Efficacy and safety of ursodeoxycholic acid versus cholestyramine in intrahepatic cholestasis of pregnancy

BACKGROUND & AIMS: Treatment of intrahepatic cholestasis of pregnancy with ursodeoxycholic acid appears promising, but data are limited so far. The aim of this randomized study was to evaluate the efficacy and safety of ursodeoxycholic acid in comparison with cholestyramine. METHODS: Eighty-four symptomatic patients with intrahepatic cholestasis of pregnancy were randomized to receive either ursodeoxycholic acid, 8-10 mg/kg body weight daily (n = 42), or cholestyramine, 8 g daily (n = 42), for 14 days. The primary end point was a reduction of pruritus by more than 50% after 14 days of treatment as evaluated by a pruritus score. Secondary end points were outcome of pregnancy, reduction of serum aminotransferase activities and serum bile acid levels, and drug safety. Intention-to-treat analysis was applied. RESULTS: Pruritus was more effectively reduced by ursodeoxycholic acid than cholestyramine (66.6% vs 19.0%, respectively; P < .005). Babies were delivered significantly closer to term by patients treated with ursodeoxycholic acid than those treated with cholestyramine (38.7 +/- 1.7 vs 37.4 +/- 1.5 weeks, respectively, P < .05). Serum alanine and aspartate aminotransferase activities were markedly reduced by 78.5% and 73.8%, respectively, after ursodeoxycholic acid, but by only 21.4%, each, after cholestyramine therapy (P < .01 vs ursodeoxycholic acid). Endogenous serum bile acid levels decreased by 59.5% and 19.0%, respectively (P < .02). Ursodeoxycholic acid, but not cholestyramine was free of adverse effects. CONCLUSIONS: Ursodeoxycholic acid is safe and more effective than cholestyramine in intrahepatic cholestasis of pregnancy.     

Ursodeoxycholic acid in the treatment of cholestasis of pregnancy: a randomized double-blind study controlled with placebo

Backgrounds/Aims: Intense pruritus and the risk of stillbirths and premature deliveries justify the search for an effective pharmacologic treatment of intrahepatic cholestasis of pregnancy. This study was designed to test the efficacy of ursodeoxycholic acid in maternal pruritus, the biochemical abnormalities and the outcome of pregnancy, in patients with intrahepatic cholestasis of pregnancy of early onset.Methods: Pregnant patients hospitalized in a secondary case-referral center with intense pruritus and abnormal serum levels of bile salts and aminotransferases, detected before week 33 of pregnancy, were randomly assigned to receive ursodeoxycholic acid, 1 g per day orally, or an identical placebo, until delivery, in a double-blind study. A 3-week trial period was chosen to compare drug and placebo effects. The follow-up was extended for 3 months after delivery.Results: Twenty-four patients entered the trial; eight had deliveries before 2 weeks of treatment and one dropped out. The study was then completed in 15 patients: eight received ursodeoxycholic acid and seven placebo. No adverse effects were detected in the mother or in their babies. After 3 week of treatment, patients receiving ursodeoxycholic acid (mean daily) dose 16 mg/kg body weight) had a significant improvement in pruritus (p<0.02), In serum bilirubin (0.36±0.19 mg/dl (mean±SD) versus 0.95±0.48 in patients receiving placebo, p<0.01), in aspartate aminostransferase (52±42 IU/l vs 98±44, p<0.05) and in alanine aminotransferase (54±50 IU/l vs 229±154, p<0.01); serum total bile salts also tended to be lower in patients receiving ursodeoxycholic acid (26.3±33.7 μmol/l vs 55.0±44.8, p N.S.). Deliveries occurred at or near term in all mothers who received ursodeoxycholic acid (mean week of pregnancy: 38), while they occurred before week 36 of pregnancy in five patients who received placebo, including one stillbirth. All babies born alive had birth weights adequate for gestational age and they were thriving normally 3 months after delivery.Conclusions: Ursodeoxycholic acid is effective and safe in patients with intrahepatic cholestasis of pregnancy of early onset, attenuating pruritus and correcting some biochemical abnormalities in the mothers. Relevant aspects of fetal outcome were also improved in patients receiving ursodeoxycholic acid compared to placebo.     

Ursodeoxycholic acid in the treatment of intrahepatic cholestasis of pregnancy. A 12-year experience.

