Per Stål

Decreased survival of subjects with elevated liver function tests during a 28‐year follow‐up

The long‐term survival of subjects with nonalcoholic fatty liver disease (NAFLD) in comparison with both individuals with elevated transaminases attributable to other causes and the general poulation is poorly characterized. This study was undertaken to determine the frequency of NAFLD in a cohort of subjects who underwent liver biopsy from 1980 to 1984 because of elevated liver enzymes, and to assess mortality among subjects with NAFLD in comparison with the general Swedish population. The 256 subjects (61% men) had a mean age of 45 ± 12 years at the inclusion. Liver biopsies were blindly scored for NAFLD and nonalcoholic steatohepatitis (NASH). Causes of death were ascertained from the national Swedish Cause of Death Registry. Fatty liver was detected in 143 of the 256 subjects, including 25 (10%) with alcoholic fatty liver disease and 118 (46%) exhibiting NAFLD. Of those, 51 (20%) were classified as NASH and 67 (26%) as nonalcoholic bland steatosis. Cirrhosis was present in 9% at inclusion. During the follow‐up period, 113 (44%) of the total population and 47 (40%) of the 118 subjects diagnosed with NAFLD died. Of the 113 deaths, 37 were of cardiovascular disease and 16 of liver diseases. Compared with the total Swedish population, adjusted for sex, age, and calendar period, subjects with NAFLD exhibited a 69% increased mortality (standardized mortality ratio [SMR] = 1.69; 95% confidence interval [CI], 1.24–2.25); subjects with bland steatosis, a 55% increase (SMR, 1.55; 95% CI, 0.98–2.32; P = 0.062); and subjects with NASH, 86% (SMR, 1.86; 95% CI, 1.19–2.76; P = 0.007). Conclusion: Patients with NASH are at increased risk of death compared with the general population. Liver disease is the third most common cause of death among patients with NAFLD. (HEPATOLOGY 2009.)

Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the Western world, strongly associated with insulin resistance and the metabolic syndrome. Nonalcoholic steatohepatitis, i.e., fatty liver accompanied by necroinflammatory changes, is mostly defined by the NAFLD activity score (NAS). The aim of the current study was to determine disease‐specific mortality in NAFLD, and evaluate the NAS and fibrosis stage as prognostic markers for overall and disease‐specific mortality. In a cohort study, data from 229 well‐characterized patients with biopsy‐proven NAFLD were collected. Mean follow‐up was 26.4 (±5.6, range 6‐33) years. A reference population was obtained from the National Registry of Population, and information on time and cause of death were obtained from the Registry of Causes of Death. NAFLD patients had an increased mortality compared with the reference population (hazard ratio [HR] 1.29, confidence interval [CI] 1.04‐1.59, P = 0.020), with increased risk of cardiovascular disease (HR 1.55, CI 1.11‐2.15, P = 0.01), hepatocellular carcinoma (HR 6.55, CI 2.14‐20.03, P = 0.001), infectious disease (HR 2.71, CI 1.02‐7.26, P = 0.046), and cirrhosis (HR 3.2, CI 1.05‐9.81, P = 0.041). Overall mortality was not increased in patients with NAS 5‐8 and fibrosis stage 0‐2 (HR 1.41, CI 0.97‐2.06, P = 0.07), whereas patients with fibrosis stage 3‐4, irrespective of NAS, had increased mortality (HR 3.3, CI 2.27‐4.76, P < 0.001). Conclusion: NAFLD patients have increased risk of death, with a high risk of death from cardiovascular disease and liver‐related disease. The NAS was not able to predict overall mortality, whereas fibrosis stage predicted both overall and disease‐specific mortality. (Hepatology 2015;61:1547–1554)

A Risk for Hepatocellular Carcinoma Persists Long-term After Sustained Virologic Response in Patients With Hepatitis C-Associated Liver Cirrhosis

