Jeffery L. Smith

Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3.

Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non‐alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade.

Importance of hepatic fibrosis in cystic fibrosis and the predictive value of liver biopsy

Cystic fibrosis liver disease (CFLD), which results from progressive hepatobiliary fibrosis, is an important cause of morbidity and mortality, but it is difficult to identify before portal hypertension (PHT) ensues. Clinical signs, serum alanine aminotransferase (ALT) levels, and ultrasound (US) are widely applied, but their value in predicting the presence of cirrhosis, the development of PHT, or adverse outcomes is undetermined. The potential gold standard, liver biopsy, is not standard practice and, notwithstanding sampling error considerations, has not been systematically evaluated. Forty patients with cystic fibrosis (median age = 10.6 years) with abnormal clinical, biochemical, and US findings were subjected to dual‐pass percutaneous liver biopsy. Clinical outcomes were recorded over 12 years of follow‐up (median = 9.5 years for survivors). Logistic regression and receiver operating characteristic analyses were applied to predict hepatic fibrosis (which was assessed by fibrosis staging and quantitative immunohistochemistry) and the occurrence of PHT. PHT occurred in 17 of 40 patients (42%), including 6 of 7 (17%) who died during follow‐up. Clinical examination, serum ALT levels, and US findings failed to predict either the presence of liver fibrosis or the development of PHT. Fibrosis staging on liver biopsy, where the accuracy was improved by dual passes (P = 0.002, nonconcordance = 38%), predicted the development of PHT (P < 0.001), which occurred more frequently and at a younger age in those with severe fibrosis. Conclusion: Clinical modalities currently employed to evaluate suspected CFLD help to identify a cohort of children at risk for liver disease and adverse outcomes but do not predict an individuals risk of liver fibrosis or PHT development. Liver fibrosis on biopsy predicts the development of clinically significant liver disease. Dual passes help to address sampling concerns. Liver biopsy has a relevant role in the management of patients with suspected CFLD and deserves more widespread application. (HEPATOLOGY 2011)

Hepatic acyl-CoA: cholesterol acyltransferase. Development of a standard assay and determination in patients with cholesterol gallstones

A standard assay was developed for human liver acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.2.26) which is more sensitive than previous methods and allows accurate activity determinations on crude microsomal fractions. ACAT activity was measured in microsomes from livers of four gallstone patients and five controls. Preincubation with exogenous cholesterol produced an increase in ACAT activity in all liver samples: gallstone samples showed a mean increase of 1.8-fold, whereas non-gallstone samples showed a mean increase of 8.2-fold. The mean ACAT activity measured in the presence of exogenous cholesterol was 52.8 +/- 22.8 (n = 4) pmol . min-1 . mg-1 for gallstone samples and 82.8 +/- 13.5 (n = 4) pmol . min-1 . mg-1 for non-gallstone samples. These results suggest that patients suffering from cholesterol gallstones have a reduced ability to esterify potentially harmful free cholesterol compared with controls. They support the proposition that cholesterol gallstone formation is related to altered hepatic cholesterol metabolism.

Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR.

ACAT (also called sterol o-acyltransferase) catalyzes the esterification of cholesterol by reaction with long-chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to β-actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n = 17), ACAT-1 transcripts were on average 9-fold (range, 1.7- to 167-fold) more abundant than ACAT-2, whereas in duodenal samples (n = 10), ACAT-2 transcripts were on average 3-fold (range, 0.39- to 12.2-fold) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine.

Endogenous ursodeoxycholic acid and cholic acid in liver disease due to cystic fibrosis

Focal biliary cirrhosis causes significant morbidity and mortality in cystic fibrosis (CF). Although the mechanisms of pathogenesis remain unclear, bile acids have been proposed as potential mediators of liver injury. This study examined bile acid composition in CF and assessed altered bile acid profiles to determine if they are associated with incidence and progression of liver injury in CF‐associated liver disease (CFLD). Bile acid composition was determined by gas–liquid chromatography/mass spectrometry in bile, urine, and serum samples from 30 children with CFLD, 15 children with CF but without liver disease (CFnoLD), and 43 controls. Liver biopsies from 29 CFLD subjects were assessed histologically by grading for fibrosis stage, inflammation, and disruption of the limiting plate. A significantly greater proportion of endogenous biliary ursodeoxycholic acid (UDCA) was demonstrated in CFnoLD subjects vs. both CFLD subjects and controls (2.4‐ and 2.2‐fold, respectively; ANOVA, P = .04), and a 3‐4 fold elevation in endogenous serum UDCA concentration was observed in both CFLD subjects and CFnoLD subjects vs. controls (ANOVA, P < .05). In CFLD, there were significant correlations between serum cholic acid and hepatic fibrosis, inflammation, and limiting plate disruption as well as the ratio of serum cholic acid/chenodeoxycholic acid to hepatic fibrosis, inflammation, and limiting plate disruption. In conclusion, elevated endogenous UDCA in CFnoLD suggests a possible protective role against liver injury in these patients. The correlation between both cholic acid and cholic acid/chenodeoxycholic acid levels with histological liver injury and fibrosis progression suggests a potential monitoring role for these bile acids in CFLD. (HEPATOLOGY 2004;39:1673–1682.)