OBJECTIVE: To assess the efficacy of ursodeoxycholic acid (UDCA) in patients with intrahepatic cholestasis of pregnancy (ICP) and in the outcome of pregnancy. METHODS: Retrospective analysis of our 12-year experience treating ICP patients with UDCA. Thirty-two patients with pruritus starting before week 34 of pregnancy and with increased serum bile salts (BS) and alanine aminotransferase (ALT) received UDCA (15 mg/kg/day) for at least 3 weeks before delivery. They were compared with 16 historical controls who did not receive UDCA. All patients were followed up until delivery and in puerperium. Newborns were followed up during 3 months. RESULTS: UDCA treatment attenuated pruritus (P < 0.05), serum bilirubin and ALT decreased (P < 0.05) and BS declined. Delivery at term (> or = 37 weeks) occurred in 65.7% of UDCA-treated patients compared with only 12.5% in controls (P < 0.01). Infants born to mothers treated with UDCA weighed a mean of 500 g more than the controls (2882+/-582 vs 2385+/-582; P < 0.01). At 3 months, all infants developed normally. Twenty-six children whose mothers received UDCA were re-examined after 1-12 years and they and their mothers were healthy. CONCLUSIONS: UDCA improved pruritus and biochemical cholestasis, and facilitated deliveries at term in ICP patients, with a higher birthweight compared with historical controls. The drug was well tolerated and no adverse effects were detected in their infants.     

Efficacy and safety of ursodeoxycholic acid in patients with intrahepatic cholestasis of pregnancy

Background and aims. Patients with intrahepatic cholestasis of pregnancy (ICP) benefit from ursodeoxycholic acid (UDCA) treatment. Since there is still certain reluctance to use UDCA in pregnant women, mainly due to warnings in the official SPC information in respective drug leaflets, our objective was to assess the efficacy and safety of UDCA during pregnancy.Material and methods. Our retrospective multicentric study was performed on 191 consecutive pregnant women with ICP treated with UDCA. Any maternal and/or fetal complications of the UDCA treatment were searched for; healthy pregnant women (n = 256) served as controls.Results. The UDCA treatment improved liver disease status in the majority of the affected women (86.1%). This treatment was well tolerated, with only negligible skin reactions (0.5%) and mild diarrhea (4.7%). No complications attributable to UDCA treatment were detected during the fetal life, delivery, or the early neonatal period.Conclusion. We confirmed the good efficacy and safety of UDCA treatment in pregnancy for both mothers and fetuses/neonates.     

44-j?hrige Patientin mit unklarer Leberwerterh?hung und famili?r geh?uftem Gallensteinleiden

"Low phospholipid associated cholelithiasis" (LPAC) syndrome is an important differential diagnosis in younger patients with biliary symptoms after cholecystectomy and concomitant elevated serum liver tests. Typical symptoms include recurrence of biliary colics after cholecystectomy, echogenic material in the intrahepatic bile ducts, intrahepatic cholestasis of pregnancy or cholestasis under hormonal contraception and a family history of gallstone disease. Patients with LPAC syndrome can be successfully treated with ursodeoxycholic acid.     

Use of ursodeoxycholic acid in liver diseases

Ursodeoxycholic acid is currently the only established drug for the treatment of chronic cholestatic liver diseases. It has cytoprotective, anti-apoptotic, membrane stabilizing, anti-oxidative and immunomodulatory effects. Prolonged administration of ursodeoxycholic acid in patients with primary biliary cirrhosis (PBC) is associated with survival benefit and a delaying of liver transplantation. There is evidence that it might even prevent progression of the histologic stage of PBC. It also has a beneficial effect on primary sclerosing cholangitis, intrahepatic cholestasis of pregnancy, liver disease associated with cystic fibrosis, chronic graft versus host disease, total parenteral nutrition associated cholestasis and various pediatric cholestatic liver diseases. In the present review the current knowledge about the mechanisms of the action and role of ursodeoxycholic acid in the treatment of various liver diseases has been discussed.     

Fetal mortality associated with cholestasis of pregnancy and the potential benefit of therapy with ursodeoxycholic acid.

