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Inflammatory bowel disease (IBD) is a multifactorial disease. A breach in the mucosal barrier, otherwise known as “leaky gut,” is alleged to promote mucosal inflammation by intensifying immune activation. However, interaction between the luminal antigen and mucosal immune system is necessary to maintain mucosal homeostasis. Furthermore, manipulations leading to deregulated gut permeability have resulted in susceptibility in mice to colitis as well as to creating adaptive immunity. These findings implicate a complex but dynamic association between mucosal permeability and immune homeostasis; however, they also emphasize that compromised gut permeability alone may not be sufficient to induce colitis. Emerging evidence further supports the role(s) of proteins associated with the mucosal barrier in epithelial injury and repair: manipulations of associated proteins also modified epithelial differentiation, proliferation, and apoptosis. Taken together, the role of gut permeability and proteins associated in regulating mucosal inflammatory diseases appears to be more complex than previously thought. Herein, we review outcomes from recent mouse models where gut permeability was altered by direct and indirect effects of manipulating mucosal barrier-associated proteins, to highlight the significance of mucosal permeability and the non-barrier-related roles of these proteins in regulating chronic mucosal inflammatory conditions.

A lcoholic liver disease (ALD) is a major cause of cirrhosis and a leading cause of death of end-stage liver disease in the United States and most of the Western world. The only means of restoring health in patients with end-stage liver disease at present is orthotopic liver transplantation. In the United States, between 3,000 and 4,000 liver transplants are performed each year, and a similar number are done yearly in Western Europe. Of the total, 20% to 25% are performed for ALD. The increasing demand for liver transplantation and lack of a similar increase in the supply of donor livers make it important to reassess the use of this precious resource on a regular basis. What criteria should be used in selecting patients with ALD for liver transplantation? How can survival and quality of life after liver transplantation for ALD be made optimal? These issues were addressed in a focused 2-day workshop, ‘‘Liver Transplantation for Alcoholic Liver Disease,’’ held under the auspices of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the National Institute for Alcohol Abuse and Alcoholism (NIAAA) in Bethesda, Maryland, December 6-7, 1996. This executive statement summarizes the issues raised at this conference and provides overall recommendations regarding treatment of patients with ALD who require liver transplantation. Most importantly, this summary focuses on important needs and directions for clinical research in liver transplantation for ALD. Alcohol is used by a majority of adults in the Western world. Population-based surveys indicate that 68% of adult Americans drink at least one alcoholic beverage per month, and 10% drink two or more per day, the usual definition used for ‘‘heavy drinking.’’ Heavy drinking is more common among men (18%) than women (3%) and among whites than blacks or Hispanics. Alcohol use is a major cause of morbidity and mortality; in the United States, 5% of deaths, approximately 100,000 per year, are either directly or indirectly attributable to alcohol abuse. Why some individuals are able to drink alcohol without dependence while others cannot is unknown. Among the factors associated with the development of alcoholism, both social and genetic factors are important. A family history of alcoholism is perhaps the major risk factor for developing alcohol dependence or abuse. The recent identification of genetic linkages to alcohol-seeking activity in laboratory animals promises to provide critical information on the genetic basis of alcoholism. Research into the nature of addiction is of central importance for the future of prevention and treatment of alcoholism. Encouragement of temperance and moderation should be the cornerstone to societal attempts to decrease alcoholism. ALD is perhaps the most widely recognized complication of chronic alcohol abuse. Nevertheless, only 15% to 20% of alcoholic subjects develop cirrhosis. The disease is more common in men than in women, and the average age of onset is in the mid-40s, typically after 10 to 20 years of heavy alcohol use. The average intake of alcohol in patients with ALD is more than 100 g of alcohol (approximately 8 drinks) per day, but the lower

