Martin Black

Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2

Acetaminophen (APAP), a widely used over-the-counter analgesic, is known to cause hepatotoxicity when ingested in large quantities in both animals and man, especially when administered after chronic ethanol consumption. Hepatotoxicity stems from APAP activation by microsomal P450 monooxygenases to a reactive metabolite that binds to tissue macromolecules, thereby initiating cellular necrosis. Alcohol consumption also causes the induction of P450IIE1, a liver microsomal enzyme that in reconstitution studies has proven to be an effective catalyst of APAP oxidation. Thus, elevated microsomal P450IIE1 levels could explain not only the known increase in APAP bioactivating activity of liver microsomes after prolonged ethanol ingestion but also the enhanced susceptibility to APAP toxicity. We therefore examined the role of P450IIE1 in human liver microsomal APAP activation. Liver microsomes from seven non-alcoholic subjects were found to convert 1 mM APAP to a reactive intermediate (detected as an APAP-cysteine conjugate by high-pressure liquid chromatography) at a rate of 0.25 +/- 0.1 nmol conjugate formed/min/nmol microsomal P450 (mean +/- SD), whereas at 10 mM, this rate increased to 0.73 +/- 0.2 nmol product/min/nmol P450. In a reconstituted system, purified human liver P450IIE1 catalyzed APAP activation at rates threefold higher than those obtained with microsomes whereas two other human P450s, P450IIC8 and P450IIC9, exhibited negligible APAP-oxidizing activity. Monospecific antibodies (IgG) directed against human P450IIE1 inhibited APAP activation in each of the human samples, with anti-P450IIE1 IgG-mediated inhibition averaging 52% (range = 30-78%) of the rates determined in the presence of control IgG. The ability of anti-P450IIE1 IgG to inhibit only one-half of the total APAP activation by microsomes suggests, however, that other P450 isozymes besides P450IIE1 contribute to bioactivation of this compound in human liver. Of the other purified P450 isozymes examined, a beta-naphthoflavone (BNF)-inducible hamster liver P450 promoted APAP activation at rates even higher than those obtained with human P450IIE1. The extensive APAP-oxidizing capacity of this hamster P450, designated P450IA2 based upon its similarity to rat P450d and rabbit form 4 in terms of NH2-terminal amino acid sequence, spectral characteristics, immunochemical properties, and inducibility by BNF, agrees with previous reports concerning the APAP substrate specificity of the rat and rabbit P450IA2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and characterization of human liver cytochrome P-450-ALC.

Cytochrome P-450-ALC, an ethanol-oxidizing form of microsomal cytochrome P-450 (P-450), has been purified from human liver. P-450-ALC (Mr = 54,000 daltons) is a low-spin ferric hemeprotein with a CO-reduced Soret maximum at 452 nm, and has an NH2-terminal amino acid sequence nearly identical to that deduced from a human P-450-ALC cDNA clone. In a reconstituted system, P-450-ALC oxidizes ethanol and aniline at turnover rates (12.2 and 7.3 nmol min-1, respectively) 10-fold greater than two other human P-450 isozymes (termed P-450-B and P-450-C) purified from the same liver. Both P-450-ALC and P-450-C effectively demethylate N-nitrosodimethylamine (NDMA) at low substrate concentrations (0.5 mM), especially in the presence of cytochrome b5. Our results provide direct evidence for a liver P-450 isozyme in humans with catalytic properties similar to the related alcohol-inducible rodent P-450s and also reveal a new human NDMA demethylase.

