Development and validation of a simple NAFLD clinical scoring system for identifying patients without advanced disease.

BACKGROUND: Clinical predictors of advanced non-alcoholic liver disease (NAFLD) are needed to guide diagnostic evaluation and treatment. METHODS: To better understand the demographics of NAFLD and risk factors for advanced disease, this study analysed 827 patients with NAFLD at two geographically separate tertiary medical centres. RESULTS: The cohort was 51% female and had a median body mass index (BMI) of 33 kg/m(2); 3% had a normal BMI. Common co-morbidities included hypertension (60%) and diabetes (35%); insulin resistance was present in 91% and advanced fibrosis in 24% of patients. When comparing patients with no fibrosis or mild fibrosis to those with advanced fibrosis, BMI > or = 28 kg/m(2), age > 50 years, and aspartate transaminase/alanine aminotransferase (AST/ALT) ratio > or = 0.8, a quantitative assessment check index (QUICKI) score < 0.294 (equivalent to homeostasis model assessment (HOMA) > 6.2) and the presence of diabetes mellitus (DM) were individually associated by univariate analysis with odds ratios (ORs) of > or = 2.4 for advanced fibrosis. Based on the results of forced entry logistic regression analysis, three variables were combined in a weighted sum (BMI > or = 28 = 1 point, AAR of > or = 0.8 = 2 points, DM = 1 point) to form an easily calculated composite score for predicting advanced fibrosis called the BARD score. A score of 2-4 was associated with an OR for advanced fibrosis of 17 (confidence interval 9.2 to 31.9) and a negative predictive value of 96%. CONCLUSIONS: Insulin resistance and its co-morbidities are often present in patients with NAFLD. An easily calculated score based on readily available clinical data can reliably exclude the presence of advanced fibrosis in these patients, particularly among non-diabetics.

Glutathione monoethyl ester ameliorates caerulein-induced pancreatitis in the mouse.

Studies in animal models suggest that oxygen radicals may be important in the pathogenesis of acute pancreatitis. Because glutathione is an essential component of the defense against radical-mediated cellular injury, we investigated whether pancreatic glutathione content is influenced by inducing acute pancreatitis and whether augmenting the intracellular supply of glutathione would alter the course of pancreatitis. Caerulein, a decapeptide cholecystokinin analogue, induces acute necrotizing pancreatitis in mice when given in high doses (50 micrograms/kg per h) over a period of 6 h. The pancreatic glutathione content (total, GSH + GSSG) in mice treated with high-dose caerulein fell to 17% of normal within 4 h of beginning caerulein and recovered toward normal after discontinuing caerulein treatment. Mice treated with glutathione monoethyl ester (20 mmol/kg 1 h before caerulein, 10 mmol/kg 3 and 7 h after starting caerulein) were found to have blunted depletion of pancreatic glutathione, diminished histologic evidence of pancreatitis (necrosis, inflammation, and vacuolization), and lower serum amylase values compared with mice treated with caerulein alone. These findings suggest that the profound depletion of pancreatic glutathione caused by hyperstimulation of the pancreas with caerulein is critically important in the pathogenesis of acute caerulein-induced pancreatitis.

Randomised clinical study: GR-MD-02, a galectin-3 inhibitor, vs. placebo in patients having non-alcoholic steatohepatitis with advanced fibrosis.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) and resultant liver fibrosis is a major health problem without approved pharmacotherapy. Pre-clinical results of GR-MD-02, a galectin-3 inhibitor, suggested potential efficacy in NASH with advanced fibrosis/cirrhosis and prompted initiation of a clinical development programme in NASH with advanced fibrosis. AIM: To evaluate the safety, pharmacokinetics and exploratory pharmacodynamic markers of GR-MD-02 in subjects having NASH with bridging fibrosis. METHODS: The GT-020 study was a first-in-human, sequential dose-ranging, placebo controlled, double-blinded study with the primary objective to assess the safety, tolerability and dose limiting toxicity of GR-MD-02, in subjects with biopsy-proven NASH with advanced fibrosis (Brunt stage 3). The secondary objectives were to characterise first-dose and multiple-dose pharmacokinetic profiles and to evaluate changes in potential serum biomarkers and liver stiffness as assessed by FibroScan. RESULTS: GR-MD-02 single and three weekly repeated of 2, 4 and 8 mg/kg revealed no meaningful clinical differences in treatment emergent adverse events, vital signs, electrocardiographic findings or laboratory tests. Pharmokinetic parameters showed a dose-dependent relationship with evidence of drug accumulation following 8 mg/kg (~twofold). CONCLUSIONS: GR-MD-02 doses were in the upper range of the targeted therapeutic dose determined from pre-clinical data and were safe and well tolerated with evidence of a pharmacodynamic effect. These results provide support for a Phase 2 development programme in advanced fibrosis due to NASH.

