Liver breath tests non-invasively predict higher stages of non-alcoholic steatohepatitis

Effectively assessing subtle hepatic metabolic functions by novel non-invasive tests might be of clinical utility in scoring NAFLD (non-alcoholic fatty liver disease) and in identifying altered metabolic pathways. The present study was conducted on 39 (20 lean and 19 obese) hypertransaminasemic patients with histologically proven NAFLD {ranging from simple steatosis to severe steatohepatitis [NASH (non-alcoholic steatohepatitis)] and fibrosis} and 28 (20 lean and eight overweight) healthy controls, who underwent stable isotope breath testing ([(13)C]methacetin and [(13)C]ketoisocaproate) for microsomal and mitochondrial liver function in relation to histology, serum hyaluronate, as a marker of liver fibrosis, and body size. Compared with healthy subjects and patients with simple steatosis, NASH patients had enhanced methacetin demethylation (P=0.001), but decreased (P=0.001) and delayed (P=0.006) ketoisocaproate decarboxylation, which was inversely related (P=0.001) to the degree of histological fibrosis (r=-0.701), serum hyaluronate (r=-0.644) and body size (r=-0.485). Ketoisocaproate decarboxylation was impaired further in obese patients with NASH, but not in patients with simple steatosis and in overweight controls. NASH and insulin resistance were independently associated with an abnormal ketoisocaproate breath test (P=0.001). The cut-off value of 9.6% cumulative expired (13)CO(2) for ketoisocaproate at 60 min was associated with the highest prediction (positive predictive value, 0.90; negative predictive value, 0.73) for NASH, yielding an overall sensitivity of 68% and specificity of 94%. In conclusion, both microsomal and mitochondrial functions are disturbed in NASH. Therefore stable isotope breath tests may usefully contribute to a better and non-invasive characterization of patients with NAFLD.

The role of genetic polymorphisms in alcoholic liver disease

Chronic alcohol consumption is a major cause of liver cirrhosis which, however, develops in only a minority of heavy drinkers. Evidence from twin studies indicates that genetic factors account for at least 50% of individual susceptibility. The contribution of genetic factors to the development of diseases may be investigated either by means of animal experiments, through linkage studies in families of affected patients, or population based case-control studies. With regard to the latter, single nucleotide polymorphisms of genes involved in the degradation of alcohol, antioxidant defense, necroinflammation, and formation and degradation of extracellular matrix are attractive candidates for studying genotype-phenotype associations. However, many associations in early studies were found to be spurious and could not be confirmed in stringently designed investigations. Therefore, future genotype-phenotype studies in alcoholic liver disease should meet certain requirements in order to avoid pure chance observations due to a lack of power, false functional interpretation, and insufficient statistical evaluation.

Alcoholic liver disease in the elderly

Although per capita alcohol consumption, and thus the prevalence of alcoholic liver disease, decreases generally with age in Europe and in the United States, recently an increase in alcohol consumption has been reported in individuals over 65 years. Reasons explaining this observation may include an increase in life expectancy or a loss of life partners and, thus, loneliness and depression. Although ethanol metabolism and ethanol distribution change with age, and an elderly person's liver is more susceptible to the toxic effect of ethanol, the spectrum of alcoholic liver diseases and their symptoms and signs is similar to that seen in patients of all ages. However, prognosis of alcoholic liver disease in the elderly is poor. In addition, chronic alcohol consumption may enhance drug associated liver disease and may also act as a cofactor in other liver diseases, such as viral hepatitis and nonalcoholic fatty liver disease.

Genetic polymorphisms of manganese-superoxide dismutase and glutathione-S-transferase in chronic alcoholic pancreatitis

Chronic alcohol consumption is a major risk factor for the development of chronic pancreatitis. However, chronic pancreatitis occurs only in a minority of heavy drinkers. This variability may be due to yet unidentified genetic factors. Several enzymes involved in the degradation of reactive oxidants and xenobiotics, such as glutathione-S-transferase P1 (GSTP1) and manganese-superoxide dismutase (MnSOD) reveal functional polymorphisms that affect the antioxidative capacity and may therefore modulate the development of chronic pancreatitis and long-term complications like endocrine and exocrine pancreatic insufficiency. Two functional polymorphisms of the MnSOD and the GSTP1 gene were assessed by polymerase chain reaction and restriction fragment length polymorphism in 165 patients with chronic alcoholic pancreatitis, 140 alcoholics without evidence of pancreatic disease and 160 healthy control subjects. The distribution of GSTP1 and MnSOD genotypes were in Hardy-Weinberg equilibrium in the total cohort. Genotype and allele frequencies for both genes were not statistically different between the three groups. Although genotype MnSOD Ala/Val was seemingly associated with the presence of exocrine pancreatic insufficiency, this subgroup was too small and the association statistically underpowered. None of the tested genotypes affected the development of endocrine pancreatic insufficiency. Polymorphisms of MnSOD and GSTP1 are not associated with chronic alcoholic pancreatitis. The present data emphasize the need for stringently designed candidate gene association studies with well-characterized cases and controls and sufficient statistical power to exclude chance observations.