Impact of weight-based ribavirin with peginterferon alfa-2b in african americans with hepatitis C virus genotype 1

WIN‐R (Weight‐based dosing of pegINterferon alfa‐2b and Ribavirin) was a multicenter, randomized, open‐label, investigator‐initiated trial involving 236 community and academic sites in the United States, comparing response to pegylated interferon (PEG‐IFN) alfa‐2b plus a flat or weight‐based dose of ribavirin (RBV) in treatment‐naive patients with chronic hepatitis C and compensated liver disease. Patients were randomized to receive PEG‐IFN alfa‐2b at 1.5 μg/kg/week plus flat‐dose (800 mg/day) or weight‐based‐dose RBV (800 mg/day for weight <65 kg, 1000 mg/day for 65‐85 kg, 1200 mg/day for >85‐105 kg, or 1400 mg/day for >105‐<125 kg). Sustained virologic response (SVR; undetectable [<125 IU/mL] hepatitis C virus [HCV] RNA at end of follow‐up) in patients ≥65 kg was the primary end point. Low SVR rates have been reported among African American individuals, in whom there is a preponderance of HCV genotype 1. This subanalysis of WIN‐R was conducted to evaluate the efficacy of weight‐based dosing among African American individuals with genotype 1 infection enrolled in the trial. Of 362 African American patients in the primary efficacy analysis, 188 received RBV flat dosing and 174 received weight‐based dosing. SVR rates were higher (21% versus 10%; P = 0.0006) and relapse rates were lower (22% versus 30%) in the weight‐based‐dose group than in the flat‐dose group. Safety and rates of drug discontinuation were similar between the 2 groups. Conclusion: Weight‐based dosing of RBV is more effective than flat dosing in combination with PEG‐IFN alfa‐2b in African American individuals with HCV genotype 1. Even with weight‐based dosing, response rates in African American individuals are lower than reported in other ethnic groups. (HEPATOLOGY 2007.)

Chronic Hepatitis with Combined Features of Autoimmune Chronic Hepatitis and Chronic Hepatitis C: Favorable Response to Prednisone and Azathioprine