Cholestasis of pregnancy is associated with increased fetal morbidity and mortality and should be treated actively. The significance attached to pruritus in pregnancy is often minimal, but it is a cardinal symptom of cholestasis of pregnancy, which may have no other clinical features. Eight women with previous cholestasis of pregnancy were referred to The Liver Unit within a 12 month period for advice concerning future pregnancies. Thirteen pregnancies had been affected by cholestasis of pregnancy and 12 had been treated expectantly with resultant perinatal morbidity or mortality in 11 (one normal delivery), including; eight stillbirths, two premature deliveries with fetal distress (one died in perinatal period), and an emergency caesarean section for fetal distress. The other pregnancy was treated actively and delivery was uncomplicated. Subsequently, three of these cases with recurrent cholestasis of pregnancy were referred while pregnant. In each, cholestasis developed with severe pruritus, gross increase of serum bile acids, and deranged liver tests. Each was treated with the choleretic agent ursodeoxycholic acid, with rapid clinical improvement and resolution of deranged biochemistry. In conclusion, cholestasis of pregnancy continues to be treated expectantly despite its association with increased morbidity and mortality and evidence suggesting improved prognosis with active treatment and the potential of reducing the associated perinatal mortality. In an uncontrolled series of three patients with cholestasis of pregnancy, ursodeoxycholic acid seemed to provide safe and effective therapy.     

Recurrent intrahepatic cholestasis of pregnancy and chain-like choledocholithiasis in a female patient with stop codon in the ABDC4-gene of the hepatobiliary phospholipid transporter

We report the case of a 40-years-old female patient with recurrent cholestatic liver disease who presented twice with severe intrahepatic cholestasis of pregnancy and pronounced choledocholithiasis between pregnancies. Bile duct stones were removed endoscopically and a laparoscopic cholecystectomy was performed after the second pregnancy. Liver histology revealed intrahepatic cholestasis with portal inflammation and fibrosis, resembling progressive familial intrahepatic cholestasis (PFIC). Molecular genetic studies identified the heterozygous mutation c.957C > T in the ABCB4 gene encoding the hepatobiliary phospholipid transporter. This is the first report of this mutation that introduces a stop codon in an index patient with intrahepatic cholestasis of pregnancy and multiple bile duct stones. In addition, we detected the ABCB11 polymorphism V 444A, which is associated with a decreased expression of the bile salt export pump. Whereas homozygous carriers of the ABCB4 mutation develop PFIC type 3, the heterozygous ABC transporter mutations represent genetic risk factors for cholelithiasis and recurrent cholestatic hepatitis upon challenge with oral contraceptives or during pregnancy. Of note, the patient presented with normal serum gamma-glutamyltranspeptidase activities during pregnancy-associated cholestatic episodes but normal liver enzymes after delivery, whereas choledocholithiasis was associated with high gamma-glutamyl transpeptidase levels. It is unknown whether ursodeoxycholic acid prevents cholestasis or gallstones in patients with ABCB4 deficiency.     

S-adenosyl-L-methionine in the treatment of patients with intrahepatic cholestasis of pregnancy: a randomized, double-blind, placebo-controlled study with negative results

S-Adenosyl-L-methionine has been reported to induce beneficial effects in intrahepatic cholestasis of pregnancy. Because cholestasis of pregnancy has a high prevalence in Chile and a deleterious effect on fetal prognosis, we decided to verify the efficacy of S-adenosyl-L-methionine in this disease. Eighteen patients with pruritus that appeared during pregnancy and with elevated serum levels of bile salts (68.1 +/- 15.9 mumol/L; mean +/- S.E.M.) and ALT (226 +/- 50 KU/L) were enrolled in a prospective double-blind study comparing the effects of the drug with a placebo. S-Adenosyl-L-methionine, 900 mg, or placebo was administered in daily intravenous infusions for 20 days. Every 5 days liver function tests were done and pruritus was assessed using a preestablished score. No significant differences in pruritus or in serum levels of bile salts, ALT, bilirubin and alkaline phosphatases were seen during or after treatment between patients who received S-adenosyl-L-methionine (n = 9) or placebo (n = 9). No relevant adverse reactions were detected. Most patients had cesarean sections because of reasons unrelated to the therapeutic trial. All newborns had Apgar scores greater than 7 and normal postnatal development. Our patients had moderately severe to severe cholestasis of pregnancy as indicated by the onset of pruritus before wk 32 of pregnancy. Seven of nine multiparous patients had a past history of recurrent cholestasis of pregnancy. In this study, the administration of S-adenosyl-L-methionine during 20 days did not improve intrahepatic cholestasis of pregnancy.     