No role of matrixmetalloproteinase-3 genetic promoter polymorphism 1171 as a risk factor for cirrhosis in alcoholic liver disease

BACKGROUND: As only a minority of alcoholics develop cirrhosis, polymorphic genes, whose products are involved in fibrosis development were suggested to confer individual susceptibility. We tested whether a functional promoter polymorphism in the gene encoding matrix metalloproteinase-3 (MMP-3; 1171 5A/6A) was associated liver cirrhosis in alcoholics. METHODS: Independent cohorts from the UK and Germany were studied. (i) UK cohort: 320 alcoholic cirrhotics and 183 heavy drinkers without liver damage and (ii) German cohort: 149 alcoholic cirrhotics, 220 alcoholic cirrhotics who underwent liver transplantation and 151 alcoholics without liver disease. Patients were genotyped for MMP-3 variants by restriction fragment length polymorphism, single strand confirmation polymorphism, and direct sequencing. In addition, MMP-3 transcript levels were correlated with MMP-3 genotype in normal liver tissues. RESULTS: Matrix metalloproteinase-3 genotype and allele distribution in all 1023 alcoholic patients were in Hardy-Weinberg equilibrium. No significant differences in MMP-3 genotype and allele frequencies were observed either between alcoholics with or without cirrhosis. There were no differences in hepatic mRNA transcription levels according to MMP-3 genotype. CONCLUSIONS: Matrix metalloproteinase-3 1171 promoter polymorphism plays no role in the genetic predisposition for liver cirrhosis in alcoholics. Stringently designed candidate gene association studies are required to exclude chance observations.

Evaluation of the transforming growth factor beta1 codon 25 (Arg-->Pro) polymorphism in alcoholic liver disease

INTRODUCTION: Liver cirrhosis develops only in a minority of heavy drinkers. Genetic factors may account for some variation in the progression of fibrosis in alcoholic liver disease (ALD). Transforming growth factor beta 1 (TGFbeta1) is a key profibrogenic cytokine in fibrosis and its gene contains several polymorphic sites. A single nucleotide polymorphism at codon 25 has been suggested to affect fibrosis progression in patients with chronic hepatitis C virus infection, fatty liver disease, and hereditary hemochromatosis. Its contribution to the progression of ALD has not been investigated sufficiently so far. PATIENTS AND METHODS: One-hundred-and-fifty-one heavy drinkers without apparent ALD, 149 individuals with alcoholic cirrhosis, and 220 alcoholic cirrhotics who underwent liver transplantation (LTX) were genotyped for TGFbeta1 codon 25 variants. RESULTS: Univariate analysis suggested that genotypes Arg/Pro or Pro/Pro are associated with decompensated liver cirrhosis requiring LTX. However, after adjusting for patients' age these genotypes did not confer a significant risk for cirrhosis requiring LTX. CONCLUSION: TGFbeta1 codon 25 genotypes Arg/Pro or Pro/Pro are not associated with alcoholic liver cirrhosis. Our study emphasizes the need for adequate statistical methods and accurate study design when evaluating the contribution of genetic variants to the course of chronic liver diseases.

Do common genetic variants in endotoxin signaling pathway contribute to predisposition to alcoholic liver cirrhosis?

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA -238A, IL1B -31T and variant IL1RN*2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-alpha and IL-1beta, respectively. Alleles CD14 -159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations. METHODS: The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease. RESULTS: Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [IL1RN 2/ 2; IL1B -31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples. CONCLUSIONS: Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations.

Ethanol-induced cytochrome P4502E1 causes carcinogenic etheno-DNA lesions in alcoholic liver disease

Oxidative stress is thought to play a major role in the pathogenesis of hepatocellular cancer (HCC), a frequent complication of alcoholic liver disease (ALD). However, the underlying mechanisms are poorly understood. In hepatocytes of ALD patients, we recently detected by immunohistochemistry significantly increased levels of carcinogenic etheno-DNA adducts that are formed by the reaction of the major lipid peroxidation product, 4-hydroxynonenal (4-HNE) with nucleobases. In the current study, we show that protein-bound 4-HNE and etheno-DNA adducts both strongly correlate with cytochrome P450 2E1 (CYP2E1) expression in patients with ALD (r = 0.9, P < 0.01). Increased levels of etheno-DNA adducts were also detected in the liver of alcohol-fed lean (Fa/?) and obese (fa/fa) Zucker rats. The number of nuclei in hepatocytes stained positively for etheno-DNA adducts correlated significantly with CYP2E1 expression (r = 0.6, P = 0.03). To further assess the role of CYP2E1 in the formation of etheno-DNA adducts, HepG2 cells stably transfected with human CYP2E1 were exposed to ethanol with or without chlormethiazole (CMZ), a specific CYP2E1 inhibitor. Ethanol increased etheno-DNA adducts in the nuclei of CYP2E1-transfected HepG2 cells in a concentration-dependent and time-dependent manner, but not in vector mock-transfected control cells. CMZ blocked the generation of etheno-DNA adducts by 70%-90% (P < 0.01). CONCLUSION: Our data support the assumption that ethanol-mediated induction of hepatic CYP2E1 leading inter alia to highly miscoding lipid peroxidation-derived DNA lesions may play a central role in hepatocarcinogenesis in patients with ALD.

