E1A, E1B double-restricted replicative adenovirus at low dose greatly augments tumor-specific suicide gene therapy for gallbladder cancer.

Combination therapy with replicative oncolytic viruses is a recent topic in innovative cancer therapy, but few studies have examined the efficacy of oncolytic adenovirus plus replication-deficient adenovirus carrying a suicide gene. We aim to evaluate whether an E1A, E1B double-restricted oncolytic adenovirus, AxdAdB-3, can improve the efficacy for gallbladder cancers (GBCs) of the replication-deficient adenovirus-based herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) therapy directed by the carcinoembryonic antigen (CEA) promoter. Cytopathic effects of AxdAdB-3 plus AxCEAprTK (an adenovirus expressing HSVtk directed by CEA promoter) or AxCAHSVtk (an adenovirus expressing HSVtk directed by a nonspecific CAG promoter) with GCV administration were examined in several GBC lines and normal cells. Efficacy in vivo was tested in severe combined immunodeficiency disease mice with GBC xenografts. Addition of AxdAdB-3 (1 multiplicity of infection, MOI) significantly enhanced the cytopathic effects of AxCEAprTK (10 MOI)/GCV on GBC cells. The augmented effect was attributable to the replication of the AxCEAprTK and also to the enhanced CEA promoter activity, which was presumably transactivated by E1A. In normal cells, AxdAdB-3 (20 MOI) plus AxCEAprTK (200 MOI)/GCV was not cytopathic, whereas AxdAdB-3 (1 MOI) plus AxCAHSVtk (10 MOI)/GCV was significantly toxic. Low-dose AxdAdB-3 (2 x 10(7) PFU, plaque-forming unit) plus AxCEAprTK (2 x 10(8) PFU)/GCV significantly suppressed the growth of GBC xenografts as compared with either AxdAdB-3 (2 x 10(7) PFU)/GCV or AxCEAprTK (2 x 10(9) PFU)/GCV alone. E1A, E1B double-restricted replicating adenovirus at low dose significantly augmented the efficacy of CEA promoter-directed HSVtk/GCV therapy without obvious toxicity to normal cells, suggesting a potential use of this combination for treating GBC and other CEA-producing malignancies.

Fatal reactivation of hepatitis B virus following cytotoxic chemotherapy for acute myelogenous leukemia: fibrosing cholestatic hepatitis.

Hepatitis B virus (HBV) is a well known pathogen that sometimes causes fulminant hepatitis in patients undergoing cytotoxic chemotherapy. Fibrosing cholestatic hepatitis (FCH) is a recently recognized unique variant of viral hepatitis, which has been occasionally reported in HBV-infected recipients of liver, renal, or bone marrow transplantation. We present here a 48-yr-old male in whom HBV was reactivated during post-remission chemotherapy for acute myelogenous leukemia, which resulted in rapidly fatal outcome. He manifested with deterioration of liver function in association with enormous replication of HBV. Liver biopsy showed marked ballooning of hepatocytes, cholestasis, and periportal fibrosis with minimum infiltrates. Immunostaining revealed that hepatocytes were strongly positive for hepatitis B surface antigen. Under the diagnosis of FCH, he was treated with lamivudine and interferon beta, which was not effective. Autopsy showed severe atrophy of the liver and marked degeneration of hepatocytes. Hematologists should be aware that FCH is a fatal complication that can develop under post-chemotherapy immunosuppressed conditions. Although there is no convincing evidence, prophylactic administration of lamivudine seems to be a reasonable strategy.

The role of previous infection of hepatitis B virus in Hbs antigen negative and anti-HCV negative Japanese patients with hepatocellular carcinoma: etiological and molecular biological study.

