Hepatic organic anion uptake in the rat.

The hepatic uptake of bilirubin (BR), indocyanine green (ICG), and sulfobromophthalein (BSP) was studied in 350 anesthetized Sprague-Dawley rats by determining the initial plasma disappearance rate (V) of various doses of unlabeled ICG, or of tracer quantities of [3H]BR or [35S]BSP injected into the jugular vein simultaneously with varying amounts of unlabeled BR or BSP. Similar studies were also performed involving the simultaneous injection of potential inhibitors of hepatic uptake. The results indicate that: (a) hepatic uptake determined by direct tissue measurement could be accurately estimated from the plasma disappearance data; (b) saturation of hepatic uptake with increasing dose was readily demonstrated for each of these three organic anions, and in each instance a plot of V versus dose took the form of a rectangular hyperbola analyzable in terms of Michaelis-Menten kinetics; (c) for BR, the saturable uptake process showed a Vmax more than 100 times the normal net transfer rate from plasma to bile; (d) hepatic uptake of BR, BSP, and ICG showed relatively selective, mutually competitive inhibition; glycoholic acid did not inhibit hepatic uptake of any of these substances; and (e) "counter-transport" could be demonstrated for each of the three test substances. These data are compatible with the existence of a carrier-mediated transport process for hepatic uptake of each of these three organic anions and clarify the relationship of hepatic BR uptake to its overall transport from plasma to bile.

Deficiency of the autologous mixed lymphocyte reaction in patients with primary biliary cirrhosis.

In this study we show that patients with primary biliary cirrhosis (PCB) have a marked deficiency in the ability to generate an autologous mixed lymphocyte reaction (AMLR) but have a normal ability to generate an allogeneic mixed lymphocyte reaction (MLR). This deficiency is not due to differences in the time-course of the proliferative response or to an altered response to variable numbers of stimulator cells. The deficiency was consistently found irrespective of the methods used to isolate autologous stimulator cells. Both responder cells and stimulator cells obtained from patients with PBC were similar to normal cells in their ability to generate an MLR in allogeneic normal human serum. In addition, serum from patients with PBC inhibited the ability of normal lymphocytes to generate both the AMLR and MLR to a similar degree, suggesting that the defect of the AMLR in PBC is not due to a serum factor. It has been shown that the responder cell population in the AMLR contains a subpopulation of cells that mediate suppression. Therefore, it is possible that the deficiency of the AMLR may be related to previously described abnormalities of suppressor function in patients with PBC.

Removing substances from blood by affinity chromatography. II. Removing bilirubin from the blood of jaundiced rats by hemoperfusion over albumin-conjugated agarose beads.

In vitro studies indicate that bilirubin and other albumin-bound substances can be efficiently removed from plasma by filtration over albumin-conjugated agarose beads. The effectiveness of this technique in vivo was investigated in rats by using a closed extracorporeal hemoperfusion system. Five Gunn rats whose endogenous bilirubin pool had been labeled with [(3)H]bilirubin and five Sprague Dawley rats with surgically created biliary obstruction were chosen as models of unconjugated and conjugated hyperbilirubinemia. Indocyanine green was injected into rats and its removal also studied. In the Gunn rats, 98% of the bilirubin was removed from plasma during the initial pass over the column as determined isotopically and chemically. Plasma bilirubin levels fell more than 70% from 8.2+/-1.6 mg/100 ml (mean+/-SD) to 2.6+/-0.5 mg/100 ml during a 1-h hemoperfusion. An average of 1,061 mug of bilirubin was recovered from the columns, representing 22.5+/-4.2% of the total exchangeable bilirubin pool and 96+/-36.4% of the plasma pool. Results were similar in the rats with biliary obstruction and in those given indocyanine green. Normal Sprague Dawley rats experience minimal changes in formed blood elements, electrolytes, and proteins as the result of hemoperfusion. When the total volume of the column did not exceed 51% of the estimated blood volume of the animal, the survival rate was 100% in 20 studies, and the procedure was without observable ill effect. Extrapolation of both in vitro and in vivo data to man suggests that extracorporeal hemoperfusion over albumin-agarose columns may be a practical means of assisting hepatic excretory function.

Hepatic accumulation and intracellular binding of conjugated bilirubin.