The evidence for an association between autoimmune forms of chronic hepatitis and hepatitis C virus (HCV) infection is controversial [1-3]. Although anti-liver-kidney microsomal antibody-positive autoimmune chronic hepatitis appears to be associated with HCV infection [4], evidence of such an association is less convincing with other subtypes of autoimmune chronic hepatitis [5]. Autoantibodies are frequently found in patients with chronic hepatitis C, although usually in low titers [6-8], which suggests that HCV elicits an immune response in the host. In a small subset of patients with chronic hepatitis C, autoantibodies are seen in high titers along with hypergammaglobulinemia, which further clouds the distinction between autoimmune chronic hepatitis and chronic hepatitis C. This has important therapeutic implications because inappropriate treatment of autoimmune chronic hepatitis with interferon- may exacerbate liver disease [9, 10]. Conversely, corticosteroid therapy for chronic hepatitis C may enhance HCV replication [11, 12], which could worsen underlying liver disease. Few data are available to show how patients with features of both autoimmune hepatitis and chronic hepatitis C should be treated. We describe two patients with combined features of autoimmune chronic hepatitis and chronic hepatitis C who showed clinical, biochemical, and histologic responses to treatment with prednisone and azathioprine. Case Reports Patient 1 A 74-year-old white woman had an 18-month history of right-sided abdominal pain, progressive fatigue, and persistently elevated serum alanine aminotransferase levels ranging from 1.18 to 1.55 kat/L. Other medical problems included insulin-dependent diabetes mellitus, hypertension, and hypothyroid disease. She reported no history of blood transfusion, intravenous drug abuse, tattoos, or excessive alcohol use. Her daily medications included 25 mg of hydrochlorothiazide, 16 units of humulin U-100 insulin, 10 mg of enalapril, 0.01 mg of synthroid, and 1 g of acetaminophen. She had no family history of liver disease. Physical examination showed no cutaneous stigmata of chronic liver disease, hepatosplenomegaly, or ascites. Laboratory data are shown in Table 1. Results of abdominal ultrasound and computed tomographic scans and upper gastrointestinal series were unremarkable. Examination of a liver biopsy specimen showed features of moderately severe chronic hepatitis with a Knodell hepatitis activity index score [13] of 16/22 (Figure 1, top). Table 1. Laboratory Findings at Diagnosis* Figure 1. Top. Bottom. Given the patients immune serologic results and hypergammaglobulinemia, she was treated with 30 mg of prednisone daily and 50 mg of azathioprine daily despite positive results for anti-HCV antibody (by recombinant immunoblot assay) and HCV RNA [as shown by results of a polymerase chain reaction]. She responded clinically and biochemically, with alanine aminotransferase levels returning to normal 8 months after therapy began. The prednisone dose was adjusted accordingly, and she has been well for 3 years while receiving 5 mg of prednisone daily and 50 mg of azathioprine daily. Examination of a liver biopsy specimen obtained 3 years after treatment showed marked improvement (Figure 1, bottom); the Knodell hepatitis activity index score was 6/22. This was accompanied by resolution of hypergammaglobulinemia. The patient remains positive for HCV RNA, with an increased quantitative level of HCV RNA as determined by the semi-quantitative method described by Ulrich and colleagues [14]. Patient 2 A 38-year-old white woman had a 19-month history of persistently elevated alanine aminotransferase levels. She had had rheumatoid arthritis since the age of 18 years and had received various nonsteroidal anti-inflammatory drugs, most recently piroxicam. Therapy with this drug had been discontinued for 6 months, with no substantial change in serum alanine aminotransferase levels, which ranged from 1.16 to 3.5 kat/L. She had no history of hepatitis or blood transfusion. She reported intravenous drug abuse between the ages of 18 and 20 years. Other medical problems included gastroesophageal reflux disease and hyperprolactinemia. She reported previous excessive use of alcohol. Her medications included 150 mg of ranitidine twice daily, 10 mg of metoclopramide three times daily, 400 mg of hydroxychloroquine sulfate daily, 2.5 mg of indapamide daily, 0.5 mg of alprazolam daily, and 5 mg of bromocriptine mesylate daily. Physical examination showed a firm, nontender liver edge 1 inch below the right costal margin, without splenomegaly, ascites, or cutaneous stigmata of chronic liver disease. Joint changes of inactive rheumatoid arthritis were observed. Laboratory data are shown in Table 1. Examination of a liver biopsy specimen showed moderately severe chronic hepatitis; the Knodell hepatitis activity index score [13] was 12/22. Results of anti-HCV tests by first-generation enzyme-linked immunosorbent assay were negative. Because of a positive antinuclear antibody titer of 1:640 and hypergammaglobulinemia, she was given 40 mg of prednisone daily and 50 mg of azathioprine daily as a therapeutic trial. She responded clinically and biochemically, with serum alanine aminotransferase levels returning to normal 5 months after the initiation of therapy. However, results of a repeat anti-HCV antibody test by second-generation enzyme-linked immunosorbent assay were positive, and HCV RNA was detected by polymerase chain reaction. Because cushingoid features developed, she discontinued prednisone therapy on her own, which resulted in a flare-up of hepatitis and an elevation of alanine aminotransferase levels to 3.3 kat/L. Therapy with prednisone and azathioprine was reinstituted, and serum alanine aminotransferase levels rapidly returned to normal. Since then, she has remained well while receiving 10 mg of prednisone daily and 50 mg of azathioprine daily. Examination of a liver biopsy specimen obtained 2 years after the initiation of therapy showed marked reduction of inflammation; the Knodell hepatitis activity index score was 7/22. She continues to be well and remains positive for HCV RNA, with no apparent change in the level of viremia as determined by the semi-quantitative methods described by Ulrich and colleagues [14]. Discussion The association between autoimmune chronic hepatitis and chronic hepatitis C remains controversial. In this context, the two cases we report provide interesting insights. Both patients had serologic and histologic features of autoimmune chronic hepatitis and chronic hepatitis C, and both had an excellent therapeutic response to immunosuppressive therapy. Although anti-liver-kidney microsomal antibody has been reported with HCV-associated autoimmune chronic hepatitis, neither of our patients had this antibody. Both patients were positive for HCV RNA by the polymerase chain reaction, which excludes the possibility of a false-positive result on the anti-HCV antibody test. Thus, HCV could have acted as a trigger for autoimmune chronic hepatitis, or the patients could have had underlying autoimmune chronic hepatitis and incidentally became infected with HCV. Determining the sequence of events is difficult. In any case, the combined features of autoimmune chronic hepatitis and chronic hepatitis C raise an important therapeutic dilemma because the therapies for each disease are believed to be mutually exclusive. Nishiguchi and associates [15] described 21 patients with steroid-responsive autoimmune hepatitis, 2 of whom tested positive for HCV RNA. In contrast, Magrin and colleagues [16] described positive recombinant immunoblot assay test results and serum HCV RNA in 15 patients whom they considered to have autoimmune hepatitis. None of their patients responded to prednisone therapy, whereas the few who were treated with interferon responded. The difficulty in interpreting their results stems from the broad criteria used to define autoimmune hepatitis. Autoantibodies are frequently found in low titers in patients with chronic hepatitis C, and most such patients who test positive for HCV RNA by the polymerase chain reaction have chronic hepatitis C and do not have autoimmune hepatitis. However, more recently the same investigators [12] described histologic improvement in 4 of 12 patients with autoimmune hepatitis treated with prednisone who tested positive for HCV RNA, and the improvement occurred despite increased serum HCV RNA titers. Two of the six patients responded to subsequent interferon treatment after not responding to prednisone therapy. Shindo and coworkers [17] reported exacerbation of liver disease during interferon- therapy for chronic hepatitis C in a patient who tested positive for both HCV RNA and antinuclear antibody but who responded partially to subsequent prednisone therapy. Both of our patients met the narrow criteria for autoimmune hepatitis; that is, they had high titers of autoantibodies and steroid-responsive chronic hepatitis. Our concern with prednisone therapy was the risk for increasing HCV replication under immunosuppression and, thus, worsening the liver disease. However, in patient 2, the degree of viremia remained stable despite immunosuppression; in addition, patient 1 showed biochemical and histologic improvement despite an apparent increase in the degree of viremia. This suggests that the risk for exacerbation of liver disease caused by HCV by immunosuppressive therapy may be substantially less than the risk for exacerbation of autoimmune chronic hepatitis by interferon- therapy. An unequivocal steroid response (that is, return of aminotransferase levels to normal and resolution of hypergammaglobulinemia) may be the only way to differentiate a disease that is primarily immune-mediated from chronic hepatitis C, in which injury reflects viral effects on the liver. This is different from the modest decreases in aminotransferase levels that are sometimes seen with