Nonalcoholic steatohepatitis.

NASH is an important form of chronic liver disease that is increasingly recognized. The diagnosis is secured by biopsy findings with similarities to alcoholic hepatitis in a patient with a confirmed history of abstinence. Obesity is a major risk factor, but the disease also occurs in the nonobese. In 20% to 40% of patients the disease can progress to various stages of fibrosis and ultimately cause cirrhosis and death from end-stage liver disease. For this reason, recognition of NASH is important, and establishing the diagnosis provides a further impetus for performing a liver biopsy as part of the evaluation of unexplained liver abnormalities. The mainstay of treatment is weight reduction in the obese. For those individuals who are not obese, continued observation is the only option currently available. Patients who develop decompensated cirrhosis should be considered for liver transplantation unless advanced age or other underlying medical illnesses are a problem. With the increasing knowledge about the pathophysiology of hepatic steatosis, it is hoped that better diagnostic tests for specific causes of NASH will be available and lead to efficacious therapy.

Glutathione regulation in rat hepatic stellate cells. Comparative studies in primary culture and in liver injury in vivo.

Lipid peroxidation accompanies many types of liver injury and is believed to promote liver fibrosis. Cellular antioxidants are likely to play an important role in modulating this process; however, little is known about antioxidants in hepatic stellate cells, the major collagen-producing cells of liver. In this study, we measured glutathione homeostasis in stellate cells isolated from rat liver. Glutathione, measured by HPLC in stellate cell homogenates, increased significantly when the cells were plated in primary culture. The rise in glutathione coincided with pretranslational up-regulation of the synthetic enzyme gamma-glutamylcysteine synthetase (GCS). Additional experiments were performed to determine whether stellate cell glutathione and GCS are similarly altered during liver injury in vivo. Two types of hepatic insults, namely, bile duct ligation (8 days) and carbon tetrachloride treatment (4 weeks), failed to provoke an increase in either stellate cell glutathione or GCS. This disparate behavior of stellate cells in culture and in vivo is unusual; the data suggest that stellate cells might respond variably to oxidants depending on their glutathione status.

Glutathione synthesis in the exocrine pancreas.

Glutathione is essential for cellular cytoprotection, and in the exocrine pancreas, it is required for digestive enzyme synthesis. The purpose of these studies was to measure the capacity of the exocrine pancreas to synthesize glutathione, determine whether the pancreatic transsulfuration pathway has a role in providing cysteine needed for glutathione synthesis, and determine whether the glutathione synthetic capacity of the pancreas responds to pathologically relevant stresses. The activity of gamma-glutamylcysteine synthetase, the key regulatory enzyme for glutathione synthesis, was 3.56 +/- 0.29 mU/mg protein in the pancreas of fed rats, compared to 31 +/- 4 in the liver and 116 +/- 5 in the kidney. Studies using dispersed rat pancreatic acinar cells showed that the exocrine pancreas synthesizes glutathione from precursor amino acids and that the transsulfuration pathway is functionally intact in the pancreas and may serve as an important source of pancreatic cysteine. In mice, pancreatic gamma-glutamylcysteine synthetase activity was induced 37% by corn oil, 77% by ethanol, and 88% by both treatments. Thus, the glutathione synthetic capacity of the pancreas is quantitatively less than that of the kidney or liver, but its key regulatory enzyme responds dynamically to pathologically relevant metabolic stresses, suggesting that glutathione is a key pancreatic cytoprotectant.