After the intravenous injection of unconjugated [(3)H]bilirubin into normal Sprague-Dawley and Wistar R rats, radiolabeled bile pigments rapidly accumulated in the liver. By 1.5 min after injection, an average of 36% of the injected isotope was present in liver homogenates. Between 3 and 15 min, 37-64% of the total intrahepatic radiolabeled bilirubin was conjugated, as demonstrated by extraction of label into the polar phase of a solvent partition system. This indicates both rapid conjugation, and accumulation of conjugated bilirubin within the liver cell. Fluorometric determination of the dissociation constants of purified bilirubin and its mono- and diglucuronides for homogeneous preparations of two human and four rat glutathione S-transferases, including ligandin, revealed avid binding of all three bile pigments to this class of proteins. Hence, the observation that the intrahepatic bile pigment pool contains substantial amounts of conjugated bilirubin can be attributed to the high binding affinities observed. Thin-layer chromatographic analysis of the (3)H-pigments produced by p-iodoaniline diazotization of homogenates and cytosol demonstrated that the intrahepatic pool of conjugated bilirubin was almost exclusively monoglucuronide. Examination of radiolabeled bilirubin conjugates excreted in bile during the first 20 min after injection of [(3)H]bilirubin showed no preferential excretion of diglucuronide. These studies indicate that (a) both bilirubin and its monoglucuronide accumulate within the liver cell as ligands with the glutathione S-transferase; and (b) bilirubin diglucuronide does not significantly accumulate within the general intrahepatocellular pool of protein-bound bile pigments. The latter observation is compatible with the formation and excretion of bilirubin diglucuronide directly from the canalicular pool of the liver cell.

Mephenytoin disposition and serum bile acids as indices of hepatic function in chronic viral hepatitis.

BACKGROUND AND OBJECTIVES: The effect of chronic viral hepatitis on liver function may vary from none to hepatic failure. Changes in function are usually the result of impaired hepatocyte function or altered vascular flow and architecture. Conventional liver function tests usually cannot distinguish contributions from these mechanisms or indicate degree of hepatic metabolic dysfunction. An alternative approach is to measure the hepatic metabolism of a highly extracted compound whose oral clearance and systemic bioavailability are dependent on both hepatocyte function and degree of portosystemic shunt. METHODS: The stereoselective metabolism of racemic mephenytoin (100 mg oral dose) was investigated in 35 patients with chronic viral hepatitis and compared with 153 healthy subjects. The mephenytoin R/S enantiomeric ratio and cumulative excretion of the 4'-hydroxymephenytoin metabolite in a 0- to 8-hour urine sample were used in addition to serum bile acid levels and pathologic examination of biopsy specimens to assess the severity of hepatic dysfunction and portosystemic shunting. RESULTS: The patients as a group excreted less 4'-hydroxymephenytoin and had a smaller R/S enantiomeric ratio of mephenytoin. The two measures were discriminatory between the patient groups classified by either serum cholylglycine level or pathologic examination of biopsy specimens. Combination of the two measures of mephenytoin metabolism allowed the patients to be classified into three groups: normal hepatocyte function without portosystemic shunt, normal hepatocyte function with portosystemic shunt, and low hepatocyte function with or without portosystemic shunt. CONCLUSION: This study has shown the potential usefulness of mephenytoin metabolism as a sensitive indicator of hepatic pathologic condition with an ability to discriminate between contributory alternative mechanisms.

Effect of corticosteroid therapy on levels of antibody to hepatitis B core antigen in patients with chronic type B hepatitis.

Serum levels of antibody to hepatitis B core antigen and IgM antibody to hepatitis B core antigen were tested in 15 patients who participated in a randomized, placebo-controlled trial of a 28-day course of prednisolone therapy. During treatment, serum levels of antibody to hepatitis B core antigen and IgM antibody to hepatitis B core antigen decreased in all 10 treated patients, but in none of five controls (p less than 0.05). Also during therapy, ALT activity decreased by an average of 50% and serum IgG levels by 30% (both p less than 0.05). Serum levels of hepatitis B virus DNA and DNA polymerase activity did not change significantly. Four to 10 weeks after discontinuation of prednisolone, a rebound of serum ALT and IgM antibody to hepatitis B core antigen levels occurred, which usually resolved within the subsequent months of follow-up evaluation. In three patients, however, there was a prolonged exacerbation of the disease following prednisolone withdrawal; in these three, levels of IgM antibody to hepatitis B core antigen and ALT remained elevated above pretreatment values. The close correlation between changes in serum ALT activity and IgM antibody to hepatitis B core antigen levels suggests that corticosteroids can modulate disease activity in chronic type B hepatitis by suppression of the host-immune response to hepatitis B virus antigens.