Use of high-resolution endoluminal sonography to measure the radius and wall thickness of esophageal varices

BACKGROUND Measurement of variceal wall tension theoretically provides the most accurate method of predicting future variceal bleeding. Using high-resolution endoluminal sonography in 45 patients with known portal hypertension, we measured and correlated the two previously unmeasured variables involved in the calculation of variceal wall tension (radius and wall thickness) by the Laplace equation. METHODS A 20 MHz 6.2F ultrasound transducer was used to image esophageal varices during standard esophagoscopy. All images were captured on videotape and later reviewed by two blinded investigators. Outer and inner variceal wall circumferences were measured at a cross section of each varix. The radius of each varix and the variceal wall thickness were calculated. The radius of each varix was then correlated with its wall thickness. The interobserver and intraobserver variabilities were measured. RESULTS The mean variceal radius was .86 +/- .34 cm for the inner radius and 1.48 +/- .41 cm for the outer radius; mean variceal wall thickness was .099 +/- 0.037 cm. Intraobserver and interobserver correlation for the radius was r = .98 and r = .97, respectively. The intraobserver and interobserver correlations for the wall thickness were r = .92 and r = .91, respectively. Variceal radius did not correlate with the wall thickness of the varix. CONCLUSIONS High-resolution endoluminal sonography provides a method for the accurate measurement of esophageal variceal radius and wall thickness. Variceal radius does not correlate with variceal wall thickness, implying that variceal wall tension cannot be accurately estimated by measurement of variceal size alone. Combining these data with measurements of variceal pressure should allow for the direct determination of wall tension and, subsequently, identification of patients at risk for variceal bleeding.