To provide health care providers, patients, and the general public with a responsible assessment of currently available data on the management of hepatitis B.A non-DHHS, nonadvocate 12-member panel representing the fields of hepatology and liver transplantation, gastroenterology, public health and epidemiology, infectious diseases, pathology, oncology, family practice, internal medicine, and a public representative. In addition, 22 experts from pertinent fields presented data to the panel and conference audience.Presentations by experts and a systematic review of the literature prepared by the Minnesota Evidence-based Practice Center, through the Agency for Healthcare Research and Quality. Scientific evidence was given precedence over anecdotal experience.The panel drafted its statement based on scientific evidence presented in open forum and on published scientific literature. The draft statement was presented on the final day of the conference and circulated to the audience for comment. The panel released a revised statement later that day at http://consensus.nih.gov. This statement is an independent report of the panel and is not a policy statement of the NIH or the Federal Government.The most important predictors of cirrhosis or hepatocellular carcinoma in persons who have chronic HBV are persistently elevated HBV DNA and ALT levels in blood. Other risk factors include HBV genotype C infection, male sex, older age, family history of hepatocellular carcinoma, and co-infection with HCV or HIV. The major goals of anti-HBV therapy are to prevent the development of progressive disease, specifically cirrhosis and liver failure, as well as hepatocellular carcinoma development and subsequent death. To date, no RCTs of anti-HBV therapies have demonstrated a beneficial impact on overall mortality, liver-specific mortality, or development of hepatocellular carcinoma. Most published reports of hepatitis therapy use changes in short-term virologic, biochemical, and histologic parameters to infer likelihood of long-term benefit. Approved therapies are associated with improvements in intermediate biomarkers, including HBV DNA, HBeAg loss or seroconversion, decreases in ALT levels, and improvement in liver histology (Table). Although various monitoring practices have been recommended, no clear evidence exists for an optimal approach. The most important research needs include representative prospective cohort studies to define the natural history of the disease and large RCTs of monotherapy and combined therapies, including placebo-controlled trials, that measure the effects on clinical health outcomes. Table. Criteria Useful in Determining for Whom Therapy is Indicated: Patients for whom therapy is indicated: Patients who have acute liver failure, cirrhosis and clinical complications, cirrhosis or advanced fibrosis and HBV DNA in serum, or reactivation of chronic HBV after chemotherapy or immunosuppression; Infants born to women who are HBsAg-positive (immunoglobulin and vaccination). Patients for whom therapy may be indicated: Patients in the immune-active phase who do not have advanced fibrosis or cirrhosis. Patients for whom immediate therapy is not routinely indicated: Patients with chronic hepatitis B in the immune-tolerant phase (with high levels of serum HBV DNA but normal serum ALT levels or little activity on liver biopsy); Patients in the inactive carrier or low replicative phase (with low levels of or no detectable HBV DNA in serum and normal serum ALT levels); Patients who have latent HBV infection (HBV DNA without HBsAg). We recommend routine screening for hepatitis B of newly arrived immigrants to the United States from countries where the HBV prevalence rate is greater than 2%. Screening will facilitate the provision of medical and public health services for infected patients and their families and provide public health data on the burden of disease in immigrant populations. The screening test should not be used to prohibit immigration.

For normal function and survival, hepatocytes require proper cell-extracellular matrix (ECM) contacts mediated by integrin receptors and focal adhesions. Previous studies have shown that chronic ethanol consumption selectively impairs perivenous (PV) hepatocyte attachment and spreading on various ECM substrates but increases expression of the beta1 integrin subunit, the common beta subunit for two major hepatocyte-ECM receptors, alpha1beta1 and alpha5beta1 integrins. This study examined the effects of ethanol treatment on the expression and cytoskeletal distribution of alpha1, alpha5, and beta1 integrin subunits, the epidermal growth factor receptor (EGF-R), and the cytoskeletal proteins focal adhesion kinase, paxillin, vinculin, and actin in periportal and PV hepatocytes.Periportal and PV hepatocytes were isolated from control and ethanol-fed rats. For expression analysis, lysates were examined by SDS-PAGE and immunoblotting procedures. For cytoskeletal distribution studies, Triton-soluble and -insoluble (cytoskeletal) fractions from hepatocytes cultured on collagen IV were analyzed by SDS-PAGE and immunoblotting.Chronic ethanol administration caused PV-specific increases in expression and cytoskeletal association of the integrin subunits. Although ethanol treatment did not affect expression of the EGF-R in either cell type, it did increase the association of the EGF-R with the cytoskeleton selectively in PV hepatocytes. Ethanol treatment had no significant effect on either the expression or the cytoskeletal distribution of focal adhesion kinase, paxillin, vinculin, or actin in either cell type.The increases in integrin expression and cytoskeletal association observed after chronic ethanol administration suggest that a process downstream of integrin-ECM interactions is impaired selectively in PV hepatocytes, possibly involving altered focal adhesion assembly or turnover, processes essential for efficient cell-ECM adhesion. Alterations in these processes could contribute to the impaired hepatocyte function and structure observed after chronic ethanol administration.