Spontaneous reactivation of chronic hepatitis B virus infection.

Studies on the natural history of chronic type B hepatitis have shown that loss of hepatitis B e antigen and seroconversion to antibody to hepatitis B e antigen are usually accompanied by remission of disease activity and improvement in serum aminotransferase levels. Twenty-five symptomatic patients with biopsy-documented chronic type B hepatitis were followed for 25 +/- 2 mo (mean +/- SEM) after disappearance of hepatitis B e antigen, hepatitis B virus-deoxyribonucleic acid, and deoxyribonucleic acid polymerase activity from the serum. Twenty-four patients developed the antibody to hepatitis B e antigen. All 25 patients demonstrated a decrease in serum aminotransferase levels, and most became asymptomatic. However, during subsequent follow-up, 8 of the 25 patients (32%) exhibited reactivation of chronic type B hepatitis manifested by abrupt elevation of serum aminotransferase levels and reappearance of serum hepatitis B virus-deoxyribonucleic acid, deoxyribonucleic acid polymerase activity, and, in 7 patients, hepatitis B e antigen. All 8 patients developed symptoms: 3 became icteric, 3 developed ascites, and 2 bled from esophageal varices. One of these patients died. Episodes of reactivation invariably occurred within 1 yr of loss of hepatitis B e antigen and lasted for up to 13 mo. These observations suggest that loss of hepatitis B e antigen and seroconversion to the antibody to hepatitis B e antigen do not necessarily imply permanent remission of chronic type B hepatitis, and subsequent spontaneous reactivation may be an important cause of progression of hepatic injury.

Acute type A hepatitis during chronic hepatitis B virus infection: association of depressed hepatitis B virus replication with appearance of endogenous alpha interferon.

Two patients with chronic type B hepatitis and intercurrent episodes of acute type A hepatitis are presented. Serological markers of hepatitis B virus replication decreased or became undetectable in both patients during the acute illness, while interferon activity was transiently detected in serum. The presence of serum leukocyte (alpha) interferon was confirmed by neutralization with specific antisera and tests of pH2 stability. These observations suggest a role for natural leukocyte (alpha) interferon in the modulation and control of hepatitis B virus infection and provide further evidence to support trials of exogenous leukocyte (alpha) interferon in the chronic infection.

Interferon alfa for patients with clinically apparent cirrhosis due to chronic hepatitis B.

BACKGROUND: The prognosis of advanced cirrhosis due to chronic hepatitis B is poor, and results of therapies, including liver transplantation, have been unsatisfactory. Little is known about the effectiveness of interferon alfa in patients with cirrhosis. METHODS: Between 1984 and 1991, 18 patients with clinically-apparent cirrhosis due to hepatitis B were treated with interferon alfa at the Clinical Center of the National Institutes of Health. RESULTS: Six treated patients (33%) had a sustained loss of hepatitis B virus DNA and hepatitis B e antigen (if present initially) and decrease of amino-transferase levels into the normal or near normal range. In follow-up, these 6 patients resolved all symptoms of cirrhosis and are alive and fully active. In contrast, the 12 patients who did not have a sustained loss of hepatitis B virus have had evidence of progressive liver disease, 6 have died and 4 underwent hepatic transplantation. Side effects of interferon were common and included bacterial infections (n = 5) and exacerbations of disease (n = 9). CONCLUSIONS: These findings indicate that interferon alfa is effective in selected patients with mildly decompensated cirrhosis due to hepatitis B.

Studies of the kinetics of purified conjugated bilirubin-3H in the rat.

Radio-labeled conjugated bilirubin was purified from rat bile by affinity chromatography. Following the intravenous injection of tracer doses of this material, the initial fractional plasma disappearance rate of conjugated bilirubin-3H in the normal Sprague-Dawley rat was found to be approximately 50% faster than that for unconjugated bilirubin-3H (0.51 +/- 0.94 [SE] vs. 0.35 +/- 0.02 min.-1; P less than .001). Hepatic recovery studies in the rat indicated that, over the first four minutes after injection, disappearance of cholephilic anions from plasma is accounted for almost entirely by hepatic uptake. Hence, these studies demonstrate that hepatic uptake of conjugated bilirubin is substantially faster than that of unconjugated bilirubin. Net hepatic clearance of conjugated bilirubin in normal rats was three-fold greater than that of unconjugated bilirubin (0.94 +/- 0.16 vs. 0.30 +/- 0.03 ml./min./100 gm. body weight; P less than .001), presumably reflecting both the more rapid hepatic uptake and the ability of the former pigment to by-pass the conjugation step. Hepatic uptake of conjugated bilirubin was shown to be inhibited by unconjugated bilirubin, sulfobromophthalein and indocyanine green but not by glycocholate. These observations cannot be explained by simple diffusion and suggest that the hepatic uptake mechanism for conjugated bilirubin is the same as that for the unconjugated pigment.

