Serum bile acid concentrations as an indicator of liver dysfunction induced during danazol therapy

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1 FERTILITY AND STERILITY Copyright The American Fertility Society Printed in U.S.A. Serum bile acid concentrations as an indicator of liver dysfunction induced during danazol therapy Jorma Heikkinen, M.D.*t Lars Ronnberg, M.D.* Pertti Kirkinen, M.D.* Eero Sotaniemi, M.D.+ University of Oulu, Oulu, Finland The effects of danazol therapy (600 mg/day) on the liver function of 16 women with endometriosis were investigated. The primary bile acids ( cholic acid [ CA] and chenodeoxycholic acid [CDCA]) were analyzed with radioimmunoassays in the fasting state and after a test meal. Also, the conventional liver function tests were performed. Ultrasonography was used to detect any possible changes in the gallbladder function. The fasting concentrations of CA increased (P < 0.05) during therapy, while those of CDCA did not change. The ratio of CA/CDCA also increased (P < 0.001). The maximal response of CA after the test meal increased (P < 0.01) during the trial. As regards the other liver function tests, only the transaminases significantly increased (P < 0.01) after 1 month of therapy but showed a tendency to decrease later during the trial. The gallbladder's volume and function did not change. All the parameters studied normalized within 1 month of cessation of danazol therapy. Danazol seems to have a rather mild effect on liver function. The analyzed parameters indicate transient cell wall injury and slight disturbances in liver cell uptake and secretion mechanism and also of synthesis activity. Fertil Steril 50: 761,1988 Danazol, a synthetic derivative of 17 a-ethinyl testosterone, has been commonly used in the treatment of several disorders, such as hereditary angioedema, benign breast diseases, and endometriosis. The drug may induce side effects such as hirsutism, voice changes, retention of fluid, increase in body weight, and disturbances of liver function.1-4 The exact mechanism ofthe liver dysfunction is yet unknown, although a cholestatic injury has been suggested.3 The determination of serum bile acids after overnight fastint- 7 and also the postprandial rise and the clearance rate of bile acids from serums-10 have been introduced as sensitive and specific liver function tests. The serum bile acid levels Received April4, 1988; revised and accepted July 13, * Department of Obstetrics and Gynaecology. t Reprint requests: Jorma Heikkinen, M.D., Department of Obstetrics and Gynaecology, University of Oulu, SF Oulu, Finland. :j: Department of Medicine. reflect many complex and interrelated variables including, in addition to intestinal factors, hepatic blood flow, sinusoidal uptake, and canalicular excretion of bile acids by the liver cell. These are, therefore, sensitive indicators of bile disturbances in liver function, especially in patients with intrahepatic and extrahepatic cholestasis The present study was undertaken to evaluate the usefulness of bile acid assay and other liver function tests in the assessment of liver function during danazol therapy and in the elucidation of the possible mechanisms of danazol-induced changes in liver function in patients with endometriosis. Patients MATERIALS AND METHODS This study was approved by the Ethical Committee of Oulu University and the subjects. Liver func- Heikkinen et al. Danazol and liver function 761

2 tion and gallbladder contractility were evaluated in 16 women with operatively (laparoscopy or laparotomy or both) verified endometriosis before, during, and after their receiving postoperative ( 4 to 6 weeks) danazol treatment. Before commencing use of this drug, all but one of the subjects were found to have normal liver function test values. One patient initially had a transaminase level of about 100 U fl. She was obese and had enlarged liver size suggesting fatty liver. No other disease processes were detected. The patients' average age (mean± standard deviation [SD]) was 31.2 ± 6.5 years (range 21 to 44 years) and their body weight 60.2 ± 7.3 kg (range 47 to 77 kg). The subjects were consecutive patients who accepted the treatment schedule. The reference values in bile acid tolerance test were determined by controls, 10 whose age and body mass did not differ from the studied patients. Methods The serum bile acid (cholic acid [CA] and chenodeoxycholic acid [CDCA]) concentrations were analyzed with radioimmunoassays. 