Many investigators have suggested that an immune reaction to acetaldehyde-protein adducts may be involved in the development and/or progression of alcohol liver disease. The most often reported acetaldehyde adduct is the reduced adduct prepared in vitro in the presence of strong reducing agents. However, the production of this adduct in vivo has been difficult to prove. Nevertheless, the detection of serum antibodies to this reduced adduct following alcohol exposure in animals and humans has been used to support the formation of this adduct in vivo. We have recently observed that when acetaldehyde-protein adducts prepared under nonreducing conditions are used to immunize animals, antibody to the reduced protein adduct is detected. Therefore, it was the purpose of this study to demonstrate that nonreduced (NR) adduct epitopes can be modified by intact cells to express reduced (R) adduct epitopes. This was accomplished using the monoclonal antibody RT1.1 that has been previously characterized by this laboratory and has been shown to recognize only R and not NR acetaldehyde adducts. In these studies, Balb/c mice were injected intraperitoneally (500 microg/animal) with either keyhole limpet hemocyanin (KLH)-NR or KLH-R adducted proteins. Immunization with KLH-NR produced significant amounts of antibodies that recognized both NR and R epitopes. In contrast, immunization with KLH-R produced antibodies to only R and not NR epitopes. Isolated peritoneal macrophages from nonimmunized mice were incubated in vitro with either KLH-NR, KLH-R, or unmodified KLH proteins, and the cell surface expression of the reduced epitope (RT1.1) and the activated macrophage marker (MAC-3) determined by double immunofluorescent staining. Activated macrophages incubated with KLH-NR expressed the R adduct on 11.5% of the cells, compared with 3.8% following incubation with unmodified KLH, and 19.4% following incubation with KLH-R. These data suggest that the NR adduct and/or the carrier protein are modified by peritoneal macrophages in vivo and present an epitope that is detected as a reduced adduct (RT1.1 positive). These observations may explain the presence of circulating antibodies to the reduced adduct that has been reported in human and animal studies.

Recent studies have shown that the alcohol metabolites malondialdehyde and acetaldehyde can combine to form a stable adduct (MAA) on proteins. This adduct has been detected in the livers of rats chronically consuming ethanol, and serum antibodies to MAA have been observed at significantly higher concentrations in ethanol-fed when compared with pair-fed or chow-fed control rats. More recently, preliminary studies have strongly suggested that the MAA adduct is capable of stimulating antibody responses to soluble proteins in the absence of adjuvants. The antibodies produced recognize either the MAA epitope or the carrier protein itself. Therefore, it was the purpose of this study to examine the potential immunogenicity of MAA-modified exogenous proteins in the absence of adjuvants. Balb/c mice were immunized in the presence or absence of adjuvant with different concentrations of unmodified or MAA-modified proteins. The antibody response to both the MAA epitope and unmodified protein epitopes were determined by ELISA. In the absence of adjuvant, significant antibody responses were induced to both the MAA epitope and nonmodified protein epitopes. Smaller immunizing doses of MAA-protein conjugate favored the production of antibodies to nonmodified proteins, whereas larger doses induced a strong anti-MAA response. In studies to begin determining a mechanism for the specificity of the response in the absence of adjuvants, peritoneal macrophages were found to bind and degrade MAA-adducted proteins through the use of a scavenger receptor. This indicated that MAA-adducted proteins may be specifically taken up and epitopes presented to the humoral immune system in the absence of adjuvants. Importantly, these are the first data showing that an alcohol-related metabolite can induce an antibody response in the absence of adjuvant and suggesting a mechanism by which antibody to the MAA adduct or its carrier (exogenous or endogenous) proteins may be generated in vivo.