BACKGROUND The long-term effect of sustained virologic response (SVR) to antiviral therapy on the risk of developing hepatocellular carcinoma (HCC), liver complications, liver-related death, and overall death in hepatitis C virus (HCV)-infected patients with liver cirrhosis is not fully known. METHODS These risks were evaluated during long-term follow-up in 351 patients with HCV-related cirrhosis. One hundred ten patients with SVR, 193 with non-SVR, and 48 who were untreated were included in a multicenter cohort that was initiated in 2001 and prospectively followed up for a mean of 5.3 (SD, 2.8) years. Complementary follow-up data from national registries were used to minimize the loss of patients during follow-up. RESULTS Six patients with SVR developed HCC at 0.04, 0.64, 2.4, 7.4, 7.4, and 7.6 years, respectively, after achieving SVR. The incidences of HCC, any liver complication, liver-related death, and overall death per 100 person-years were significantly lower in SVR time with 1.0, 0.9, 0.7, and 1.9, compared to 2.3, 3.2, 3.0, and 4.1 in non-SVR and 4.0, 4.9, 4.5, and 5.1 in untreated time. The long-term consequences did not decline significantly after >3 years versus during the first 3 years of follow-up. CONCLUSIONS The risk for HCC, liver decompensation, and death in patients with liver cirrhosis related to HCV was markedly reduced after SVR, but a long-term risk of developing HCC remains for up to 8 years. Cirrhotic patients with HCV who achieve SVR should therefore maintain long-term surveillance for HCC. Future studies aimed to better identify those with remaining long-term risk for HCC are needed.

Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta‐analysis

Liver fibrosis is the most important predictor of mortality in nonalcoholic fatty liver disease (NAFLD). Quantitative risk of mortality by fibrosis stage has not been systematically evaluated. We aimed to quantify the fibrosis stage–specific risk of all‐cause and liver‐related mortality in NAFLD. Through a systematic review and meta‐analysis, we identified five adult NAFLD cohort studies reporting fibrosis stage–specific mortality (0‐4). Using fibrosis stage 0 as a reference population, fibrosis stage–specific mortality rate ratios (MRRs) with 95% confidence intervals (CIs) for all‐cause and liver‐related mortality were estimated. The study is reported according to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses statement. Included were 1,495 NAFLD patients with 17,452 patient years of follow‐up. Compared to NAFLD patients with no fibrosis (stage 0), NAFLD patients with fibrosis were at an increased risk for all‐cause mortality, and this risk increased with increases in the stage of fibrosis: stage 1, MRR = 1.58 (95% CI 1.19‐2.11); stage 2, MRR = 2.52 (95% CI 1.85‐3.42); stage 3, MRR = 3.48 (95% CI 2.51‐4.83); and stage 4, MRR = 6.40 (95% CI 4.11‐9.95). The results were more pronounced as the risk of liver‐related mortality increased exponentially with each increase in the stage of fibrosis: stage 1, MRR = 1.41 (95% CI 0.17‐11.95); stage 2, MRR = 9.57 (95% CI 1.67‐54.93); stage 3, MRR = 16.69 (95% CI 2.92‐95.36); and stage 4, MRR = 42.30 (95% CI 3.51‐510.34). Limitations of the study include an inability to adjust for comorbid conditions or demographics known to impact fibrosis progression in NAFLD and the inclusion of patients with simple steatosis and nonalcoholic steatohepatitis without fibrosis in the reference comparison group. Conclusion: The risk of liver‐related mortality increases exponentially with increase in fibrosis stage; these data have important implications in assessing the utility of each stage and benefits of regression of fibrosis from one stage to another. (Hepatology 2017;65:1557‐1565).

Iron increases ethanol toxicity in rat liver

Clinical evidence indicates that patients with iron overload are more susceptible to liver cell damage from alcohol than persons with normal iron stores. Iron may act as a co-factor to catalyze the lipid peroxidation induced by hepatotoxic compounds such as alcohol. To elucidate the role of iron in ethanol-induced hepatocellular damage, we developed a new experimental model in the rat. Following dietary carbonyl iron feeding for 8 weeks, animals were pair-fed a liquid ethanol diet for 4 weeks. In iron-fed animals the liver iron content was 6.4 vs. 0.5 micrograms Fe/mg protein in the controls. Blood alcohol concentrations were similar in all ethanol-fed animals. Serum alanine aminotransferase (ALT) levels were elevated to 269 +/- 49 U/l in the iron+alcohol group compared to 52 +/- 6 U/l in the other groups. There was a strong correlation between ALT levels and hepatic iron content in the ethanol-fed animals. Morphologically, the alcohol-fed rats displayed hepatic steatosis, whereas occasional inflammation and iron in Kupffer cells was seen in the iron+alcohol animals. Ultrastructurally, necrotic hepatocytes and cells phagocytosed by Kupffer cells were only encountered in the iron+alcohol group. Compared to controls, the liver content of hydroxyproline was significantly increased in the iron+alcohol group. No morphological evidence of fibrosis was noted. The present study demonstrates biochemical and morphological evidence of increased hepatocellular damage following the combination of iron and ethanol.