Interdependence of serum concentrations of vitamin K1, vitamin E, lipids, apolipoprotein A1, and apolipoprotein B: Importance in assessing vitamin status

Vitamin E (alpha-tocopherol) and vitamin K1 (phylloquinone) are fat-soluble vitamins and are important nutrients in health and disease. In this study serum concentrations of vitamin E and vitamin K1, lipids and apolipoproteins A1 and B were measured in neonates, normal and hyperlipidaemic individuals in an attempt to establish their interrelationships. A high degree of correlation was observed between the concentrations of the vitamins and those of lipids and apolipoproteins (r ranged from 0.42 to 0.92; p<0.001). Stepwise linear regression methods determined that serum concentrations of both vitamin E and vitamin K1 could best be predicted by using equations excluding lipids but containing only apolipoprotein A1 and B concentrations. Correlation coefficients between predicted and measured values were 0.89 for serum vitamin E, and 0.83 for serum vitamin K1 concentrations. To test the validity of the derived formulae, measured and estimated vitamin K1 and vitamin E concentrations in serum were determined in another group of neonates, normal adults and hypercholesterolemic adults and the comparisons were shown to be very good. These results indicate that the serum levels of both vitamins depend critically on the concentration of the lipoprotein carriers, apolipoproteins A1 and B. Hence, in order to identify variations in serum vitamin K1 and vitamin E concentrations, which are independent of variations in carrier concentration, it will be necessary to express these serum vitamins as ratios of vitamins to apolipoprotein A1 and B carriers.

Effects of simvastatin on hepatic cholesterol metabolism, bile lithogenicity and bile acid hydrophobicity in patients with gallstones

Background and Aims : There is limited information available on the effects of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors on hepatic and biliary cholesterol metabolism in patients with gallstones. The aims of this study were to determine the effect of simvastatin on the regulatory elements of cholesterol metabolism that determine the concentrations of cholesterol in plasma and bile.

Evidence for a sub-morphological inflammatory process in the liver in haemochromatosis

BACKGROUND/AIMS The role of cytokines in hepatic injury has been examined for many liver diseases however little is known of the cytokine involvement in haemochromatosis. The aim of the current study was to examine the hepatic gene expression of potential proinflammatory and profibrogenic cytokines in haemochromatosis. METHODS Interferon-gamma, interleukin-10, transforming growth factor-beta(1) and tumor necrosis factor-alpha mRNA expression was assessed in liver tissue from 20 haemochromatosis patients, eight controls and eight chronic hepatitis C patients. To assess the immunophenotype of the inflammatory infiltrate in haemochromatosis, liver sections were subjected to immunohistochemistry using markers for macrophages (CD68, HAM56, MAC387) or T cells (CD3 and CD45RO). RESULTS Interferon-gamma mRNA was increased in both haemochromatosis (0.29+/-0.08%, P=0.01) and hepatitis C patients (1.02+/-0.32%, P=0.03) compared to controls (0.04+/-0.01%). Interleukin-10 mRNA was significantly decreased in both haemochromatosis and hepatitis C patients (0.01+/-0.003%, P=0.008 and 0.03+/-0.015%, P=0.02, respectively) compared to controls (0.12+/-0.01%). CD3 positive T-cell number was significantly correlated with increasing hepatic iron concentration (r=0.56, P=0.03). CONCLUSIONS This study has demonstrated a distinct pattern of cytokine gene expression in haemochromatosis, which resembles that of inflammatory conditions such as chronic hepatitis C. These factors may play a role in the development of iron-induced hepatic fibrosis in haemochromatosis.

Lecithin-cholesterol acyltransferase activity in normocholesterolaemic and hypercholesterolaemic roosters: modulation by lipid apheresis.

Lipid apheresis, a recently described procedure for the elimination of lipid but not apolipoproteins from plasma, was applied to normocholesterolaemic and hypercholesterolaemic roosters. Lipid apheresis resulted in an immediate reduction in plasma unesterified cholesterol concentration, which was sustained for 150 min. The reduction in unesterified cholesterol concentration was higher in the normocholesterolaemic animals than in the hypercholesterolaemic animals. Lipid apheresis induced changes in the ratio of plasma unesterified to total cholesterol in normocholesterolaemic animals but not in hypercholesterolaemic animals. In hypercholesterolaemic animals, lecithin–cholesterol acyltransferase (LCAT) activity was not affected by lipid apheresis, whereas in normocholesterolaemic animals LCAT activity was acutely reduced for 150 min after lipid apheresis. Saturated LCAT kinetics occurred in the hypercholesterolaemic animals but not in the normocholesterolaemic animals. LCAT obeyed Michaelis–Menten kinetics. After lipid apheresis, there was a pool of unesterified cholesterol that was available as substrate for LCAT to a greater extent in hypercholesterolaemic animals than in normocholesterolaemic animals. These observations may have important implications for lipid apheresis as a treatment for atherosclerosis.

Lipid apheresis in an animal model causes in vivo changes in lipoprotein electrophoretic patterns

Lipid apheresis, a new extracorporeal procedure based on plasma delipidation and showing promise as a possible treatment for atherosclerosis, was recently reported for the first time from this laboratory [Cham et al., J Clin Apheresis 10:61–69. 1995]. In the present study lipid apheresis was applied to hypercholesterolemic and normocholesterolemic roosters to examine its effect on plasma lipoprotein particles. This procedure resulted in conspicuous changes in electrophoretic patterns of plasma lipoproteins. The electrophoretic mobilities of all the lipoprotein fractions had changed considerably. Lipid stainable material was present in at least three bands in the α‐globulin area. In particular, changes in the electrophoretic region of high‐density lipoproteins were observed. Lipid apheresis markedly induced the anti‐atherogenic pre‐β‐high‐density lipoproteins. The observed changes induced by lipid apheresis were more pronounced in the hyperlipidemic animals compared with the normocholesterolemic controls. A novel pre‐α‐lipoprotein band was observed soon after lipid apheresis. This lipoprotein band had a density larger than 1.21. At approximately 150 minutes after lipid apheresis, the electrophoretic pattern had almost returned to its original base pattern. Lipid apheresis results in plasma lipoprotein changes which may induce reverse cholesterol transport and shows promise as a possible treatment of atherosclerosis.