The field of liver transplantation has evolved slowly over the past 5 years. The most important new additions to this field include new immunosuppressive agents and greatly broadened recipient selection criteria. The most compelling problem in transplantation today remains the lack of suitable donors. Innovative surgical techniques, such as auxiliary liver transplantation, extracorporeal support and living-related donation, represent new and important additions to the approach to patients with liver disease.

The purpose of this study was to review our experience with Epstein-Barr virus (EBV) hepatitis after liver transplantation.During a 68-month period, we performed 668 liver transplants and 585 patients. We identified 11 patients (2 percent), including 5 adults and 6 children with EBV hepatitis after liver transplantation. The diagnosis of EBV hepatitis was established by evaluating allograft biopsies. The histology was confirmed by the use of polymerase chain reaction technology.The average time of diagnosis after liver transplantation was 45 days. Eight of eleven cases occurred within the first six months after transplantation. After the diagnosis of EBV hepatitis, treatment consisted of a decrease in immunosuppression plus antiviral therapy and intravenous immunoglobulin. The one-year actuarial survival for patients with EBV hepatitis, was 73 percent (8 of 11). Two patients died of progressive multi-organ EBV involvement. To determine the risk of developing EBV hepatitis, we reviewed our experience with the administration of antilymphocyte preparations in 585 patients. The patients found to have a significantly greater risk of developing EBV hepatitis included those receiving more than one course of antilymphocyte therapy or greater than a total dose of 70 milligrams of OKT3 in a single course.EBV hepatitis after liver transplantation is an infrequent event, which may be treated successfully. The occurrence of EBV hepatitis appears closely linked to the use of antilymphocyte preparations.

Although patients with cirrhosis have an increased susceptibility for bacterial infections, endocarditis complicating cirrhosis has been reported only infrequently. In this study, our objective was to determine whether, bacterial endocarditis is, in fact, a complicating factor in cirrhosis.We retrospectively studied all cases of bacterial endocarditis that occurred over the last 15 yr in patients with known cirrhosis.Ten patients (three males, seven females) were identified, whose mean age was 55 yr (range 29-65 yr). Bacterial organisms included Staphylococcus aureus, coagulase-positive (eight patients), Peptostreptococcus (one patient), and Enterococcus (one patient). Underlying liver disease consisted of alcoholism (five patients), autoimmune chronic active hepatitis (two), cryptogenic cirrhosis (two), and primary biliary cirrhosis (one). Distribution of heart valves affected were mitral valve (six), aorta (two), and there were two involving both mitral and aortic valves. Echocardiograms revealed vegetation in 50% of the patients. Laboratory studies were markedly abnormal, with mean values of albumin 2.4 mg/dl, creatinine 2.5 mg/dl, BUN 76.5 mg/dl, and total bilirubin 8.2 mg/dl. Potential associated sources of infection were upper gastrointestinal bleeding (four), pneumonia (two), and one each of spontaneous bacterial peritonitis, hip replacement, heart catheterization, and abdominal abscess. The outcome was poor, with death in eight of 10 patients.Bacterial endocarditis may complicate cirrhosis, may be more frequent in females, typically involves the mitral valve, and probably is due to Staphylococcus aureus.

Six transplantations of the liver were performed over a period of six years in five adult patients with Type I diabetes mellitus (DM). The diabetic group included two males and three females with a mean age of 36 years and a mean duration of DM of 20 years. Primary diseases of the liver included two instances of primary biliary cirrhosis, two instances of sclerosing cholangitis and one instance of autoimmune chronic hepatitis. Three patients also received a simultaneous whole organ pancreatic transplant. All patients were managed with cyclosporine and prednisone immunosuppression with selective OKT3 induction. Patient and hepatic allograft survival rates were 80 and 67 percent, respectively, after a mean follow-up period of 4.7 years. One of the three pancreatic grafts was successful and resulted in euglycemia for two years. Three patients have subsequently undergone successful renal transplantation at one, two and one-half, and six and one-half years after hepatic transplantation. Although transplantation of the liver can be performed safely in carefully selected patients with Type I DM, these patients are still at risk for the development of progressive nephropathy. Renal transplantation is an acceptable therapeutic alternative when this occurs.