Ductopenia and cirrhosis in a 32-year-old woman with progressive familial intrahepatic cholestasis type 3: A case report and review of the literature

Progressive familial intrahepatic cholestasis type 3 is caused by a mutation in the ATP-binding cassette, subfamily B, member 4 (ABCB4) gene encoding multidrug resistance protein 3. A 32-year-old woman with a history of acute hepatitis at age 9 years was found to have jaundice during pregnancy in 2008, and was diagnosed as having intrahepatic cholestasis of pregnancy. In 2009, she underwent cholecystectomy for gallstones and chronic cholecystitis. However, itching and jaundice did not resolve postoperatively. She was admitted to our hospital with fatigue, jaundice, and a recently elevated γ-glutamyl transpeptidase level. Liver biopsy led to the diagnosis of biliary cirrhosis with ductopenia. Genetic testing revealed a pathogenic heterozygous mutation, ex13 c.1531G A (p.A511 T), in the ABCB4 gene. Her father did not carry the mutation, but her mother's brother carried the heterozygous mutation. We made a definitivediagnosis of familial intrahepatic cholestasis type 3. He symptoms and liver function improved after 3 mo o treatment with ursodeoxycholic acid.     

The effectual level of ursodeoxycholic acid in therapy for non-advanced chronic cholestasis is fifty percent of total serum bile acids.

Ursodeoxycholic acid therapy (600 mg/day) was evaluated in twelve patients with non-advanced chronic cholestasis. Within four months, ursodeoxycholic acid replaced more than 50% of total bile acids in 8 patients and the reduction of serum gamma-glutamyltranspeptidase, alkaline phosphatase and transaminases averaged 30% or more. The serum levels of chenodeoxycholic acid depend on those of ursodeoxycholic acid, but are not related to those of biochemical parameters. Drug therapy was continued in three poor responders for 2-3 four-month periods. In one case an increase of the serum proportion of ursodeoxycholic acid was associated with a reduction in biochemical parameters. The other two cases had high serum levels of chenodeoxycholic acid and/or cholic acid throughout the entire course of treatment. While the treatment of chronic cholestasis requires an effectual serum proportion of ursodeoxycholic acid, it is important to distinguish endogenous persistent hyper-bile-acidemia from ursodeoxycholic acid-related acidemia.     

The effectual level of ursodeoxycholic acid in therapy for non-advanced chronic cholestasis is fifty percent of total serum bile acids

Ursodeoxycholic acid therapy (600 mg/day) was evaluated in twelve patients with nonadvanced chronic cholestasis. Within four months, ursodeoxycholic acid replaced more than 50% of total bile acids in 8 patients and the reduction of serum γ-glutamyltranspeptidase, alkaline phosphatase and transaminases averaged 30% or more. The serum levels of chenodeoxycholic acid depend on those of ursodeoxycholic acid, but are not related to those of biochemical parameters. Drug therapy was continued in three poor responders for 2–3 four-month periods. In one case an increase of the serum proportion of ursodeoxycholic acid was associated with a reduction in biochemical parameters. The other two cases had high serum levels of chenodeoxycholic acid and/or cholic acid throughout the entire course of treatment. While the treatment of chronic cholestasis requires an effectual serum proportion of ursodeoxycholic acid, it is important to distinguish endogenous persistent hyper-bile-acidemia from ursodeoxycholic acid-related acidemia.     

Failure of ursodeoxycholic acid to prevent a cholestatic episode in a patient with benign recurrent intrahepatic cholestasis: a study of bile acid metabolism

