of fluormetrically estimated serum bile acids in liver disease. Clin Chim Acta 1977; 75: 81-90. 26. Nicholas JC, Chaintreuil J, Descomps B, Crastes De Paulet A. Enzymatic microassay of serum bile acids: increased sensitivity with an enzyme amplification technique.
Analyt Biochem 1980; 103: 170-75. 27. Simmonds WJ, Korman MG, Go VLW, Hofmann A. Radioimmunoassay of conjugated cholyl bile acids in serum. Gastroenterology 1976; 65: 705-11. 28. Ross PE, Pennington CR, Bouchier IAD. Gas liquid chromatographic assay of serum bile acids. Analyt Biochem 1977; 80: 458-65. 29. Roda A, Kricke LJ, De Luca M. Bioluminescent measurement of bile acids using immobilised 7-hydroxysteroid dehydrogenase: application to serum bile acids. J Lipid Res (in press). 30. Kaplowitz N, Kok E, Javitt NB. Postprandial serum bile acid for the detection of hepatobiliary disease JAMA 1973; 225: 292-93. 31. Barnes S, Gallo GA, Trash DB, Morris JS. Diagnostic value of serum bile acid estimations in liver disease. J Clin Pathol 1975; 28: 506-09. 32. Fausa O, Gjone E. Serum bile acid concentrations in patients with liver disease. Scand J Gastroenterol 1976; 11: 537-43. 33. Thjodleifsson B, Barnes S, Chitranukroh A, Billing B, Sherlock S. Assessment of the plasma disappearance of cholyl-114 C-glycine as a test of hepatocellular disease. Gut 1977; 18: 697-702. 34. Jones MB, Wemstock S, Koretz RL, Lewin KJ, Higgins J, Gitnick GL. Clinical value of serum bile acid levels in chronic hepatitis. Dig Dis 1981; 26: 978-83. 35. Monroe PS, Baker AL, Schneider JF, Krager PS, Klein PD, Schoeller D. The aminopyrine breath test and serum bile acids reflect histologic severity in chronic hepatitis. Hepatology 1982; 2: 317-22. 36. Pennington CR, Ross PE, Bouchier IAD. Serum bile acids in the diagnosis of hepatobiliary disease. Cut 1977, 18: 903-08. 37. Davidson GP, Corey M, Morad-Hassel F, Sondheimer JM, Crozier D, Forstner GG. Immunoassay of serum conjugates of cholic acid in cystic fibrosis. J Clin Pathol 1980; 33: 390-94. 38. Tobiasson P, Boeryd B. Serum cholic and chenodeoxycholic acid conjugates and standard liver function tests in various morphological stages of alcoholic liver disease. Scand J Gastroenterol 1980; 15: 657-63.
not a1l36-39 workers believe the postprandial level to be more discriminant. However, since the postprandial rise is governed largely by non-hepatic factors,4° fasting levels are probably more reproducible in the assessment of liver disease. The requirements of any new liver test are that it should surpass existing tests in simplicity, sensitivity, or
specificity. Serum bile acid measurement with good clearly not simple. Regarding sensitivity,
resolution is
most,30,31,35,37,38,41 but not all,32-34,36,39,42 recent studies indicate that, with an adequate assay, serum bile acid levels are superior to conventional liver function tests in detecting minor hepatobiliary dysfunction. As to specificity, if fasting or postprandial serum bile acid levels are raised, then a hepatobiliary defect is likely to be responsible (original reports of raised serum bile acid levels in stagnant loop syndrome4’ and hyperlipidaemia44 either remain unconfirmed or have been contradicted45). However, other serum bile acid abnormalities-for instance, absence of postprandial rise,46,47 or abnormal ratios of individual bile acids47 -may be associated with disorders of the alimentary limb of bile acid metabolism. Are serum bile acid measurements helpful in differentiating one type of liver disorder from another? It seems that total bile acids are not, whereas the ratios of individual acids or the degree of sulphation may separate cholestatic from hepatocellular lesions.48 These complex tests, however, may be no more informative than modern imaging techniques such as ultrasound. If serum bile acid levels are more sensitive than conventional liver tests (and the case is not uncontested), are they really useful? The answer would be yes only if detection of slight liver abnormalities aided diagnosis or prognosis, obviated a more invasive test, or caused a change in treatment. The condition best studied in this context is chronic hepatitis where differentiation between the persistent and aggressive forms can, by convention, be judged only by histological examination. Several groups 34,35,41,49 have claimed that serum bile acid levels are more closely
39.