We have used extracorporeal liver perfusion (ECLP) to aid in the management of three patients with fulminant hepatic failure (FHF). Organs were used for ECLP only if they would have gone otherwise unused through United Network for Organ Sharing. In all three patients treated, serial serum bilirubin and arterial ammonia values trended toward the normal range. The neurologic examinations improved dramatically in two patients, and metabolic function of the extracorporeally perfused livers was unequivocally demonstrated by the clearance of theophylline in the last two patients. Two patients ultimately had successful liver transplants, whereas the third patient failed to improve neurologically despite evidence of metabolic function by the extracorporeally perfused liver, and died 7 days after ECLP was discontinued, from pulmonary and renal failure. These studies suggest that, 30 yr after initial clinical trials, ECLP can be applied safely without the need for arterial access 1) as a bridge to transplantation, 2) to assess whether patients in FHF will benefit from improved hepatic function and therefore transplantation, and 3) potentially, to evaluate the "usability" of questionable donor organs.

The results of orthotopic liver transplantation (OLTx) in patients with diabetes mellitus (DM) are not well defined.Between 1985 and 1991, 45 adult patients with pretransplantation DM (5 type I, 40 type II) underwent OLTx at our center as identified by retrospective chart review. We compared this diabetic recipient group to a case-control nondiabetic group matched for age, gender, primary liver disease, weight, and timing of OLTx. A total of 30 variables were collected and analyzed with McNemar's test for categorical data, paired t tests for continuous data, and survival and repeated measures analysis for longitudinal data.No differences between diabetic and nondiabetic recipients were noted in patient or graft survival, the incidence or severity of rejection, blood transfusions, operative complications, readmissions, major infections, or number of hospital days after OLTx. However, the incidence of minor bacterial (p = 0.046) and minor fungal (p = 0.035) infections were higher in the DM group. Serum blood urea nitrogen (p = 0.02) and creatinine (p = 0.03) levels were also higher in patients with diabetes versus control patients during the first year after OLTx.In carefully selected patients with pretransplantation DM, OLTx can be accomplished with results similar to nondiabetic recipients in spite of a higher incidence of minor infections and renal dysfunction.

Previous studies have shown that the assembly and properties of the hepatocyte plasma membrane are altered by ethanol administration, indicating possible changes in the receptor-mediated binding of the plasma membrane to extracellular matrix substrates. In the present study, the effects of chronic ethanol consumption on the ability of hepatocytes to attach to various components of the extracellular matrix were investigated.Rats were pair-fed for 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. The effects of ethanol treatment on hepatocyte-extracellular matrix interactions was ascertained by determining the ability of isolated hepatocytes to attach to various extracellular matrix substrates.The attachment of hepatocytes, isolated from the ethanol-fed rats, to laminin-coated plates was significantly decreased compared with hepatocytes from chow-fed or pair-fed controls. Greater decreases in attachment were seen when higher numbers of hepatocytes were seeded in the plates. Similar inhibitions of attachment were also observed when fibronectin or type I collagen were used as matrices. Time-course cell attachment assays indicated that the maximum extent of attachment rather than the rate of attachment was primarily altered by chronic ethanol feeding. Hepatocytes from the ethanol-fed rats also detached more readily from the matrix-coated plates than those from the controls. A reduced number of functional surface receptors for matrix components is likely the most important factor that accounts for the ethanol-induced impairment of hepatocyte attachment.These results indicate that chronic ethanol administration impairs the interactions of hepatocytes with their extracellular matrix and that this defect could lead to alterations of hepatocyte structure and function.

Jejunoileal bypass operation was originally done to promote weight loss for treatment of morbid obesity. We used such a model to determine if dietary vitamin absorption is compromised by such an operation. Six rats were subjected to a jejunoileal bypass, 6 control rats were pair-fed to bypassed rats; and 6 were fed ad libitum. Vitamin content of folic, B6, riboflavin, nicotinate, pantothenate, thiamin, biotin, B12, vitamins A, E, and carotene in blood and liver was determined after 8 postoperative weeks. Aside from riboflavin, blood vitamin levels were significantly depressed in bypassed rats. The deepest depression was seen for B12, carotene and vitamin E. Liver vitamin stores of folate, riboflavin, thiamin, B12, clearly were significantly depressed in the bypassed animals compared to the pair-fed and ad libitum-fed controls. This model can serve for rapidly studying micronutrient depletion due to malabsorption without dietary manipulation or antibiotics for gut sterilization.