12 The activities of alanine aminotransferase (AL T), aspartate aminotransferase (AST), alkaline phosphatase (AP), and gamma-glutamyltranspeptidase ( -y-gt) and the total bilirubin content and serum albumin concentrations were measured by standard automatic analysis techniques. The volume of the gallbladder was measured by real-time ultrasonography, calculated by ellipsoid approximation. 13 The dosage of danazol (Danatrol, Farmos LTD, Turku, Finland) used was 600 mg daily for 6 months, and the treatment was initiated within 1 to 2 months of the diagnosis being established. A test meal to contract the gallbladder was routinely used by the radiologists, this being a standard meal (Sorbitol 8.9 g and dried egg yolk 10.0 g) (Biloptin, Schering AG, Berlin, FRG), 20 g dissolved in water. Protocol Blood samples for bile acid estimations and for other liver function tests were drawn after an overnight fast. At the same time, the first ultrasonography of the gallbladder in the fasting state was performed. Each patient then drank the Biloptin test meal dissolved in water. Plasma samples were collected by venipuncture for bile acid estimations 1 and 3 hours after loading. Ultrasonography measurements were carried out 1 and 3 hours after the test meal. All these analyses were performed before 762 Heikkinen et al. Danazol and liver function CA ~ z 5.0 ;. i 0 :: -~~ ~ 05 t: t " ~ 0.2 i.. MONTHS ON DANAZOL TREATMENT HOURS AFTER THE TEST MEAL Figure 1 The individual cholic acid (CA) concentrations before, during, and after danazol treatment. The mean levels are linear. The scale is logarithmic. *P < 0.05; **P < 0.01 and after 1, 3, and 6 months of danazol treatment and 1 month after cessation of the therapy. Statistical analyses of the liver function test results were performed with the Student's t-test and random block design of variance analyses and of the gallbladder volumes with the Mann-Whitney U-test and linear regression. RESULTS Bile Acid Concentrations Before Treatment in Patients with Endometriosis The fasting levels of bile acids varied within the normal range. 10 The maximal response expressed by an elevation of the CA level after the test meal was higher (P < 0.05) in the endometriosis patients (mean ± SEM; 2.67 ± 0.34) than in the healthy controls (1.43 ± 0.16). 10 The patient with mild fatty liver changes and increased transaminase levels had normal fasting and postprandial bile acid concentrations. Effects of Danazol The Fasting Levels of Bile Acids Danazol therapy was accompanied by a significant increase (P < 0.05) (mean± SEM; from 0.42 ± 0.06 to 0.90 ± 0.19) in the fasting CA levels after 6 months of treatment, while those of CDCA showed a tendency to decrease (Fig. 1). Thus, the ratio of CA/CDCA increased (P < o-.001) during the trial. The fasting levels of bile acids normalized within 1 month of cessation of the treatment. Fertility and Sterility

3 20.0 ::::: 15.0 COCA 2!'~~At 'It I ~ a:: 20 ~ I I ~ ~.: " ~ UJ MONTHS ON OANAZOL TREATMENT 1MONTH AFTER.1\f t 1\f A~ "":... : : ~ :. : : ; ; ~ i~ f I ~ 1. : :,. 5o r :.. I I ; HOURS AFTER THE TEST MEAl Figure 2 The individual chenodeoxycholic acid ( CDCA) concentrations are presented before, during, and after danazol treatment. The means are linear. The scale is logarithmic. Bile Acids After the Test Meal The maximal response of CA was significantly higher (P < 0.01) after 6 months of treatment (mean± SEM; 6.54 ± 1.14) if correlated with the initial values (2.67 ± 0.34) (Fig. 1). The responses of CDCA again showed a tendency to decrease (Fig. 2). The ratio of CA/CDCA at 1 hour (maximal response) increased (P < 0.01) during the treatment follow-up period. The Other Liver Function Tests Activities of the transaminases increased (P < 0.01) transiently in one month of danazol therapy (Table 1) and showed a tendency to normalize later during the follow-up period and normalized completely after therapy. The patient with fatty liver changes had no increase in the transaminase levels during the therapy. At the 1-month assessment, seven cases demonstrated pathologically increased ALT activities, while at the 3-month assessment only three patients and at 6 months five patients, respectively, showed such changes. AST activities were increased in fewer cases. No correlation was found between bile acid and transaminase levels. The bilirubin levels and the activities of alkaline phosphatase and "(-glutamyltransferase did not change during the trial. The serum albumin concentrations had decreased (P < 0.05) at 1- month control during the therapy but normalized later (Table 1). The Gallbladder Function The gallbladder fasting volumes and conractility values did not change during danazol treatment. The volume of gallbladder as cm 3 (mean± SD) before treatment at fasting state was 17.6 ± 5.6 and after contraction in 1 hour 6.9 ± 3.2. Three hours after the administration of the test meal, the volume of the gallbladder had returned to the fasting volume. DISCUSSION Primary bile acids are synthetized in the liver from cholesterol. The liver cells secrete these into the canaliculi and bile ducts, and later they are carried through the gallbladder to the intestine, where the secondary bile acids are formed Hepatic uptake, intracellular transport, biotransformation, and canalicular secretion of bile acids form a rather complex system, and there are several different mechanisms involved. 16 The impairment of any of these steps leads to disturbance in the enterohepatic circulation of the bile acids and thus to changes in the serum levels of these compounds. In the present study, the bile acid levels of the endometriosis patients in the fasting state were similar to those of healthy controls. 10 On the other hand, their responses to the test meal were enhanced compared with those of the healthy nonpregnant controls. 10 All other investigated hepatic function parameters were, however, normal in the endometriosis patients. The subjects' gallbladder function showed no changes when compared to that of the healthy controls. 17 Perhaps the endometriosis itself might slightly affect the liver uptake Table 1 The Conventional Liver Function Tests (mean ± SEM) Presented Before, During, and After Treatment Time (months) ALTU/L ASTU/L Bil. total umol/l -y-gtu/l APU/L Albumin Gr/L Before after 23.3± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.4b 12.3 ± ± ± ± ± ± ± ± ± 0.9 P< b P<0.05. Heikkinen et al. Danazol and liver function 763

4 mechanism and, therefore, the maximal increase is higher after the test meal. The divergence from the results obtained for the healthy controls is, however, slight and perhaps without any clinical significance. Danazol significantly affected both the fasting and the posttest meal levels of bile acids. During the treatment, the serum levels of CA increased, while those of CDCA showed a tendency to decrease. The synthesis of CA occurs mainly in the microsomal fraction in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen. 18 CDCA is synthesized in the microsomal, as well as in the mitochondrial, fraction. Synthesis in the mitochondrial fraction is stimulated by 26-hydroxylase, which requires cytochrome P Danazol has a decreasing effect on the cytochrome P-450 content and function, 20 and thus CDCA synthesis might be diminished. It has been previously noted that CA levels increase relatively more than CDCA levels in cholestatic-type cases, 7 21 indicating an impairment of the liver cell uptake mechanism. Perhaps the same mechanism is present also during danazol treatment. Responses following the test meal also produced significant effects on bile acid levels. At 1 hour after the test meal, the CA level increased significantly and the CDCA value showed a tendency to decrease. The disposal rate of bile acids from serum is in direct correlation to the absorption capacity of the liver. 22 In the present study, there were significant increases in transaminase levels after 1 month of therapy. This signifies hepatocyte cell wall injury. This increase was highest at the 1-month assessment and showed a tendency to decrease subsequently during therapy. After ending the treatment, the values normalized fully. This indicates that the cell wall injury is transient and very similar to that seen during use of contraceptive steroids. One subject with mild fatty liver changes showed no increase in transaminase levels during the follow-up period, and therefore it was possible to continue the therapy as planned. All in all it seems that the hepatic side effects during danazol treatment seem to be mild and are partly transient in character. Ultrasonography evaluations showed that there were only minimal changes in the gallbladder fasting volumes as well as in contractility and ejection volumes during danazol therapy. It has previously been observed that contraceptive steroids do not affect gallbladder function. 