Nonalcoholic steatohepatitis.

NASH is a form of chronic liver disease that is defined by biopsy findings and has the appearance of alcoholic hepatitis. Although this disease was once thought to be a problem of women, diabetics, and the obese, more recent studies have identified a significant proportion of patients who do not fit these risk factors. In a fraction of patients, the disease can progress to various stages of fibrosis leading ultimately to cirrhosis and death from end-stage liver disease. For this reason, recognition of NASH is important and provides a further impetus for performing a liver biopsy as part of the evaluation of unexplained liver biochemical abnormalities. The mainstay of treatment is weight reduction in the obese. For those individuals who are not obese, continued observation is the only available option at this point. With increasing knowledge about the pathophysiology of hepatic steatosis, perhaps more specific diagnostic tests for the cause of the disease in specific patients will be available and will guide appropriate therapy.

Nonalcoholic steatohepatitis: an evolving diagnosis.

Nonalcoholic steatohepatitis (NASH) is a histological diagnosis applied to a constellation of liver biopsy findings that develop in the absence of alcohol abuse. Steatosis, a mixed cellular inflammatory infiltrate across the lobule, evidence of hepatocyte injury and fibrosis are the findings that can be seen. This entity is often identified during evaluation of elevated aminotransferases after exclusion of viral, metabolic and other causes of liver disease. Obesity is a major risk factor for NASH. The role of diabetes is less certain, although evidence is accumulating that hyperinsulinism may play an important pathophysiological role. Patients sometimes suffer from right upper quadrant abdominal pain and fatigue; examination may reveal centripetal obesity and hepatomegaly. Although patients are often discovered because of persistent aminotransferase elevations, these enzymes can be normal in NASH. When they are elevated, the alanine aminotransferase level is typically significantly greater than the aspartate aminotransferase level. This can be particularly helpful for excluding occult alcohol abuse. Imaging studies identify hepatic steatosis when the amount of fat in the liver is significant; however, imaging does not distinguish benign steatosis from NASH. Ultimately a liver biopsy is needed to diagnose NASH. The biopsy may be useful for establishing prognosis based on the presence or absence of fibrosis and for excluding other unexpected causes of liver enzyme elevations. Weight loss is the mainstay of treatment for obese patients. About 15% to 40% of NASH patients develop fibrosis; how many of these cases progress to cirrhosis is unknown, but about 1% of liver transplants are performed with a pretransplant diagnosis of NASH.

Diurnal variability of cysteine and glutathione content in the pancreas and liver of the mouse.

The sensitivity of organs such as the liver to injury by certain drugs is modulated by the endogenous capacity to synthesize glutathione during periods of increased demand. Recent experimental evidence suggests that glutathione availability could also play an important role in preventing pancreatic injury as well. To better understand the role of cysteine availability in regulating glutathione homeostasis in the pancreas and liver under normal conditions, the diurnal variation in cysteine in mouse pancreas and liver was measured and compared with corresponding measurements of organ glutathione content. Pancreatic cysteine varied significantly over a 24-hr period, dropping to 21 nmol/g at 2 P.M. and rising to 68 nmol/g at 10 P.M. Fasting prevented this diurnal variation in pancreatic cysteine. Pancreatic glutathione was at its lowest at 10 P.M. and rose sharply to a peak at 2 A.M. Fasting had no effect on this diurnal pattern. In contrast to the pancreas, fasting did not prevent the diurnal change in liver cysteine, whereas it caused substantial depletion of liver glutathione. Together, these findings suggest that under normal conditions, pancreatic and liver glutathione content are not determined solely by tissue cysteine availability. Moreover, basal glutathione content is under differing homeostatic mechanisms in the pancreas and the liver.

Fatty liver and nonalcoholic steatohepatitis.