Douglas JG, Beckett GJ, Nimmo IA, Finlayson NDC, Percy-Robb IW. Clinical value
of bile salt tests in anicteric liver disease. Gut 1981; 22: 141-48. 40. Gilmore IT, Hofmann AF. Altered drug metabolism and elevated serum bile acids in liver disease: a unified pharmacokinetic explanation. Gastroenterology 1980; 78: 177-79. 41. Matsui A, Psacharopoulos HT, Mowat AP, Portmann B, Murphy GM. Radioimmunoassay of serum glycocholic acid, standard laboratory tests of liver function and liver biopsy findings: comparative study of children with liver disease. J Clin Pathol 1982; 35: 1011-17. 42. Milton Mishler J, Barbosa L, Mihalko LJ, McCarter H. Serum bile acids and alanine aminotransferase concentrations. JAMA 1981; 246: 2340-44. 43. Lewis B, Panveliwalla D, Tabaqchali S, Wooton IDP. Serum bile acids in the stagnant loop syndrome. Lancet 1969; i: 219-20. 44. Pennington CR, Ross PE, Bateson MC, Bouchier IAD. Serum bile acids m patients with hyperlipidaemia. J Clin Pathol 1978; 31: 58-62. 45. Beckett GJ, Douglas-JG, Finlayson NDC, Percy-Robb IW. Are serum bile salt concentrations raised in hyperlipidaemia? Gut 1980; 21: 219-22 46. Suchy FS, Balistreri WF. Ileal dysfunction in Crohn’s disease assessed by the postprandial serum bile acid response. Gut 1981; 22: 948-52. 47. Aldini R, Roda A, Festi D, Mazzella G, Morselli AM, Sama C, Roda E, Scopmaro N, Barbara L. Diagnostic value of serum primary bile acids in detecting bile acid malabsorption. Gut 1982; 23: 829-34. 48. Bouchier IAD, Pennington CR. Serum bile acids in hepatobiliary disease. Gut 1978; 19: 492-96. 49. Korman MG, Hofmann AF, Summerskill WHJ. Assessment of activity in chronic active liver disease. N Engl J Med 1974; 290: 1339-1402.
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linked to changes in the histological appearance of chronic hepatitis than are the results of conventional tests. In none of these reports, however, was the correlation between serum bile acid levels and histological findings so close that liver biopsy could be deemed unnecessary. Rather than a marker of disease, serum bile acid levels may be of more value in ruling it out; thus, where Gilbert’s syndrome is suspected because of lone unconjugated hyperbilirubinaemia, the finding of normal serum bile acid levels would make that diagnosis more certain. 50 In screening for liver disease serum bile acid levels are perhaps more sensitive than any other individual blood test, but probably not more so than the standard combination of liver tests offered by most laboratories. The assay systems for serum bile acid measurement do not lend themselves to auto-analysis. In view of the questionable clinical relevance of an extra, albeit more sensitive and specific, liver function test, measurement of serum bile-acid levels is likely to remain a research tool, or at least be restricted to centres with a special interest.
MORE ABOUT HUMAN ONCOGENES
ONCOGENES-specific segments of DNA which confer the properties of malignancy’ -have now been isolated from several human cancer cell lines. There is fierce competition to be the first to determine a precise mechanism for human cancer, and progress has been remarkable. Two separate groups have now found the difference between a human
bladder-cancer oncogene and its corresponding normal gene.2,3 The result is amazingly simple: the swapping (transversion) of a base pair from guanosine-cytosine to adenine-thymine at a defined position in the gene. Over the past two years several groups have isolated pieces of human DNA with oncogenic potential by means of the transfection assay. Here pieces of DNA are precipitated with calcium phosphate and mixed with an indicator cell line such as NIH 3T3, a non-transformed mouse fibroblast. Oncogenic DNA changes the growth pattern of the cells from a confluent monolayer into clumps of piled up, transformed fibroblasts. These cells now grow as tumours in mice whereas the NIH 3T3 cells do not. The beauty of this technique is that the oncogenic DNA can come from any species including man. By cloning of genes in suitable plasmid or bacteriophage vectors, unlimited quantities of defined pieces of DNA can be produced. The combination of gene cloning and the transfection assay provides a powerful tool by which human cancer genes can be isolated and analysed. In the newly reported experiments from the United States, DNA was prepared from two human bladder-carcinoma lines, EJ and T24. Restriction enzymes were used to JM, Berk PD, Hofmann AF, Martin JF, Wolkoff AW, Scharschmidt BF. fasting-state levels of serum cholyl conjugated bile acids in Gilbert’s syndrome: an aid to the diagnosis Hepatology 1982; 2: 340-43.