We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde reacts with a variety of proteins to form both stable and unstable adducts. Unstable adducts are readily reversible and are largely represented by Schiff bases. Stable adducts are essentially irreversible products that are characterized by their resistance to various treatments. The structures of stable adducts have not been established, but it appears that the epsilon-amino group of certain lysines and the amino group of N-terminal amino acids participate in aldehyde binding. Stable adducts can form in the absence of a reducing agent, such as sodium cyanoborohydride, and are distinctly different than the ethylated amino groups formed under reductive conditions. Upon prolonged incubation, stable binding of acetaldehyde results in the formation of fluorescent products, and Schiff bases serve as intermediates of these advanced stable products. It is likely that several acetaldehyde molecules and perhaps at least two amino groups participate in the formation of these stable fluorescent products. Such a reaction mechanism could account for both inter-and intra-molecular cross-linking of proteins. Furthermore, it is likely that stable adducts are represented by multiple products whose structures may vary, depending upon the particular target protein. A reaction mechanism involving an aldol condensation and generation of a crotonaldehyde Schiff base is proposed as an essential step in stable adduct formation.

Both acute and chronic ethanol administration inhibit the secretion of albumin and glycoproteins from the liver. Impairment of posttranslational steps of the secretory process are mainly involved in this secretory defect, although in some instances altered synthesis of the protein moiety may be a factor. Decreased secretion following ethanol administration results in the intrahepatic retention of export proteins. The secretory defect is a consequence of the metabolism of ethanol and is likely mediated via acetaldehyde, although more conclusive proof is still required. The manner by which acetaldehyde impairs the secretory process is unknown, but may be related to its high reactivity with hepatocellular proteins. The specific posttranslational steps or processes involved in the secretory defect are still unclear; however, it appears that the final steps of secretion (post-Golgi events) may be the primary site of impairment. Impaired secretion of proteins from the liver could contribute to altered levels of plasma proteins and hepatomegaly as well as to the liver injury observed in the alcoholic.

Ribonuclease A was reacted with [1-13C,1,2-14C]acetaldehyde and sodium cyanoborohydride in the presence or absence of 0.2 M phosphate. After several hours of incubation at 4 degrees C (pH 7.4) stable acetaldehyde-RNase adducts were formed, and the extent of their formation was similar regardless of the presence of phosphate. Although the total amount of covalent binding was comparable in the absence or presence of phosphate, this active site ligand prevented the inhibition of enzymatic activity seen in its absence. This protective action of phosphate diminished with progressive ethylation of RNase, indicating that the reversible association of phosphate with the active site lysyl residue was overcome by the irreversible process of reductive ethylation. Modified RNase was analysed using 13C proton decoupled NMR spectroscopy. Peaks arising from the covalent binding of enriched acetaldehyde to free amino groups in the absence of phosphate were as follows: NH2-terminal alpha amino group, 47.3 ppm; bulk ethylation at epsilon amino groups of nonessential lysyl residues, 43.0 ppm; and the epsilon amino group of lysine-41 at the active site, 47.4 ppm. In the spectrum of RNase ethylated in the presence of phosphate, the peak at 47.4 ppm was absent. When RNase was selectively premethylated in the presence of phosphate, to block all but the active site lysyl residues and then ethylated in its absence, the signal at 43.0 ppm was greatly diminished, and that arising from the active site lysyl residue at 47.4 ppm was enhanced. These results indicate that phosphate specifically protected the active site lysine from reaction with acetaldehyde, and that modification of this lysine by acetaldehyde adduct formation resulted in inhibition of catalytic activity.