Fatty liver is a relatively common incidental finding on imaging studies. Although generally a benign condition, fat in the liver can be troubling for clinicians because it can cause persistently elevated liver enzyme levels. The finding of fatty liver may also indicate the presence of nonalcoholic steatohepatitis (NASH). NASH is a histologic diagnosis applied to a constellation of liver biopsy findings that appear similar to alcoholic liver disease but are found in the absence of alcohol abuse. NASH is typically identified during the evaluation of elevated aminotransferase levels after exclusion of viral, metabolic, and other causes of liver disease. Obesity is a major risk factor; the role of diabetes is less certain, although evidence is accumulating that hyperinsulinism may play an important pathophysiologic role. About 15% to 40% of NASH patients develop hepatic fibrosis, a precursor to cirrhosis. Exactly how many patients with NASH progress to cirrhosis is unknown, but 1% to 2% of liver transplants are now performed because of a pretransplant diagnosis of NASH. Specific and effective treatments are needed but until the pathogenesis of this common liver disease is better understood, weight loss will remain the mainstay of treatment for obese patients.

Common blood tests for liver disease. Which ones are most useful?

Prompt diagnosis is important in patients who have elevated serum liver enzyme levels or biochemical markers. With understanding of the advantages and pitfalls of serologic tests commonly used for evaluation of liver disease, diagnosis can be approached in a rational and cost-effective manner. This article focuses on interpretation of results of the most useful tests. Discussion includes the nuances of laboratory evaluation of jaundice and current refinements in the use of liver enzyme measurements. Diagnostic tests for viral hepatitis also are discussed, with emphasis on recent developments in the evaluation of hepatitis B and C.

Vitamin E and changes in serum alanine aminotransferase levels in patients with non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a common cause of serum alanine aminotransferase (ALT) elevations and chronic liver disease, but it is unclear how well ALT elevations reflect the liver injury. AIM: To assess how well changes in ALT elevations reflect improvements in liver histology in response to vitamin E therapy. METHODS: The vitamin E and placebo arms of the Pioglitazone vs. Vitamin E vs. Placebo in Non-alcoholic Steatohepatitis (PIVENS) trial were reassessed for associations among changes in ALT levels, body weight and liver histology. An ALT response was defined as a decrease to ≤40 U/L and by ≥30% of baseline. Liver biopsies taken before and after treatment were scored for non-alcoholic fatty liver disease activity (NAS) and fibrosis. RESULTS: ALT responses were more frequent among vitamin E (48%) than placebo (16%) recipients (P < 0.001). Among vitamin E recipients, ALT responses were associated with decreases in NAS (P < 0.001), but not fibrosis scores (P = 0.34), whereas among placebo recipients, ALT responses were associated with significant decreases in both (P < 0.05). Weight loss (≥2 kg) was also associated with ALT response (P < 0.001), improvements in NAS (P < 0.001) and fibrosis (P < 0.02), but vitamin E had an added effect both with and without weight loss. Weight gain (≥2 kg) was associated with lack of ALT response and worsening NAS and fibrosis scores in patients not on vitamin E. CONCLUSIONS: Decrease of ALT levels to normal in patients with NASH is usually associated with improved histological activity. Management should stress the value of weight loss and strongly discourage weight gain. Vitamin E can improve both ALT levels and histology with and without weight loss. CLINICAL TRIAL NUMBER: NCT00063622.

Organ interactions in the hepatorenal syndrome.

An increasing number of patients with severe liver dysfunction are admitted to the ICU for stabilization and organ-specific support, including liver transplantation. Global impairment of hepatic performance frequently results in pathologic organ interactions that limit the potential for recovery. One of the most notable of these interactions is the hepatorenal syndrome, an otherwise uniformly fatal complication of end-stage liver disease characterized by the progressive development of oliguria and low urine sodium excretion. The syndrome can occur in the setting of either acute or chronic liver disease, and portal hypertension may be important in the pathogenesis. The patient with the hepatorenal syndrome also has a number of systemic circulatory abnormalities induced by liver disease and/or portal hypertension, but the exact pathologic role of these abnormalities in the development of oliguria is uncertain. It is reasonably well established that diminished systemic BP characteristic of liver failure is not the primary cause of renal insufficiency. Rather, intrarenal preglomerular vasoconstriction mediated by unknown stimuli is the major defect in the hepatorenal syndrome, manifested by relative ischemia. Current data point to abnormal renal sympathetic innervation as one of the more likely major causes of this vasoconstriction. After exclusion of systemic intravascular volume depletion and other causes of oliguria, dialytic therapy is indicated when liver transplantation or recovery of liver function is anticipated; terminal supportive care is appropriate when these outcomes are not options.