Quinidine pharmacokinetics in patients with cirrhosis or receiving propranolol

Quinidine pharmacokinetics (half-life, volume of distribution, and clearance) as well as protein binding were evaluated following a single 200 mg. oral dose of quinidine sulfate in eight control patients, in eight patients with moderate to severe cirrhosis, and in seven patients receiving 40 to 400 mg./day of propranolol. Patients with cirrhosis had a significantly longer quinidine half-life (9 +/- 1 hr; p less than .01) when compared to control patients (6 +/- 0.5h). This was not related to a reduced quinidine clearance rate but rather to an increase in quinidine volume of distribution (4.1 +/- .4 L./Kg. in cirrhotic patients vs 2.6 +/- 1 L./Kg. in control patients; p less than .01). Abnormal quinidine binding (greater than 25 per cent unbound fraction) was noted in seven of the eight cirrhotic patients. In contrast, patients receiving propranolol had a normal quinidine half-life of 6 +/- 0.5 hr. However, these patients had a significantly reduced quinidine clearance (3.3 +/- .7 ml./min./Kg. vs. 5.3 +/- .5 ml./min./Kg. in controls; p less than .05) and higher peak concentrations (1.25 +/- .20 micrograms/ml. vs. .80 +/- .5 micrograms/ml. in controls; p less than .05). Therefore in patients receiving propranolol, quinidine levels may be higher than expected shortly after dosage, and therefore a potential for transient toxicity exists in these patients. Maintenance quinidine dosage may have to be reduced in patients with moderate to severe hepatic cirrhosis, but not in patients receiving propranolol. Total quinidine concentration measurement underestimate free quinidine concentrations in most cirrhotic patients.

Possible Ranitidine Hepatotoxicity

Excerpt Ranitidine (Zantac; Glaxo Incorporated, Research Triangle Park, North Carolina) is a newly introduced H2-receptor antagonist approved by the Food and Drug Administration for short-term oral...

Risk of Esophageal Variceal Bleeding Based on Endoscopic Ultrasound Evaluation of the Sum of Esophageal Variceal Cross-Sectional Surface Area

OBJECTIVE:The aim of this study was to evaluate the risk of future variceal bleeding, based on the endoscopic ultrasound measurement of the sum of the cross-sectional surface area (CSA) of all of the esophageal varices in the distal esophagus.METHODS:Twenty-eight patients with portal hypertension and esophageal varices, but no prior history of variceal bleeding, were evaluated using endoscopic ultrasound (20-MHz ultrasound probe, Microvasive, Boston, MA; Olympus, Tokyo, Japan). The entire esophagus was imaged, and an image was selected at a point where the varices appeared the largest. This image was digitized, and the sum of the CSA of all of the varices was measured (Image Pro Plus, Silver Springs, MD) by an investigator blinded to the patients’ clinical status. The follow-up time for each patient was calculated (time to first bleed, time to liver transplantation, time to death, or time to the end of study). The Cox Proportional Hazards Model was used to determine if there was a significant difference between the sums of the CSA in the patients who bled compared with those who did not bleed. An OR was calculated to determine the risk of future variceal bleeding based on the sum of the CSA as measured in cm2/month. Positive and negative predictive values were calculated for future variceal bleeding.RESULTS:Six of 28 patients (21%) experienced esophageal variceal bleeding on follow-up. The mean CSA ± SEM of the sum of the esophageal varices in these patients was 0.77 cm2 plusmn; 0.31 cm2 (range 0.07–2.09 cm2). The mean time to bleeding was 15.5 months ± 4.95 months (range 1–29 months). Twenty-two of 28 patients (79%) did not experience variceal bleeding. The mean CSA ± SEM of the sum of the varices in these patients was 0.36 cm2 plusmn; 0.08 cm2 (range 0.02–1.19 cm2). The mean time to follow-up was 35.7 months ± 6.69 months (range 1.2–103.2 months). The sum of the CSA between the patients who bleed and those who did not bleed was significantly different at the p < 0.018 level. The OR for the risk of variceal bleeding for each one cm2 difference in the sum of the CSA per month was 6.34. Using a cutoff of 0.45 cm2, the sensitivity and specificity for future variceal bleeding was 83% and 75%, respectively.CONCLUSIONS:There is a significant difference (p < 0.018) in the sum of the esophageal variceal CSA between those patients who will experience future variceal bleeding and those who will not. There is a 76-fold increase per year in the risk of future variceal bleeding for each one cm2 increase in variceal CSA. Using a cutoff value for the CSA of 0.45 cm2, the sensitivity and specificity for future variceal bleeding above and below this point is 83% and 75%, respectively.