The aim of this study is to elucidate the important role of the previous infection of HBV, and the relations among HBV genome integration and p53 gene mutation, telomerase activity and genetic instability in liver tissue with HBsAg-negative (NB) and anti-HCV negative (NC) hepatocellular carcinoma (HCC). We examined the backgrounds of 34 NB and NC (NBNC) Japanese patients with chronic liver disease (CLD) patients not associated with HCC and 26 NBNC CLD patients with HCC. HBV genome integration into host cell genome, p53 gene mutation telomerase activity and genetic instability were examined in 6 with NBNC HCC (NBNC-HCC) tumorous tissue (T) and non-tumorous tissues (NT). In the NBNC group, HBV-related antibody positive patients with HCC are significantly more than the patients without HCC. Moreover, concerning the stage of the coexisted liver diseases, in NBNC CLD, LC patients with HCC is 19 of 26 (73.1%) , on the other hand, LC patients without HCC is 16 of 34 (47.1%). LC patients with HCC group is significantly more than that without HCC. Three (50%) of 6 in T and 3 cases (50% ) in NT were found to integrated genome of HBV. p53 gene mutation was observed in 3 (50%) of T. Concerning the telomerase activity, 3 of 6 cases (50%) in T and 1 case in NT was recognized. There was no genetic instability (LOH or RER) of D2S123, D3S1067 and TP 53 in T and NT. Finally in T of NBNC HCC cases, TTVDNA was detected in 3 of 5. Even in the HBsAg-negative and anti-HCV negative HCC cases, CLD coexisting with LC, previous HBV infection and HBVDNA integration were observed. There were a few cases with HBVDNA integration, p53 gene mutation, telomerase activity and genetic instability, simultaneously in HCC tissue, and in some cases, the coexistence with TTVDNA were concurrently confirmed. It is speculated that the important role of the previous infection of HBV may have also been proposed for HCC oncogentic progression in NBNC CLD [corrected].

Long-term ursodeoxycholic acid therapy is associated with reduced risk of biliary pain and acute cholecystitis in patients with gallbladder stones: a cohort analysis.

Whether ursodeoxycholic acid (UDCA) therapy alters the long-term clinical course of gallstones (GS) without stone dissolution remains unknown. We aimed to clarify the relationship between long-term UDCA therapy and risks of biliary pain or acute cholecystitis in GS patients. We also aimed to identify factors affecting the natural course, and to explore a simple patient selection criteria for UDCA therapy. A cohort of 527 uncomplicated GS patients with or without UDCA (600 mg/d) followed for up to 18 years was analyzed. Patients who had frequent attacks or were complicated with cholecystitis were converted to cholecystectomy. History and UDCA therapy were identified on Cox analysis as 2 factors affecting the long-term clinical course. In patients without therapy, history was the only predictor of biliary pain among various patient or stone characteristics; biliary pain was rare in asymptomatic patients, while frequent in symptomatic patients (P <.001). UDCA therapy was associated with reduced risk for biliary pain in both symptomatic (62% vs. 92% in untreated patients at 10 years; P <.001; relative risk, 0.19; 95% CI, 0.10-0.34) and asymptomatic patients (6% vs. 12% in untreated patients at 10 years; P =.037; relative risk, 0.19; 95% CI, 0.04-0.91). Risk for the conversion was also reduced in UDCA-treated symptomatic patients (26% vs. 88% in untreated patients at 10 years, P <.001; relative risk, 0.08; 95% CI, 0.03-0.22). These effects were independent of stone dissolution. Three factors were identified on Cox analysis as affecting GS dissolution: radiolucency, small size (<10 mm) of stones, and visualized gallbladder (GB) on cholecystogram. A selection criteria based on these appears to exhibit high sensitivity (74%) and specificity (95%) for dissolution. UDCA therapy might be considered in symptomatic patients fulfilling these criteria, and also in patients who have significant surgical risk, because the longterm therapy is clearly associated with reduced risk of biliary pain and acute cholecystitis.

Plasma levels of mevalonate and 7alpha-hydroxy-4-cholesten-3-one in chronic liver disease.