50. Vierling
Normal
1. Editorial. Human oncogenes Lancet 1982; ii: 195-96. 2. Tobin CT, Bradley SM, Borgmann CI, Weinberg RA, Papageorge AG, Scolnick EM, Dhor R, Lowry DR, Chang EG. Mechanism of activation ofa human oncogene. Nature 1982; 300: 143-49. 3. Reddy EP, Reynolds RK, Santos E, Barbacid M A point mutation is responsible for the acquisition of transforming properties by the T24 human bladder carcinoma oncogenes Nature 1982; 300: 149-52. 4. Weinberg RA. Use of transfection to analyse genetic information and malignant transformation Biochem Biophys Act 1981, 651: 25-35.
fragment the DNA, and the smallest piece with oncogenic potential in a transfection assay was immortalised by gene cloning. This turned out to be a sequence of 6600 nucleotide base pairs. The sequence showed remarkable homology to an RNA tumour virus oncogene (v-Ha-ras) which causes in rats.s The oncogene codes for a 21 000 dalton protein, p21. The cloned human oncogene was then compared with the corresponding DNA sequence isolated from normal bladder epithelium. By breaking and recombination of these two DNA sequences the site of the difference was identified and the DNA sequence determined. A single base-pair transversion was noted at position 35 in the functional p21 gene. Such a transversion causes the twelth aminoacid of p2l to be changed from glycine to valine. Several key questions are outstanding. How does p21 result in the alteration of a cell’s growth-control apparatus to cause malignant change? How universal is the mechanism involved? Perhaps most important of all is, how can this discovery be harnessed to reverse the changes of malignancy in patients? Biopsy material from patients with common solid tumours often contains DNA sequences related to the oncogenes of RNA tumour viruses, suggesting that these intriguing results are not artifacts of cell lines but provide a promising new avenue of research in clinical oncology. There are vast sums of money available to fund clinical cancer research but a dearth of new ideas. The time has come to group young clinicians able and willing to grasp the impact of modern molecular biology with energetic scientists in order to bring these developments to the bedside of the cancer sarcomas
patient as soon as possible. TOPICAL DILEMMAS IN ACNE TREATMENT THE mainstay in management of moderate or severe acne is long-term, low-dose antibiotic therapy with either erythromycin or one of the original tetracyclines such as tetracycline or oxytetracycline. Most patients so treated will respond well, but for maximum benefit the treatment must be continued for at least six months.6 In patients who do not respond the dose of the original antibiotic can be increased from the usual 0 - 5 g daily to 1 or even 2 g daily in severe cases, or the oral antibiotic can be changed to co-trimoxazole, minocycline, or clindamycin. Oral clindamycin is particularly effective in acne but when taken orally it occasionally causes antibiotic-associated colitis. What about the possibility of treating acne, and in particular minor degrees of acne, with topical antibiotics? Topical preparations of tetracycline, clindamycin, and erythromycin are all available in the United States and have been approved by the Food and Drugs Administration;but many dermatologists believe that topical antibiotics are generally less effective than conventional oral treatment.’ Three papers by Cunliffe and his co-workers 8-10 raise important points about topical application of antibiotics in 5. Prodha LF, Tobin CJ, Shih C, Weinberg RA. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus rat gene. Nature 1982; 297: 474-78. 6. Cunliffe WJ, Clayden AD, Gould D, Simpson MB. Acne vulgaris—its aetiology and treatment. A review. Clin Exp Dermatol 1981, 6: 461-69. 7. Stoughton RB. Topical antibiotics for acne vulgaris. Current uses. Arch Dermatol 1979; 115: 486-89. 8. Eady EA, Holland KT, Cunliffe WJ. The use of antibiotics in acne therapy oral or topical administration? J Antimicrob Chemother 1982; 10: 89-115. 9. Eady EA, Holland KT, Cunliffe WJ. Topical antibiotics in acne therapy.J Am Acad Dermatol 1981; 5: 455-56. 10. Eady EA, Holland KT, Cunliffe WJ. Should topical antibiotics be used for the treatment of acne vulgaris? Br J Dermatol 1982; 107: 235-46