Attachment to collagen by isolated hepatocytes from rats with induced hepatic fibrosis.

The attachment to and synthesis of collagen by hepatocytes isolated from rats with and without hepatic fibrosis were studied. Attachment of normal hepatocytes was enhanced fourfold in the presence of serum, whereas attachment of hepatocytes from CCl4-treated rats with severely fibrotic livers was enhanced only twofold by serum. Fibronectin promoted hepatocyte attachment to collagen to the same degree as serum, whereas serum from which the fibronectin had been removed did not. Thus hepatocytes adhere to collagen via fibronectin, a known adhesion protein for fibroblasts. In the absence of serum or fibronectin, hepatocytes attached best to basement membrane collagen (type IV). This enhanced attachment was abolished by periodate oxidation of the collagen, an observation which suggested that carbohydrate residues are involved in hepatocyte attachment to this collagen. Collagen synthesis was determined after hepatocytes were cultured for 24 hr. No difference was found in the amount of collagen synthesized by cultures of hepatocytes derived from control rats and from rats with hepatic fibrosis. By immunofluorescence, the cell responsible for the synthesis of collagen appeared to have a fibroblastic morphology.

Hepatitis virus infection in an isolated Canadian Inuit (Eskimo) population.

The epidemiology of Hepatitis A virus (HAV) and hepatitis B virus (HBV) infection was studied in a northern Canadian Inuit (Eskimo) settlement. Sera from 720 of the 850 inhabitants of Baker Lake, Canada, were tested for markers of HAV and HBV infection. Anti-HAV was present in 71% of the residents and its prevalence increased with age. Serologic evidence of HBV infections was found in 27% of residents. The prevalence increased with age, being uncommon under the age of 20 (6%) and almost universal over the age of 60 (93%). Among the 29 hepatitis B surface antigen (HBsAg) carriers identified, all were adults, all had low levels of HBsAg, and all were negative for hepatitis B e antigen (HBeAg) and DNA polymerase but positive for antibody to HBeAg. These data demonstrate a high prevalence of HAV and HBV infection in this population. Further, they suggest that a dramatic decrease in the transmission of HBV infection has occurred over the past 20-30 years.

Peroxidase-anti-peroxidase detection of hepatitis B surface and core antigen in liver biopsy specimens from patients with chronic type B hepatitis.

Liver biopsy specimens from 58 American patients with chronic type B hepatitis were investigated for the presence and distribution of the hepatitis B core (HBcAg) and surface (HBsAg) antigens by peroxidase-anti-peroxidase techniques. HBsAg was detected in 43 (77%) and HBcAg in 52 (90%) patients. HBcAg was present in 50 of 51 (98%) patients with hepatitis B e antigen (HBeAg) but in only two of seven (29%) of patients with antibody to HBeAg (anti-HBe). There was no correlation between severity of hepatitis or height of aminotransferase activities and the amount of HBsAg or HBcAg in hepatocytes but there was a positive correlation between amount of HBcAg and height of HBV-DNA and DNA polymerase activity in serum. Follow-up liver biopsies, taken 1 to 3 yr later, were available from 39 patients. HBcAg remained detectable in 25 of 26 patients with persistence of HBeAg but disappeared in 12 patients who had lost HBeAg. In nine patients, HBcAg was cytoplasmic as well as nuclear in distribution. Seven of these patients had an intense lobular hepatitis with marked elevations in aminotransferase activities. These findings indicate that the amount of HBcAg in liver correlates with the amount of serum hepatitis B virus as quantified by serum levels of DNA polymerase and HBV-DNA. The amount of nuclear HBcAg does not correlate with the severity of the liver disease, but the presence of cytoplasmic HBcAg usually reflects an active and severe ongoing hepatitis.

Hepatitis B virus deoxyribonucleic acid in liver of chronic carriers. Correlation with serum markers and changes associated with loss of hepatitis B e antigen after antiviral therapy.