23 On the other hand, during pregnancy and particularly in patients with intrahepatic cholestasis of pregnancy, the gallbladder's volume is double or even more than that of nonpregnant women. 17 It seems that danazol has no significant effect on the extrahepatic circulation of bile acids. Perhaps danazol has no role in the reduction or disturbance of absorption of bile acids from the intestine. After discontinuation of danazol therapy, every parameter normalized within 1 month. The transaminases showed a tendency to remain at the same level or to decrease after 1 month of treatment, while changes in the bile acids' levels progressed during therapy. This indicates that the effect of cell wall injury must be partly transient but that the effects on other structures or functions are potentially more significant and progressive. It is obvious that there were no permanent alterations of liver function. On the basis of the present findings, we can postulate that 1) endometriosis itself seems to have a very mild effect on liver function, 2) danazol causes partially transient hepatocyte wall damage and impairment of the hepatocyte uptake mechanism, and 3) danazol did not significantly affect the function of the extrahepatic biliary pathways. Because danazol seems to influence liver function it is indicated to follow the liver function tests, e.g., transaminase and bile acid evaluations, during the treatment. Special care should be taken if the patient treated has a history of intrahepatic cholestasis or any other liver disease. REFERENCES 1. Wynn V: Metabolic effects of danazol. J Int Med Res 5(Suppl3):25, Ronnberg L, Ylostalo P, Jarvinen PA: Effects of danazol in the treatment of severe endometriosis. Postgrad Med J 55: 21, Pearson K, Zimmerman HJ: Danazol and liver damage. Lancet 2:645, Holt JP, Jr, Keller D: Danazol treatment increases serum enzyme levels. Fertil Steril41:70, Pennington CR, Ross PE, Bouchier lad: Serum bile acids in the diagnosis of hepatobiliary disease. Gut 18:903, Stiehl L, Ast E, Czygan P, Frohling W, Raedsch R, Stiehl A, Kommerell B: Serum bile acids in patients with hepatobiliary diseases: a sensitiye indicator of hepatocellular damage or cholestasis. 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5 intravenous cholic acid in subjects with and without chronic liver disease. Gut 21:123, Heikkinen J: Effect of standard test meal on serum bile acid levels in healthy nonpregnant and pregnant women and in patients with intrahepatic cholestasis of pregnancy. Ann Clin Res 15:183, Bouchier lad, Pennington CR: Serum bile acids in hepatobiliary disease. Gut 19:492, Miientausta 0, Jiinne 0: Radioimmunoassay of conjugated cholic acid, chenodeoxycholic acid and deoxycholic acid from human serum with use 125!-labeled ligands. Clin Chern 25:264, Yliistalo P, Kirkinen P, Heikkinen J, Miientausta 0: Gall bladder volume and serum bile acids in cholestasis of pregnancy. Br J Obstet Gynaecol89:59, Hanson RF, Pries JM: Synthesis and enterohepatic circulation of bile salts. Gastroenterology 73:611, Hofmann AF: Chemistry and enterohepatic circulation of bile acids. Hepatology 4:4, Reuben A: Bile formation: sites and mechanisms. Hepatology 4:15, Kirkinen P, Yliistalo P, Heikkinen J, Miientausta 0: Gallbladder function and maternal bile acids in intrahepatic cholestasis of pregnancy. Eur J Obstet Reprod Biol 18:29, Hanson RF, Klein PD, Williams GC: Bile acid formation in man: Metabolism of 7a-hydroxy-4-cholesten-3-one in bile fistula patients. J Lipid Res 14:50, Bjiirkheim I, Danielsson H, Wikkvall K: Hydroxylation of bile acids by reconstituted systems from rat liver microsomes. J Biol Chern 240:6439, Betz G, Miller HH, Hales DB: Actions of danazol in vivo on cytochrome P-450 and steroidogenic enzymes in rat testis and liver microsomal preparations. Am J Obstet Gynecol141:962, Heikkinen J, Miientausta 0, Yliistalo P, Jiinne 0: Changes in serum bile acid concentrations during normal pregnancy, in patients with intrahepatic cholestasis of pregnancy and in pregnant women with itching. Br J Obstet Gynaecol 88: 240, Ng PY, Hofmann AF: Tissue distribution of cholylglycine- 1-14C in rats and hamsters with a bile fistula or bile duct ligation. Proc Soc Exp Biol Med 154:134, Braverman DZ, Johnson ML, Kern Jr F: Effects of pregnancy and contraceptive steroids on gallbladder function. N Engl J Med 302:362, 1980 Heikkinen et al. Danazol and liver function 765