Mild iron overload in patients carrying the HFE S65C gene mutation: a retrospective study in patients with suspected iron overload and healthy controls

Background and aims: The role of the HFE S65C mutation in the development of hepatic iron overload is unknown. The aim of the present study was: (A) to determine the HFE S65C frequency in a Northern European population; and (B) to evaluate whether the presence of the HFE S65C mutation would result in a significant hepatic iron overload. Patients and methods: Biochemical iron parameters and HFE mutation analysis (for the C282Y, H63D, and S65C mutations) were analysed in 250 healthy control subjects and collected retrospectively in 296 patients with suspected iron overload (elevated serum ferritin and/or transferrin saturation). The frequency of patients having at least mild iron overload, and mean serum ferritin and transferrin saturation values were calculated for each HFE genotype. For patients carrying the S65C mutation, clinical data, liver biopsy results, and amount of blood removed at phlebotomy were determined. Results: The HFE S65C mutation was found in 14 patients and eight controls. In controls, the S65C allele frequency was 1.6%. The S65C allele frequency was enriched in non-C282Y non-H63D chromosomes from patients (4.9%) compared with controls (1.9%) (p<0.05). Serum ferritin was significantly increased in controls carrying the S65C mutation compared with those without HFE mutations. Fifty per cent of controls and relatives having the S65C mutation had elevated serum ferritin levels or transferrin saturation. The number of iron overloaded patients was significantly higher among those having HFE S65C compared with those without any HFE mutation. Half of patients carrying the S65C mutation (7/14) had evidence of mild or moderate hepatic iron overload but no signs of extensive fibrosis in liver biopsies. Screening of relatives revealed one S65C homozygote who had no signs of iron overload. Compound heterozygosity with S65C and C282Y or H63D did not significantly increase the risk of iron overload compared with S65C heterozygosity alone. Conclusions: The HFE S65C mutation may lead to mild to moderate hepatic iron overload but neither clinically manifest haemochromatosis nor iron associated extensive liver fibrosis was encountered in any of the patients carrying this mutation.

Biliary dysplasia, cell proliferation and nuclear DNA‐fragmentation in primary sclerosing cholangitis with and without cholangiocarcinoma

Abstract. Bergquist A, Glaumann H, Stål P, Wang G‐S, Broomé U (Huddinge University Hospital, Huddinge; and Karolinska Institute, Stockholm; Sweden). Biliary dysplasia, cell proliferation and nuclear DNA‐fragmentation in primary sclerosing cholangitis with and without cholangiocarcinoma. J Intern Med 2001; 249: 69–75.

Hepatotoxicity induced by iron overload and alcohol: Studies on the role of chelatable iron, cytochrome P450 2E1 and lipid peroxidation

BACKGROUND/AIMS Clinical experience and studies with experimental animal models indicate a synergistic hepatotoxic effect of dietary iron overload and chronic alcohol ingestion. In order to elucidate the mechanism underlying this synergism, we examined the hepatic levels of ethanol-inducible cytochrome P450 2E1, glutathione and malondialdehyde, and the effect of iron chelation with desferrioxamine, in livers from rats treated with iron and/or ethanol. METHODS Animals received diets with or without 2.5-3% carbonyl iron for 6-9 weeks, followed by an ethanol-containing diet or a liquid control diet for 5-9 weeks. Desferrioxamine was administered subcutaneously with mini-osmotic pumps. Alanine aminotransferase activity in serum and hepatic contents of glutathione and malondialdehyde were determined. The hepatic level of cytochrome P450 2E1 was determined with Western Blotting using a specific polyclonal antibody. RESULTS The combination of iron and alcohol led to a marked increase in serum alanine aminotransferase activity as compared with all other treatment groups, and iron chelation with desferrioxamine reversed these increases. Treatment with alcohol alone led to slightly increased aminotransferases compared with controls. The level of cytochrome P450 2E1 was significantly elevated in microsomes isolated from ethanol-treated rats, but neither additional iron supplementation nor desferrioxamine influenced this level significantly. Glutathione contents were increased in the livers of animals treated with iron and/or ethanol. Malondialdehyde values were increased in iron-treated animals, whereas neither ethanol nor desferrioxamine altered malondialdehyde levels significantly. CONCLUSIONS The toxic effects exerted by the combination of iron overload and chronic ethanol feeding on rat liver are dependent on a pool of chelatable iron. The hepatic level of cytochrome P450 2E1 is markedly induced by ethanol but not further altered by iron overload. Neither increased lipid peroxidation nor depletion of hepatic glutathione levels can explain the synergistic hepatotoxic effects of iron and ethanol in this model.