BACKGROUND AND AIMS: Cholesterol levels in blood tend to be preserved despite hepatic impairment, in contrast to albumin levels and other markers of liver function in patients with liver cirrhosis (LC). We reported previously that the levels of plasma mevalonate (MVA) and 7alpha-hydroxy-4-cholesten-3-one (7alpha3one) closely reflect hepatic synthetic rates of cholesterol and bile acids. The aim of this study was to examine the association between hepatic cholesterogenesis and bile acid synthesis in hepatocellular impairment using these indices. METHODS: The plasma indices were measured in patients with LC (n = 38) or chronic hepatitis (CH; n = 11) and in normal controls (n = 22). The severity of LC was assessed by the Child-Pugh score. RESULTS: There were no significant differences in plasma MVA levels between CH, LC and control groups. However, plasma 7alpha3one levels were significantly lower in LC than in CH and control groups (P< 0.01). While MVA levels did not correlate with the Child-Pugh score, there was a significant correlation between 7alpha3one level and Child-Pugh score (P< 0.005). The plasma 7alpha3one level in controls correlated positively with MVA levels (P< 0.01); however, there was no significant correlation between these indices in CH and LC. CONCLUSION: In chronic liver disease, there was a tendency for hepatic cholesterogenesis to be sustained in the face of hepatocellular impairment, while bile acid synthesis declined in parallel with the severity of impairment.

Multiple regression analysis for assessing the growth of small hepatocellular carcinoma: the MIB-1 labeling index is the most effective parameter.

The aim of this study was to clarify whether histological parameters reflected tumor aggressiveness in patients with hepatocellular carcinoma (HCC). The tumor volume doubling times (TVDTs) of 21 HCCs, less than 3 cm in diameter at the start of the observation period, were calculated in 21 patients in whom the natural progression of the lesion was observed by ultrasonography. Paraffin-embedded sections were prepared from samples obtained by ultrasound-guided fine-needle liver biopsy at the end of the observation period. The histological parameters examined were the MIB-1 labeling index (LI), for which we performed immunohistochemical staining with the MIB-1 monoclonal antibody, using an antigen retrieval method; the nucleo-cytoplasmic (N/C ratio), cellularity, and the nuclear form factor (NFF), were calculated with an imaging analyzer. We performed multiple regression analysis for estimating the growth of small HCCs. With the N/C ratio (0.154 +/- 0.068; mean +/- SD), cellularity (453 +/- 21.8 cells/10(4) microm2), NFF (1.150 +/- 0.096), and degree of HCC differentiation as independent variables, only the MIB-1 LI (11.8 +/- 6.1%) showed a significant correlation with TVDT (207.5 +/- 162.6 days) (r = -0.658; P < 0.05). Compared to the conventional indices of histological atypism tested, i.e., N/C ratio, cellularity NFF, and degree of HCC differentiation, only MIB-1 LI was significantly correlated with small HCC growth rate. The MIB-1 LI may therefore be a simple and useful index of tumor aggressiveness.

Short-term effects of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor on cholesterol and bile acid synthesis in humans.

Competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase improve hypercholesterolemia. However, reports about the effects of these agents on bile acid synthesis, the metabolic pathway of cholesterol, are conflicting. We studied the short-term effect on one of these agents, pravastatin, on bile acid synthesis. Six male volunteers were given 40 mg of pravastatin. Plasma mevalonate level (which reflects cholesterol synthesis) and 7 alpha-hydroxy-4-cholesten-3-one level (which reflects bile acid synthesis) were measured every 2 h for 8 h. These plasma levels were compared to those of the same volunteers without pravastatin. Plasma mevalonate level after 2 h was lower than control (3.0 +/- 1.1 ng/mL vs. 6.7 +/- 2.5, mean +/- SD; P < 0.05). This decrease continued for 8 h (2.5 +/- 0.8 vs. 5.2 +/- 1.5; P < 0.05). On the other hand, plasma 7 alpha-hydroxy-4-cholesten-3-one level did not change until after 6 h; then at 8 h it was lower than control (15.7 +/- 11.8 ng/mL vs. 24.7 +/- 16.9; P < 0.05). According to three-way layout analysis of variance, mevalonate level was influenced by both pravastatin treatment (P < 0.01) and time-course (P < 0.01). On the other hand, the 7 alpha-hydroxy-4-cholesten-3-one level was affected by both individual difference (P < 0.01) and time course (P < 0.01), but pravastatin treatment did not influence this compound. This indicates that bile acid synthesis was influenced by pravastatin, although cholesterol synthesis was inhibited. The short-term inhibition of cholesterol synthesis did not affect bile acid synthesis.