Cerulein-induced pancreatic cysteine depletion: prevention does not diminish acute pancreatitis in the mouse.

BACKGROUND/AIMS: Induction of acute necrotizing pancreatitis with cerulein causes profound pancreatic glutathione depletion in the mouse. Because cysteine availability can be rate-limiting for glutathione synthesis, the pancreatic content of cysteine during cerulein treatment was measured and the potential benefit of augmenting pancreatic cysteine was determined. METHODS: Female Swiss-Webster mice were treated with cerulein with and without simultaneous administration of the cysteine prodrug, L-2-oxothiazolidine 4-carboxylic acid. Pancreatic cysteine and glutathione content were measured, and serum amylase levels and pancreatic histology were assessed. RESULTS: Pancreatic cysteine content decreased to 42% of normal after 4 hours of cerulein treatment. L-2-oxothiazolidine 4-carboxylic acid more than doubled pancreatic cysteine content at 4 hours and prevented pancreatic cysteine depletion when administered with cerulein. Cerulein caused pancreatic glutathione depletion despite this normalization of cysteine. Moreover, the biochemical and histological evidence of cerulein-induced pancreatitis was unaltered by preventing cysteine depletion. CONCLUSIONS: Pancreatic cysteine is depleted during induction of acute pancreatitis with cerulein in parallel with depletion of pancreatic glutathione. Because preventing pancreatic cysteine depletion with L-2-oxothiazolidine 4-carboxylic acid did not prevent glutathione loss, causes of pancreatic glutathione depletion other than insufficiency of this critical precursor must also play an important role in cerulein-induced glutathione depletion.

Manipulation of glutathione stores in rat hepatic stellate cells does not alter collagen synthesis.

Hepatic stellate cells are resistant to the fibrogenic effects of lipid hydroperoxides in primary culture. Recent studies from our laboratory suggest that antioxidants, particularly glutathione, play a role in this resistance. We have observed that glutathione accumulates rapidly in stellate cells during primary culture; in the current study, we investigated whether glutathione modulates stellate cell collagen synthesis. Stellate cells from normal rat liver were plated in primary culture and maintained for 7 days. From day 4 through day 7, the cells were treated with L-buthionine sulfoximine (BSO) to deplete glutathione stores. BSO profoundly diminished stellate cell glutathione but had no effect on morphology, viability, or basal levels of collagen synthesis and gene expression. When cultured stellate cells were incubated with the putative fibrogenic mediator 4-hydroxynonenal or iron/ascorbate, little or no increase in collagen synthesis occurred regardless of glutathione content. In contrast, iron/ascorbate induced collagen synthesis by cultured fibroblasts. The data indicate that stellate cells strongly resist oxidant- and lipid peroxide-induced collagen synthesis in primary culture. They demonstrate that the mechanism of this resistance does not involve glutathione.

Chemotactic activity for human PMN generated during ethanol metabolism by rat hepatocytes: role of glutathione and glutathione peroxidase.

Infiltration of the liver by polymorphonuclear leukocytes is a characteristic feature of alcoholic hepatitis. We have previously shown that liver cytosol metabolizing ethanol generates chemoattractant activity for polymorphonuclear leukocytes and that hydroxyl radical scavengers inhibit this process. To investigate the possibility that endogenous glutathione and glutathione peroxidase also inhibit this process, we evaluated chemoattractant activity production by glutathione and glutathione peroxidase deficient rat liver cytosol during ethanol metabolism. Incubation of cytosol deficient in both glutathione and glutathione peroxidase with 10 mM ethanol for 1 hour resulted in a 500-fold increase in chemoattractant activity when compared to cytosol with normal glutathione and glutathione peroxidase content. These findings provide further evidence for a role of oxygen free radicals in the generation of chemotactic activity and they also suggest that the ethanol-induced decrease in hepatic glutathione and glutathione peroxidase reported by others may have a significant potentiating effect on the recruitment of pro-inflammatory cells into the liver.

Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione-orthophthalaldehyde adduct.