Patients with chronic hepatitis B receiving antiviral or immunomodulatory therapy were prospectively studied to determine the relationship between the state of hepatitis B virus deoxyribonucleic acid in liver and the levels of serum markers of hepatitis B virus infection. Hepatitis B virus deoxyribonucleic acid was quantitated and the molecular forms determined using molecular hybridization in two separate liver specimens taken at least 1 yr apart. All 30 patients initially had hepatitis B surface antigen and e antigen in serum and all had hepatitis B virus deoxyribonucleic acid, including replicative intermediate forms, present in liver. The amount of viral deoxyribonucleic acid in liver correlated significantly with the e antigen titer in serum. At the time of the second liver biopsy, e antigen was no longer detectable in the serum of 12 patients although all except 1 patient still had detectable hepatitis B surface antigen. In this group, the amount of hepatitis B virus deoxyribonucleic acid in liver had decreased significantly and replicative viral intermediates had disappeared. In contrast, among patients who remained e antigen-positive, the amount of viral deoxyribonucleic acid did not change appreciably and replicative intermediate forms were still detectable. These findings imply that in chronic hepatitis B, loss of e antigen is usually associated with resolution of hepatitis B virus replication.

The effect of repeated phlebotomy on bilirubin turnover, bilirubin clearance and unconjugated hyperbilirubinaemia in the Crigler-Najjar syndrome and the jaundiced Gunn rat: application of computers to experimental design.

1. A multicompartmental model of erythrokinetics and bilirubin production has been developed to predict the consequences of chronic phlebotomy on daily bilirubin turnover. 2. Control values for four physiological variables including bilirubin turnover were determined in a 20-year-old woman with type I congenital nonhaemolytic jaundice (Crigler-Najjar syndrome). With these base-line data, the model predicted the following changes during phlebotomy: a 34% fall in bilirubin turnover; a 240% increase in the haemoglobin content of bone-marrow erythroid precursors; a 25% fall in the half-life of 51Cr-labelled erythrocytes; a characteristic alteration of the erythrocyte survival curve after labelling with [2-14C]glycine. 3. On the assumption, previously validated in normal volunteer subjects and patients with Gilbert's syndrome, that hepatic bilirubin clearance was independent of turnover and would therefore remain unchanged, a fall in plasma unconjugated bilirubin concentration during phlebotomy from 436 to 282 mumol/1 was expected. 4. Accordingly, the patient underwent phlebotomy 350 ml/week for 2 months, and 500 ml/week during an additional 3 months. Appropriate studies during phlebotomy confirmed each of the predictions in paragraph 2 above. In particular, turnover fell by 31%. Unexpectedly, plasma unconjugated bilirubin remained essentially unchanged. Analogous results were observed in phlebotomized jaundiced Gunn rats. 5. Kinetic studies in both the patient and the rats demonstrated that the failure of plasma unconjugated bilirubin to fall in parallel with bilirubin turnover resulted from a prolongation of the terminal half-life of radioactively labelled bilirubin and a fall in bilirubin clearance in every instance. 6. These studies indicate that (a) in congenital non-haemolytic jaundice, bilirubin clearance is uniquely influenced by bilirubin turnover and (b) compartmental modelling and kinetic studies are useful for predicting and interpreting the results of both physiological experiments and experimental therapeutic regimens.

The significance of antibody to hepatitis C virus in patients with chronic hepatitis B.

We assessed the prevalence and clinical significance of antibodies to hepatitis C virus among a cohort of 148 patients with chronic hepatitis B virus infection. Sixteen patients (11%) had anti-hepatitis C virus detectable by enzyme-linked immunoassay. The results from eight of these patients were positive by recombinant immunoblot assay. The results of recombinant immunoblot assay testing were not consistent; therefore the analysis of the patients' data was based on anti-hepatitis C virus enzyme-linked immunoassay results. Patients with chronic hepatitis B with anti-hepatitis C virus were more likely to be cirrhotic (44% vs. 21%) and to have decompensated liver disease (24% vs. 6%). Hepatitis B virus replication appeared to be suppressed in patients with both infections as measured by hepatitis B virus-associated DNA polymerase activity (mean = 2,055 vs. 2,555 cpm). Human immunodeficiency virus infection was more common in the anti-hepatitis C virus positive group (36% vs. 11%). Thus hepatitis C virus appears to suppress hepatitis B virus replication and to cause more severe liver disease in patients with chronic hepatitis B infection.