[Preventive effects of eicosapentaenoic acid (EPA) on cholesterol gallstone formation in hamsters].

Polyunsaturated fatty acids (PUFA) regulate various biological functions and are involved in a variety of diseases. Eicosapentaenoic acid (20:5, EPA), one of the omega-3 PUFA, has been reported a number of actions including suppression of platelet aggregability and decrease in serum lipids and is well known to be useful in preventing the atherosclerotic diseases. In this paper, we demonstrated that highly purified ethyl eicosapentaenoate (EPA-E) prevents cholesterol (CH) gallstone formation in hamster model. Repeated administration of EPA-E to animals fed a lithogenic diet for 6 weeks decreased the incidences of both CH crystal and CH gallstone formations in gallbladder bile. Contrary, bezafibrate, one of fibric acid derivative which were reported to increase CH saturation index (CSI) in bile, significantly increased the incidences of CH crystal and gallstone formations. EPA-E did not affect the CSI, but markedly increased biliary phospholipid concentration. In the same model, ethyl palmitate (16:0), ethyl oleate (18:1), ethyl linolate (18:2, omega-6) and ethyl arachidonate (20:4, omega-6) had no effects both on biliary lipids composition and CH gallstone formation. These result suggest the benefit of EPA-E in the prevention of CH gallstone.

Trimethadione as a probe drug to estimate hepatic oxidizing capacity in humans.

Trimethadione (TMO) has the properties required of probe drugs for the evaluation of hepatic drug-oxidizing capacity in humans in vivo. TMO is demethylated to dimethadione (DMO), its only metabolite, in the liver after oral administration. Involvement of two cytochrome P450's--CYP2C9 and 3A4--in TMO metabolism has been seen in humans, but involvement of 1A2 is not clearly established. In humans with various types of liver disease and hepatectomy, the serum DMO/TMO ratios, which were measured on blood samples obtained by a single collection 4 hr after oral administration of TMO, correlated well with the degree of hepatic damage. This finding suggests that TMO may be used as a probe drug in the rapid determination of the functional reserve mass of the liver as well as hepatic drug-oxidizing capacity in humans in vivo.

Multivariate analysis of risk factors for hepatocellular carcinoma in patients with hepatitis C virus-related liver cirrhosis.

To elucidate the risk factors for hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)-related liver cirrhosis (LC), we examined 204 cirrhotic patients negative for hepatitis B surface antigen and positive for HCV antibodies. The independent influence of various clinical characteristics in these patients was analyzed by multiple logistic regression, and the risk factors for HCC were identified. Multiple logistic regression analysis identified and ranked the following four risk factors: male sex (P < 0.001), habitual heavy drinking (P < 0.005), hepatitis B virus antibody positivity (anti-HBs and/or anti-HBc, P < 0.05), and age greater than 60 years (P < 0.05). The odds ratio of HCC was 4.20 (95% confidence interval; CI, 1.80-9.78) in male patients, 3.27 (95% CI, 1.46-7.30) in habitual heavy drinkers, 2.01 (95% CI, 1.01-3.99) in patients positive for hepatitis B virus antibodies, and 2.06 (95% CI, 1.00-4.23) in patients older than 60 years. The cumulative occurrence rates of HCC after blood transfusion were significantly higher in habitual heavy drinkers (4.8%, 49.4%, and 74.7% at 10, 20, and 30 years, respectively) than in non-drinkers (0%, 21.0%, and 23.3% at 10, 20, and 30 years, respectively, P < 0.0003). The mean interval for progression to LC after blood transfusion was significantly shorter in the habitual heavy drinkers than in the non-drinkers (22.4 +/- 4.4 years vs 28.4 +/- 3.9 years; P < 0.0003). This multivariate analysis revealed that habitual heavy drinking and hepatitis B virus antibody positivity are significant risk factors for HCC in HCV-related liver cirrhosis.