Metabolomic tissue signature in human non-alcoholic fatty liver disease identifies protective candidate metabolites

Background Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries, yet its pathophysiology is incompletely understood. Small-molecule metabolite screens may offer new insights into disease mechanisms and reveal new treatment targets. Methods Discovery (N = 33) and replication (N = 66) of liver biopsies spanning the range from normal liver histology to non-alcoholic steatohepatitis (NASH) were ascertained ensuring rapid freezing under 30 s in patients. 252 metabolites were assessed using GC/MS. Replicated metabolites were evaluated in a murine high-fat diet model of NAFLD. Results In a two-stage metabolic screening, hydroquinone (HQ, pcombined = 3.0 × 10−4) and nicotinic acid (NA, pcombined = 3.9 × 10−9) were inversely correlated with histological NAFLD severity. A murine high-fat diet model of NAFLD demonstrated a protective effect of these two substances against NAFLD: Supplementation with 1% HQ reduced only liver steatosis, whereas 0.6% NA reduced both liver fat content and serum transaminase levels and induced a complex regulatory network of genes linked to NALFD pathogenesis in a global expression pathway analysis. Human nutritional intake of NA equivalent was also consistent with a protective effect of NA against NASH progression. Conclusion This first small-molecular screen of human liver tissue identified two replicated protective metabolites. Either the use of NA or targeting its regulatory pathways might be explored to treat or prevent human NAFLD.

The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al ., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of C-14-ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.

A common polymorphism in the NCAN gene is associated with hepatocellular carcinoma in alcoholic liver disease

BACKGROUND & AIMS: The genetic background of alcoholic liver diseases and their complications are increasingly recognized. A common polymorphism in the neurocan (NCAN) gene, which is known to be expressed in neuronal tissue, has been identified as a risk factor for non-alcoholic fatty liver disease (NAFLD). We investigated if this polymorphism may also be related to alcoholic liver disease (ALD) and hepatocellular carcinoma (HCC). METHODS: We analysed the distribution of the NCAN rs2228603 genotypes in 356 patients with alcoholic liver cirrhosis, 126 patients with alcoholic HCC, 382 persons with alcohol abuse without liver damage, 362 healthy controls and in 171 patients with hepatitis C virus (HCV) associated HCC. Furthermore, a validation cohort of 229 patients with alcoholic cirrhosis (83 with HCC) was analysed. The genotypes were determined by LightSNiP assays. The expression of NCAN was studied by RT-PCR and immunofluorescence microscopy. RESULTS: The frequency of the NCAN rs2228603 T allele was significantly increased in patients with HCC due to ALD (15.1%) compared to alcoholic cirrhosis without HCC (9.3%), alcoholic controls (7.2%), healthy controls (7.9%), and HCV associated HCC (9.1%). This finding was confirmed in the validation cohort (15.7% vs. 6.8%, OR=2.53; 95%CI: 1.36-4.68; p=0.0025) and by multivariate analysis (OR=1.840; 95%CI: 1.22-2.78; p=0.004 for carriage of the rs2228603 T allele). In addition, we identified and localised NCAN expression in human liver. CONCLUSIONS: NCAN is not only expressed in neuronal tissue, but also in the liver. Its rs2228603 polymorphism is a risk factor for HCC in ALD, but not in HCV infection.

Metabolic inflexibility and insulin resistance in obese adolescents with non-alcoholic fatty liver disease.

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is a comorbidity of childhood obesity. OBJECTIVE We examined whole-body substrate metabolism and metabolic characteristics in obese adolescents with vs. without NAFLD. SUBJECTS Twelve obese (BMI ≥ 95th percentile) adolescents with and without NAFLD [intrahepatic triglyceride (IHTG) ≥5.0% vs. <5.0%] were pair-matched for race, gender, age and % body fat. METHODS Insulin sensitivity (IS) was assessed by a 3-h hyperinsulinemic-euglycemic clamp and whole-body substrate oxidation by indirect calorimetry during fasting and insulin-stimulated conditions. RESULTS Adolescents with NAFLD had increased (p < 0.05) abdominal fat, lipids, and liver enzymes compared with those without NAFLD. Fasting glucose concentration was not different between groups, but fasting insulin concentration was higher (p < 0.05) in the NAFLD group compared with those without. Fasting hepatic glucose production and hepatic IS did not differ (p > 0.1) between groups. Adolescents with NAFLD had higher (p < 0.05) fasting glucose oxidation and a tendency for lower fat oxidation. Adolescents with NAFLD had lower (p < 0.05) insulin-stimulated glucose disposal and lower peripheral IS compared with those without NAFLD. Although respiratory quotient (RQ) increased significantly from fasting to insulin-stimulated conditions in both groups (main effect, p < 0.001), the increase in RQ was lower in adolescents with NAFLD vs. those without (interaction, p = 0.037). CONCLUSION NAFLD in obese adolescents is associated with adverse cardiometabolic profile, peripheral insulin resistance and metabolic inflexibility.