The effects of chronic ethanol feeding on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Liver slices from rats fed ethanol for 4-5 wk showed a decreased ability to incorporate [14C]glucosamine into medium trichloracetic acid-precipitable proteins when compared to the pair-fed controls; however, the labeling of hepatocellular glycoproteins was unaffected by chronic ethanol treatment. Immunoprecipitation of radiolabeled secretory (serum) glycoproteins with antiserum against rat serum proteins showed a similar marked inhibition in the appearance of glucosamine-labeled proteins in the medium of slices from ethanol-fed rats. Minimal effects, however, were noted in the labeling of intracellular secretory glycoproteins. Protein synthesis, as determined by measuring [14C]leucine incorporation into medium and liver proteins, was decreased in liver slices from ethanol-fed rats as compared to the pair-fed controls. This was the case for both total proteins as well as immunoprecipitable secretory proteins, although the labeling of secretory proteins retained in the liver slices was reduced to a lesser extent than total radiolabeled hepatic proteins. When the terminal sugar, [14C]fucose, was employed as a precursor in order to more closely focus on the final steps of hepatic glycoprotein secretion, liver slices obtained from chronic ethanol-fed rats exhibited impaired secretion of fucose-labeled proteins into the medium. When ethanol (5 or 10 mM) was added to the incubation medium containing liver slices from the ethanol-fed rats, the alterations in protein and glycoprotein synthesis and secretion caused by the chronic ethanol treatment were further potentiated. The results of this study indicate that liver slices prepared from chronic ethanol-fed rats exhibit both impaired synthesis and secretion of proteins and glycoproteins, and these defects are further potentiated by acute ethanol administration.

Acetaldehyde production and the radiolabeling of hepatic proteins were determined in rat liver slices incubated with 14C-ethanol (10 mmol/L). Significant labeling of hepatic proteins occurred in the presence of protein synthesis inhibitors, indicating that, under these conditions, the radiolabeling of protein did not occur via de novo protein synthesis. Additional experiments indicated that the major source of protein-bound radioactivity derived from 14C-ethanol oxidation was the formation of 14C-acetaldehyde adducts with proteins. This conclusion was made from observations that pyrazole, an inhibitor of ethanol oxidation and, therefore, acetaldehyde formation, decreased radiolabeling of protein, whereas cyanamide, which elevated hepatic acetaldehyde levels, markedly increased the labeling of protein. Furthermore, L-cysteine, which can bind acetaldehyde and, therefore, act as an acetaldehyde trap, substantially reduced protein-bound radioactivity. It was also demonstrated that acetaldehyde formed both stable and unstable adducts with hepatic proteins and that unstable adducts may undergo conversion to form stable adducts during incubation.

Drugs that block the assembly of microtubules inhibit protein secretion by mature hepatocytes. In this study, we ascertained the effects of these drugs on hepatic protein secretion during development. Slices prepared from adult and late-gestation (day 21) fetal rat livers were incubated with colchicine, vinblastine, or podophyllotoxin, and the secretion of [14C]leucine-labeled proteins was then determined. Colchicine (10 microM) decreased trichloroacetic acid-precipitable protein secretion by 65% in adult liver, but only by 31% in fetal liver. A higher (50 microM) concentration of colchicine caused minimal further impairment of secretion in both age groups. Colchicine (10 microM) impaired the secretion of immunoprecipitable albumin to about the same degree as trichloroacetic acid-precipitable proteins in both adult and fetus. Determinations of [3H]colchicine uptake into slices indicated that reduced hepatocellular levels of the drug were not the cause of the diminished effect on protein secretion observed in the fetus. Like colchicine, various concentrations of vinblastine and podophyllotoxin inhibited trichloroacetic acid-precipitable protein and albumin secretion consistently less from the fetal liver (30%-50%) than from the adult liver (50%-75%). These results indicate a reduced dependence on normal microtubule function for protein secretion in the developing liver.