Severe acute hepatitis A associated with acute pure red cell aplasia.

A rare case of severe acute hepatitis A complicated by pure red cell aplasia (PRCA) is reported. A 60-year-old man with jaundice and hepatomegaly was diagnosed as having acute hepatitis A by positive IgM anti-hepatitis A antibody (anti-HAV). Severe anemia rapidly developed 3 weeks after admission, and the patient was diagnosed with PRCA by both bone marrow smears and erythrocyte survival study. The anemia was transient and bone marrow recovered within 1 week. However, concomitant with bone marrow recovery, the hepatitis worsened. He became drowsy and disoriented and severe jaundice, ascites, prolonged prothrombin time, increased transaminase levels, and abnormal electroencephalogram (EEG) were exhibited. Plasma exchange transfusion and glucagon-insulin (GI) therapy improved the consciousness level, but bilirubin, transaminase levels, and IgM anti-HAV titer remained high. Intravenous administration of lipophilized prostaglandin E1 (lipo-PGE1) was added to the GI therapy. Bilirubin and transaminase levels were normalized in the 8th week after the initiation of this combination therapy (17 weeks after admission). The combined use of lipo-PGE1 with plasma exchange and GI therapy appeared to be useful for the prolonged severe hepatitis in this patient.

The role of previous hepatitis B virus infection and heavy smoking in hepatitis C virus-related hepatocellular carcinoma.

OBJECTIVE: Worldwide epidemiological studies have demonstrated that hepatitis C virus (HCV) probably is a causative agent of hepatocellular carcinoma (HCC). However, there are no available reports that clearly identify the risk factors for the development of HCC in HCV-related chronic liver disease (CLD). The aim of the present study is to explore the risk factors for hepatocarcinogenesis in HCV-related CLD. METHODS: We prospectively observed 412 patients with anti-HCV-positive CLD but without co-infection of hepatitis B virus (232 patients with chronic hepatitis and 180 with liver cirrhosis) for between 0.5 and 15.8 yr (median: 4.9 yr). Risk factors for hepatocarcinogenesis were identified with a Cox proportional-hazard model. RESULTS: Sixty-three patients (15.3%) developed HCC during the observation period; the cumulative occurrence rates at the end of the 5th, 10th, and 15th yr was 3.7%, 12.1%, and 12.1%, respectively, for chronic hepatitis patients and 23.3%, 49.4%, and 90.7%, respectively, for 180 cirrhotic patients. The Cox proportional-hazard model showed that the risk of hepatocarcinogenesis increased almost 5-fold in cirrhotic patients (risk ratio, 5.14; 95% confidence interval, 2.52-10.46, p = 0.0001), 2-fold in patients with positive antibodies against hepatitis B surface antigen and/or antibodies against hepatitis B core antigen (risk ratio, 2.14; 95% confidence interval, 1.13-4.07, p = 0.0201), and 2.5-fold in heavy smokers (risk ratio, 2.46; 95% confidence interval, 1.11-5.49, p = 0.0276). CONCLUSION: These epidemiological results indicate that previous infection with hepatitis B virus and heavy smoking (in addition to liver cirrhosis, a known risk factor) play important roles as risk factors for carcinogenesis in HCV-related CLD.

Clinical significance of the trimethadione tolerance test in chronic hepatitis: a useful indicator of hepatic drug metabolizing capacity.