Liver cell damage and lysosomal iron storage in patients with idiopathic hemochromatosis: A light and electron microscopic study

Eleven patients with idiopathic hemochromatosis were subjected to percutaneous liver biopsy, and seven were rebiopsied after repeated phlebotomies. The liver tissue was examined by light and electron microscopy. Ultrastructural morphometry was also performed and iron content was determined. Initially, all biopsies displayed large iron-laden lysosomes in the hepatocytes. Lysosomal volume density was increased and correlated well with liver iron content (r = 0.84, p less than 0.005). Neither mitochondria nor the endoplasmic reticulum showed any ultrastructural changes, except in the necrotic cells of biopsies with the highest liver iron content. In these cases, iron-laden lysosomes were also encountered in the Kupffer cells. Following treatment, liver iron content and lysosomal volume density were normalized. More specifically, iron content was 14.1 +/- 2.1 micrograms Fe/mg protein before and 1.3 +/- 0.3 micrograms Fe/mg protein after treatment (p less than 0.001). Lysosomal volume density was 6.1 +/- 0.8% before and 1.8 +/- 0.2% after treatment (p less than 0.001). Hence, in the precirrhotic stage of idiopathic hemochromatosis, the first evident ultrastructural changes are in the lysosomal compartment. These changes correlate well with the iron overload, also in advanced stages of the disease, and are reversed after iron removal.

Fibrosis stage but not NASH predicts mortality and time to development of severe liver disease in biopsy-proven NAFLD

BACKGROUND & AIMS Non-alcoholic fatty liver disease (NAFLD) is very common in the general population, but identifying patients with increased risk of mortality and liver-specific morbidity remains a challenge. Non-alcoholic steatohepatitis (NASH) is thought to enhance this risk; therefore, resolution of NASH is a major endpoint in current pharmacologic studies. Herein, we aim to investigate the long-term prognosis of a large cohort of NAFLD patients, and to study the specific effect of NASH and fibrosis stage on prognosis. METHODS We conducted a retrospective cohort study of 646 biopsy-proven NAFLD patients. Each case was matched for age, sex and municipality to ten controls. Outcomes on mortality and severe liver disease, defined as cirrhosis, liver decompensation/failure or hepatocellular carcinoma, were evaluated using population-based registers. Cox regression models adjusted for age, sex and type 2 diabetes were used to examine the long-term risk according to fibrosis stage. Likelihood ratio tests were used to assess whether adding NASH to these models increased the predictive capacity. Laplace regression was used to estimate the time to severe liver disease according to stage of fibrosis. RESULTS During a follow-up of mean 20years (range 0-40) equivalent to 139,163 person-years, 12% of NAFLD patients and 2.2% of controls developed severe liver disease (p<0.001). Compared to controls, the risk of severe liver disease increased per stage of fibrosis (hazard ratio ranging from 1.9 in F0 to 104.9 in F4). Accounting for the presence of NASH did not change these estimates significantly (likelihood ratio test >0.05 for all stages of fibrosis). Similar results were seen for overall mortality. The lower end of the 95% confidence interval for the 10th percentile of time to development of severe liver disease was 22-26years in F0-1, 9.3years in F2, 2.3years in F3, and 0.9years to liver decompensation in F4. CONCLUSIONS In this, the largest ever study of biopsy-proven NAFLD, the presence of NASH did not increase the risk of liver-specific morbidity or overall mortality. Knowledge of time to development of severe liver disease according to fibrosis stage can be used in individual patient counselling and for public health decisions. LAY SUMMARY Non-alcoholic fatty liver disease (NAFLD) is very common in the general population, but reaching an accurate prognosis remains challenging. We investigate the long-term prognosis of a large cohort of NAFLD patients. In this, the largest ever study of biopsy-proven NAFLD, the presence of NASH did not increase the risk of liver-specific morbidity or overall mortality. Knowledge of time to development of severe liver disease according to fibrosis stage can be used in individual patient counselling and for public health decisions.