Acetaldehyde reacts with lysyl residues of proteins to form unstable and stable adducts. The effects of adduct formation on the biological activity of certain proteins were investigated. The covalent binding of acetaldehyde to lysine-dependent enzymes selectively inhibited their catalytic activity. Acetaldehyde adduct formation with tubulin also decreased its ability to assemble into microtubules. In addition, certain lysine residues in the alpha-chain of the tubulin dimer had a selective and enhanced reactivity toward acetaldehyde. This selectivity of binding to the alpha-chain occurred for stable adducts but not unstable adducts, and impaired tubulin assembly correlated with stable adduct formation. These studies indicate that acetaldehyde may preferentially bind to certain key lysine amino groups in proteins, and the resultant binding can cause marked alterations in biological function.

The effects of chronic ethanol intoxication on hepatic microtubules are controversial. We, therefore, designed a morphometric study to examine ethanol-induced alterations in microtubules in vivo and in vitro. Using a computer-assisted point-counting system we determined the volume, surface, and length densities of hepatic microtubules, in addition to those of other hepatic organelles. Consistent with previous qualitative descriptions, chronic ethanol feeding caused increases in the volume and surface densities of mitochondria and microsomes. These changes account, in part, for the increased protein content of hepatocytes after chronic ethanol consumption. However, there was no effect on the volume, surface, or length densities of hepatic microtubules, nor on their calculated radii. The structure of isolated bovine brain microtubules after polymerization in vitro was not changed by ethanol or its metabolites, acetaldehyde and acetate. These data indicate that neither chronic ethanol intoxication nor the presence of ethanol, or its metabolites, affects the number or structure of microtubules.

Determination of total and polymerized tubulin in the liver by the [3H]colchicine-binding assay is hampered by rapid loss of binding sites. One solution, which has been proposed to overcome this problem, is the use of a "time-decay" corrected assay that extrapolates to an initial binding capacity based on the results of testing multiple aliquots of liver supernatants over time. If such an approach is valid, it follows that the extrapolated initial value should be independent of the rate of time-decay. To test this relationship, we modulated the half-life (T1/2) for time-decay of a fixed amount of hepatic microtubule-derived tubulin using various concentrations of organic acids, sodium glutamate and glucose-1-phosphate. We found that the initial binding capacity based on simple first order decay did vary with the T1/2. Similar results were also obtained with bovine brain tubulin added to hepatic supernatants. These results indicate that the quantification of hepatic tubulin derived by extrapolated time-decay values is inaccurate and may overestimate the actual amount of tubulin present, especially when time-decay is rapid.

The in vivo effects of acute ethanol administration on the secretion of plasma glycoproteins and albumin were studied. Ethanol (6 g/kg body wt) was administered intragastrically to nonfasted rats, while control animals received isocaloric glucose. Hepatic protein and glycoprotein secretion in these animals was assessed by determining the rate of appearance of [3H]leucine- and [14C]fucose-labeled proteins into the plasma. Ethanol treatment decreased the secretion of both leucine- and fucose-labeled proteins into the plasma. This inhibition of secretion was accompanied by a corresponding increase in the hepatic retention of both leucine- and fucose-labeled immunoprecipitable secretory proteins. Pretreatment of the ethanol-treated animals with pyrazole, an inhibitor of ethanol metabolism, prevented the inhibition of hepatic protein secretion. These in vivo studies confirm our earlier in vitro findings that acute ethanol administration inhibits the secretion of plasma proteins, resulting in their hepatocellular retention.

Homogenates of rat liver incubated with 10 mM [14C]ethanol were analyzed for acetaldehyde production and for both stable and unstable radiolabeled acetaldehyde adducts with proteins. During incubation, formation of acetaldehyde and of 14C-labeled proteins both increased with time in a parallel manner. Acetaldehyde generation and subsequent formation of radiolabeled proteins were potentiated by supplementation of the cell-free system with NAD+ (1 mM). Cycloheximide (0.1 mM) caused no significant reduction in protein-bound radioactivity, whereas the addition of strong nucleophiles L-cysteine (5 mM) and reduced glutathione (5 mM) each decreased radiolabeling by 50 to 60%. Preheating of crude homogenates at 90 degrees C, prior to incubation with [14C]ethanol profoundly decreased subsequent production of acetaldehyde and formation of 14C-labeled proteins. The results indicate that the major source of protein-bound radioactivity derived from [14C]ethanol oxidation in this system is due to binding of enzymatically derived [14C]acetaldehyde to hepatic proteins.