Trimethadione (TMO) was chosen as an indicator of quantitative hepatic microsomal function, and its pharmacokinetics were studied in 52 patients with chronic hepatitis. Findings in these patients were compared with those for 26 healthy subjects and 13 patients with renal failure. Patients with chronic hepatitis, but not those with renal failure, showed significant reduction in clearance (CL) and prolongation of half-life (t1/2), and the extent of abnormalities was found to reflect the severity of histologic changes in liver tissue. The serum dimethadione (DMO)/TMO ratio 4 h after the administration of TMO altered in parallel with the CL and t1/2 of TMO, and abnormalities in this simple ratio were also related to the histologic severity of changes in the liver tissue. A low DMO/TMO ratio (< 0.4) was associated with advanced histologic changes (chronic active hepatitis with bridging or chronic active hepatitis with cirrhosis), whereas a high DMO/TMO ratio (> 0.4) was associated with mild histologic changes (chronic persistent hepatitis or chronic active hepatitis) (sensitivity, 0.81; specificity, 0.86). These results indicate that the DMO/TMO ratio, which can be obtained from a single blood sampling, reflects the histologic severity of changes in tissue liver, and that the TMO tolerance test is a useful indicator of quantitative liver function.

Biliary alpha 1-acid glycoprotein concentrations in gallstone-free controls and in patients with multiple or solitary cholesterol gallstones.

We recently identified a promoting glycoprotein in the concanavalin A-bound fraction of gallbladder bile as a biliary form of alpha 1-glycoprotein (AAG). The concentration of biliary AAG appears to exert an important promoting effect on the speed of cholesterol nucleation in many patients with cholesterol gallstone disease. In the current study, we provide information about the biliary concentration of AAG as well as the amount and comparative potency of its subfractions in patients with and without cholesterol gallstone disease. The amount of total biliary AAG and the amounts of its different isoforms separated by concanavalin A affinity chromatography were measured by ELISA. Estimates of absolute concentrations of AAG for each sample were normalized to the sample total protein content to give relative AAG values. The promoting activity (potency) of immunopurified biliary AAG from gallstone patients and gallstone-free controls on cholesterol crystallization was compared by a crystal growth assay. The mean absolute concentration of AAG in gallstone-free controls was not significantly different from multiple stone patients. The relative concentration of AAG (micrograms per milligram total protein) was significantly increased in patients with multiple stones when compared to controls (P < 0.05), and both the absolute and relative concentrations of AAG (micrograms per milligram bile), were three- and to five fold higher in a number of these patients. The functional activity and distribution of AAG in different subfractions was similar in gallstone patients and gallstone-free controls. The relative concentration of biliary AAG is significantly greater in cholesterol gallstone patients with multiple stones than in gallstone-free controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between biliary alpha 1-acid glycoprotein concentration and cholesterol crystal nucleation time in gallstone disease.

A biliary form of the alpha 1-acid glycoprotein (AAG) promotes cholesterol crystallization in the lower-molecular-weight, concanavalin A-bound fraction of gallbladder bile. In addition, bile AAG concentration is higher in cholesterol gallstone patients with multiple stones than in control patients without gallstone disease. In this study we sought to determine whether the increased biliary concentration of AAG in cholesterol gallstone patients is accompanied by a more rapid nucleation time in patients with multiple stones. AAG concentration in native biles was measured by ELISA. Nucleation time was measured using a standard microscopy method. The concentration of biliary AAG was then related to nucleation time in biles from the same patients. Nucleation times were significantly shorter (< or = 5 days) in cholesterol gallstone patients with raised AAG concentrations (P < 0.03). There was a significant (P = 0.004) negative correlation (r = -0.53) between nucleation time and the AAG concentration in cholesterol gallstone patients with multiple stones. The concentration of biliary AAG appears to exert an important influence on the speed of cholesterol nucleation in bile in many patients with cholesterol gallstone disease.

Randomized control trial of lipo-prostaglandin E1 in patients with acute liver injury induced by lipiodol-targeted chemotherapy.