Results of steroid-based therapy for the hepatitis C-autoimmune hepatitis overlap syndrome.

OBJECTIVE:Overlap syndromes in which persons manifest clinical, histological, or immunological features of both hepatitis C infection and autoimmune hepatitis are well described. The discordant forms of treatment for hepatitis C and autoimmune hepatitis have made medical management of these patients difficult. We report our experience in using corticosteroids as first line therapy for the hepatitis C–autoimmune hepatitis overlap syndrome.METHODS:Seven patients with this overlap syndrome (diagnosis based on the presence of serum hepatitis C antibody by RIBA and serum hepatitis C RNA by polymerase chain reaction, and serum hypergammaglobulinemia, elevated ANA or ASMA titers, or histological findings consistent with autoimmune hepatitis) were treated with prednisone with or without azathioprine or cyclosporine, and followed for a median duration of 44.5 months.RESULTS:Five patients (71%) showed improvement of median serum ALT level from 162 U/L to 38 U/L (p = 0.04) and median serum γ-globulin from 2.1 g/dl to 1.4 g/dl (p = 0.04) by 6 months of therapy. The mean modified histological activity index score also decreased from 11.4 ± 2.5 to 6.6 ± 2.6 (p = 0.04) by at least 1 yr of therapy. One patient discontinued prednisone while taking azathioprine and experienced a rebound elevation of serum ALT that did not respond to retreatment with prednisone. Antiviral therapy was subsequently administered and resulted in biochemical and virologic response. Hepatitis C virus RNA remained detectable in all other patients.CONCLUSION:Corticosteroids are beneficial as a first line therapy for some patients with the hepatitis C–autoimmune overlap syndrome, resulting in appreciable biochemical and histological response but without viral eradication.

Identification of a human liver cytochrome P-450 exhibiting catalytic and immunochemical similarities to cytochrome P-450 3a of rabbit liver

Immunoblot analysis of liver microsomes from nine patients demonstrated that each contained a cytochrome P-450 that reacted with an antibody directed against the ethanol-inducible rabbit liver cytochrome, P-450 3a. Two of the liver specimens exhibited high concentrations of the immunoreactive protein, high rates of aniline hydroxylation and N-nitrosodimethylamine demethylation, and extensive inhibition of activity in the presence of antibody to P-450 3a. One other liver specimen exhibited a very low rate of aniline hydroxylation with significantly less antibody inhibition. The variability witnessed was independent of the alcohol history of the individual patients, suggesting that the human cytochrome may be under some other environmental, dietary or genetic regulation. Its inducibility by ethanol was not directly studied in this investigation. However, we conclude that there is a cytochrome P-450 present in human liver which is immunochemically and catalytically similar to the ethanol-inducible P-450 of rabbit liver.