Glutathione reacts with orthophthalaldehyde to form a stable, highly fluorescent tricyclic derivative which is easily separated and quantitated by high-performance liquid chromatography. Separation of the glutathione adduct is achieved by isocratic elution over a reverse-phase column with 7.5% methanol/92.5% 0.15 M sodium acetate, pH 7.00. The adduct is detected fluorometrically and quantitated by integration of peak area. Detection of 0.1 to 200 pmol glutathione produces a linear response and the recovery of reduced and oxidized glutathione from rat liver homogenate, bile, and plasma is quantitative. The chemical identity of the adduct was confirmed by mass spectrometry.

Nafcillin-associated hepatotoxicity. Report of a case and review of the literature.

Nafcillin is a semisynthetic penicillin that is generally well tolerated with few side effects. Hepatic complications are rare but have a potential for serious liver dysfunction. This unusual complication causes a predominantly cholestatic injury, which can persist for prolonged periods even after discontinuing the medication. The pathophysiology may include direct cytotoxicity or an immune-mediate hypersensitivity. Treatment is generally supportive, except for severely symptomatic patients who may require steroids. We report a case of nafcillin-associated hepatotoxicity and review the literature of this disorder.

Nonalcoholic steatohepatitis: an expanded clinical entity.

BACKGROUND/AIMS: In the past, nonalcoholic steatohepatitis has been described mostly in obese women with diabetes. The aim of this study was to describe a series of patients with nonalcoholic steatohepatitis with a different clinical profile. METHODS: The clinical, biochemical, and histological features of 33 patients with nonalcoholic steatohepatitis seen from July 1990 to June 1993 were analyzed. RESULTS: The mean age was 47 years. All patients were antibody to hepatitis C virus-negative. Nineteen of 33 (58%) were men, 20 of 33 (61%) were nonobese, 26 of 33 (79%) had normal glucose levels, and 26 of 33 (79%) had normal lipid levels. Fourteen of 33 (42%) had normal glucose and lipid levels and were not obese. Thirteen of 33 (39%) had pathological increases in fibrosis, 5 of whom had micronodular cirrhosis. Of these 13 with severe, progressive disease, 8 (62%) were women, 8 (62%) were obese, 4 (31%) were diabetic or had an elevated glucose level, and 3 (23%) had hyperlipidemia. Although serum iron studies (transferrin saturation and ferritin) were abnormal in 18 of 31 (58%), no patient had hemochromatosis. CONCLUSIONS: Nonalcoholic steatohepatitis can be a severe, progressive liver disease leading to the development of cirrhosis. It should no longer be considered a disease predominantly seen in obese women with diabetes.

Cholestatic liver injury down-regulates hepatic glutathione synthesis.

Hepatocellular injury caused by cholestasis may be caused in part by oxidant stress. The purpose of this study was to establish how acute cholestasis might alter hepatic glutathione homeostasis and to determine whether injured hepatocytes are capable of reverting to normal glutathione homeostatic mechanisms. Acute cholestasis was achieved by surgical ligation of the common bile duct in rats. Bile duct ligation induced a 3.7-fold increase in hepatic glutathione content over 4 days. This increase was not due to increased hepatic activity of gamma-glutamylcysteine synthetase (GCS); on the contrary, whole-liver GCS activity was substantially diminished in the bile duct-ligated liver to 34 and 11% of normal after 4 and 7 days, respectively. To determine if hepatocytes removed from the cholestatic environment maintained these changes in glutathione homeostasis, hepatocytes were isolated from bile ductligated livers and established in primary culture. In cells isolated after 4 days of bile duct ligation, the elevated hepatocyte glutathione content decreased and the low GCS activity increased over 2 days in culture. More importantly, the ability of postcholestatic hepatocytes to substantially increase their glutathione synthetic capacity by increasing GCS activity in response to stress was preserved. This compensatory increase was due primarily to new protein synthesis. Together, these observations suggest that acute cholestasis impairs the ability of the liver to synthesize glutathione by down-regulating the key regulatory enzyme for its synthesis in response to acutely elevated glutathione levels and that the impaired glutathione synthetic capacity is corrected after cells are removed from the cholestatic environment.