Ursodeoxycholic acid was administered to a patient with benign recurrent intrahepatic cholestasis to prevent cholestatic episodes. A detailed study of bile acid metabolism in this patient was carried out in the anicteric and icteric phases before and after ursodeoxycholic acid (750 mg/day) administration. Urinary, biliary and serum bile acids were measured by gas chromatography-mass spectrometry and by high-performance liquid chromatography techniques. During the anicteric phase the daily urinary excretion and serum concentrations of bile acids were within normal ranges, indicating normal hepatic uptake and secretion of bile acids during the cholestasis-free period. Only slight qualitative differences from normal individuals were observed; the relative proportions of deoxycholic acid in the bile and serum were higher, and 12-oxo-lithocholic acid was the predominant urinary bile acid. During the icteric phase a marked increase in the urinary excretion of primary bile acids and C-1, C-2, C-4 and C-6 hydroxylated metabolites was found. Serum bile acid concentrations increased before the rise in bilirubin, suggesting an acute disturbance in bile acid transport at the onset of the cholestatic attack. After ursodeoxycholic acid administration in the anicteric phase, bile became enriched with the exogenous bile acid, but little qualitative change was found in the other metabolites present in the urine, serum or bile during the anicteric or icteric phases. Prolonged administration of ursodeoxycholic acid failed to prevent recurrence of a cholestatic episode, suggesting that in benign recurrent intrahepatic cholestasis, oral ursodeoxycholic acid may be of little benefit in the treatment or prevention of cholestasis despite marked enrichment of the bile acid pool with this hydrophilic bile acid.     

Benign recurrent intrahepatic cholestasis (BRIC) in an adult

Benign recurrent intrahepatic cholestasis is a rare hereditary disorder characterised by recurrent episodes ofcholestasis. We report the case of a young male patient with benign recurrent intrahepatic cholestasis who presented to us with recurrent cholestatic jaundice and pruritus with negative work up for all possible aetiologies and a liver biopsy consistent with intrahepatic cholestasis. He improved on treatment with ursodeoxycholic acid and ondansterone and is doing well on follow up.     

Prolonged cholestasis following successful removal of common bile duct stones： Beware patients on estrogen therapy

There are various well described forms of chronic cholestatic jaundice in adults, such as autoimmune cholangitis, drug-induced cholangitis and intrahepatic cholestasis of pregnancy. We present two cases of prolonged cholestasis following removal of gallstones at endoscopic retrograde cholangiopancreatography （ERCP） and subsequent clear cholangiography. Both patients were taking oral estrogens at the time of presentation, which were subsequently withdrawn. The first case responded rapidly to corticosteroid treatment, and the second case had a much slower resolution with ursodeoxycholic acid. Both cases highlighted the significance of estrogen-induced cholestasis in female patients with protracted jaundice following ERCP and removal of intra-ductal stones. After oral estrogens are discontinued, a short course of steroids needs to be considered.     

住院患者药物性肝损伤病因分析及熊去氧胆酸协同治疗疗效观察

目的 观察熊去氧胆酸协同治疗药物性肝损伤（DILI）患者的临床疗效,分析我院引起DILI的药物种类及其发生比例。方法 纳入我院200例药物性肝损伤患者,分析引起DILI的药物种类。将患者分成两组,对照组给予异甘草酸镁、谷胱甘肽和多烯磷酯酰胆碱中的1~3种药物治疗,对伴有高胆红素血症者加用腺苷蛋氨酸;治疗组在对照组治疗的基础上,加用熊去氧胆酸,两组疗程均为4 w,观察治疗后肝功能变化和临床疗效。结果 本组引起DILI的首位药物是免疫抑制剂（34.0%）,其次为中药（28.5%）,其后依次为抗生素（13.0%）、抗结核药物（7.5%）、抗肿瘤化疗药（7.5%）和解热镇痛药（4.5%）;治疗组在肝功能改善方面明显优于对照组（P<0.01）,尽管两组患者肝功能均有明显改善。结论 引起我院住院患者DILI的最常见的药物为免疫抑制剂,可能与我院开展器官移植较多有关。熊去氧胆酸在甘草酸制剂等的基础上可以较快地改善DILI患者肝功能。     

Ursodeoxycholic acid therapy in cystic fibrosis-associated liver disease: a dose-response study.