OBJECTIVE: The aim of this study was to prove whether lipo-prostaglandin E1 (PGE1)/glucagon insulin therapy combination could prevent the acute liver dysfunction induced by Lipiodol (iodized oil)-targeted chemotherapy for hepatocellular carcinoma. METHODS: This study was a randomized control trial. Patients in two groups (groups A and B: n = 29) were each given an intravenous injection of 10 units of insulin and 1 mg glucagon every 12 hours for 1 week after Lipiodol-targeted chemotherapy. Patients in group B (n = 11) were each given an intravenous injection of 20 micrograms lipo-PGE1 every 12 hours over 1 week. Several items, including conventional liver function tests, were evaluated at the start of the study and on the days 1, 2, 4, 7, and 14 after Lipiodol-targeted chemotherapy. RESULTS: Combined lipo-PGE1/glucagon-insulin therapy can prevent the elevation of serum ALT level and total bilirubin level after Lipiodol-targeted chemotherapy. In the group A (glucagon-insulin therapy only), the maximum level in the follow-up interval was statistically higher than that in the pretreatment level (p < 0.05), whereas there was no significant difference in group B (treated with combined glucagon-insulin therapy and lipo-PGE1). Changes of ALT level in group B tend to be lower than in group A; however, there was no significant statistical difference. There were few episodes of side effects in both groups. CONCLUSION: Combined lipo-PGE1/glucagon-insulin therapy may be a safe and effective treatment for the prevention of acute hepatic failure.

Identification of human biliary alpha 1-acid glycoprotein as a cholesterol crystallization promoter.

BACKGROUND/AIMS: We have recently outlined the biochemical features of a human 42-kilodalton biliary glycoprotein that shows concentration-dependent cholesterol crystallization-promoting activity. The goal in this work was to establish its identity and to examine some aspects of its biochemical properties relative to its activity. METHODS: Internal amino acid sequencing following tryptic digestion was performed. Based upon this result, immunoreactivity against the 42-kilodalton glycoprotein was examined using a relevant antibody. With the same antibody, the 42-kilodalton glycoprotein was isolated from bile and assayed for activity. Sequential enzymatic deglycosylation of successive terminal glycans of the purified glycoprotein was performed, and the effects on both reductions in molecular radius (M(r)) and on comparative promoter activities were examined. RESULTS: Both amino acid sequence and immunochemical data identify the 42-kilodalton glycoprotein as a biliary form of alpha 1-acid glycoprotein. When purified by immunoaffinity chromatography, potent promoting activity shown was proportionately reduced by successive removal of terminal glycans that also reduced the M(r)s. CONCLUSIONS: The 42-kilodalton cholesterol crystallization-promoting glycoprotein is now identified as a biliary form of alpha 1-acid glycoprotein. Further, some aspects of the important role of glycans in this extensively glycosylated protein have been explored.

Cholesterol crystallization-promoters in human bile: comparative potencies of immunoglobulins, alpha 1-acid glycoprotein, phospholipase C, and aminopeptidase N1.

Concanavalin A (Con A)-binding glycoproteins accelerate the rate of cholesterol crystal formation as a prelude to gallstone formation. Immunoglobulins (IgM, IgA, and IgG), aminopeptidase N (APN), phospholipase C (pcPLC), and alpha 1-acid glycoprotein from this Con A fraction have all been proposed as candidate promoters. We immunopurified each of the six putative promoters and examined their comparative effects by adding equal amounts to a cholesterol crystal growth assay. The effects of immunoabsorptive removal of each of the specific candidate promoters from native bile were also compared. In additional studies, the potency of these proteins was in the following order: IgM > IgA = AAG > IgG. APN and pcPLC showed no effect on cholesterol crystal growth at their apparent physiological concentrations. In subtractive experiments, only a minor loss (< 10%) of net promoting activity from that of the whole Con A-bound fraction was observed after immunoabsorptive removal of pcPLC, APN, or immunoglobulins. Total removal of AAG, however, showed a far greater loss (/33%) of the net promoting activity. These data indicate that AAG accounts for the greatest portion of net biliary Con A-bound promoting activity derived from currently defined and well-identified glycoproteins. However, more than 60% of total Con A-binding promoting activity remains unaccounted for, indicating the presence of other important and still unidentified promoters in human bile.