Previous studies from our groups have demonstrated improvements in biochemical markers of liver function when cystic fibrosis patients with associated liver disease were administered oral ursodeoxycholic acid. The magnitude of the response was somewhat less than that found when comparable doses (10 to 15 mg/kg body wt/day) of ursodeoxycholic acid are given to other liver disease patients; this may be explained by the bile acid malabsorption that is characteristic of the disease. For this reason a dose-response study was carried out in nine cystic fibrosis patients with liver disease to establish whether improved efficacy could be obtained with higher doses. Ursodeoxycholic acid in doses of 5, 10 and 15 mg/kg body wt/day was given orally for consecutive 2-mo periods in a replicated Latin-square design. After this, all patients received 20 mg/kg body wt/day. Liver function, individual serum bile acids and biliary bile acid composition were determined at entry and at the end of each treatment period. Our data demonstrate that the magnitude of the biochemical improvement in serum liver enzymes was significantly greater with higher doses of ursodeoxycholic acid; at 20 mg/kg body wt/day it was similar to that reported for patients with other liver diseases administered lower doses. Biliary ursodeoxycholic acid enrichment increased with increasing doses, attaining 42% +/- 6% of the total biliary bile acids with the highest dose. Fasting serum ursodeoxycholic acid concentrations increased during ursodeoxycholic acid administration but were variable and correlated poorly with the dose of ursodeoxycholic acid administered, whereas no correlation was found between serum ursodeoxycholic acid concentration and the proportion of ursodeoxycholic acid in bile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ursodeoxycholic acid for treatment of primary sclerosing cholangitis: a placebo-controlled trial.

The efficacy and safety of ursodeoxycholic acid for the treatment of primary sclerosing cholangitis were evaluated in a prospective, randomized, double-blind, placebo-controlled trial. Fourteen patients with primary sclerosing cholangitis documented by cholestatic serum enzyme pattern, liver histological appearance and endoscopic retrograde cholangiography were included in the trial. Six patients received ursodeoxycholic acid (13 to 15 mg/kg body wt/day), and eight patients received placebo. Two patients had to be withdrawn from the study, one because of UDCA-related diarrhea and the other because of worsening of the disease during placebo treatment. Patients in the ursodeoxycholic acid group improved significantly during 1 yr of treatment with respect to serum levels of bilirubin (median = -50%), alkaline phosphatase (median = -67%), gamma-glutamyltransferase (median = -53%), AST (median = -54%) and ALT (median = -36%) compared with the placebo group, but not with respect to serum levels of hydrophobic bile acids. During ursodeoxycholic acid treatment, histopathological features also improved significantly, as evaluated by multiparametric score. Expression of human leukocyte antigen class I molecules appeared to be markedly reduced on liver cells after ursodeoxycholic acid treatment. We conclude that ursodeoxycholic acid is beneficial in reducing disease activity in patients with primary sclerosing cholangitis.     

腺苷蛋氨酸和熊去氧胆酸治疗妊娠期肝内胆汁淤积症的回顾性分析

目的比较S-腺苷蛋氨酸（SAMe）、熊去氧胆酸（UDCA）以及二者联合用药对于妊娠期肝内胆汁淤积症（ICP）的治疗效果。方法根据药物治疗方案的不同,将138例ICP患者分成3组：A组43例,单用SAMe1g＋5%葡萄糖500mL,静脉滴注;B组56例,单用UDCA每日15mg/kg,口服;C组39例,联合使用SAMe1g和UDCA每日15mg/kg治疗。疗程均为2周,观察疗效。结果三组患者治疗后的瘙痒评分、总胆汁酸（TBA）、ALT和AST均较治疗前有所下降,差异均具有统计学意义（P〈0.01）,其中以C组下降最显著;而总胆红素（TBil）水平治疗前后差异无统计学意义（P〉0.05）。C组中直接胆红素（DBil）的下降较治疗前差异有统计学意义（P〈0.05）,但在A组和B组,DBil治疗前后的变化差异无统计学意义（P〉0.05）。在改善妊娠结局方面,三种治疗方法的效果差异无统计学意义（P〉0.05）。结论 SAMe和UDCA均是治疗ICP的有效药物,联合用药效果最佳。     

Pregnancy and liver disease

Although liver disease does not occur frequently during pregnancy, when it does, it can cause significant morbidity and mortality for both fetus and mother. Recent advances in molecular genetics have provided some insight into the pathogenesis of diseases such as intrahepatic cholestasis of pregnancy (IHCP) and acute fatty liver of pregnancy. Newer studies linking serum bile acid levels with fetal outcome support the importance of this marker in IHCP. Randomized trials with ursodeoxycholic acid have shown promising results in reducing maternal symptoms and serum bile acid levels in IHCP. Although further studies must be performed, early strategies aimed at reducing viral loads in mothers may reduce perinatal transmission of hepatitis B.