Noninvasive Detection of Nonalcoholic Steatohepatitis Using Clinical Markers and Circulating Levels of Lipids and Metabolites.

BACKGROUND & AIMS: Use of targeted mass spectrometry (MS)-based methods is increasing in clinical chemistry laboratories. We investigate whether MS-based profiling of plasma improves noninvasive risk estimates of nonalcoholic steatohepatitis (NASH) compared with routinely available clinical parameters and patatin-like phospholipase domain-containing protein 3 (PNPLA3) genotype at rs738409. METHODS: We used MS-based analytic platforms to measure levels of lipids and metabolites in blood samples from 318 subjects who underwent a liver biopsy because of suspected NASH. The subjects were divided randomly into estimation (n = 223) and validation (n = 95) groups to build and validate the model. Gibbs sampling and stepwise logistic regression, which fulfilled the Bayesian information criterion, were used for variable selection and modeling. RESULTS: Features of the metabolic syndrome and the variant in PNPLA3 encoding I148M were significantly more common among subjects with than without NASH. We developed a model to identify subjects with NASH based on clinical data and PNPLA3 genotype (NASH Clin Score), which included aspartate aminotransferase (AST), fasting insulin, and PNPLA3 genotype. This model identified subjects with NASH with an area under the receiver operating characteristic of 0.778 (95% confidence interval, 0.709-0.846). We then used backward stepwise logistic regression analyses of variables from the NASH Clin Score and MS-based factors associated with NASH to develop the NASH ClinLipMet Score. This included glutamate, isoleucine, glycine, lysophosphatidylcholine 16:0, phosphoethanolamine 40:6, AST, and fasting insulin, along with PNPLA3 genotype. It identified patients with NASH with an area under the receiver operating characteristic of 0.866 (95% confidence interval, 0.820-0.913). The NASH ClinLipMet score identified patients with NASH with significantly higher accuracy than the NASH Clin Score or MS-based profiling alone. CONCLUSIONS: A score based on MS (glutamate, isoleucine, glycine, lysophosphatidylcholine 16:0, phosphoethanolamine 40:6) and knowledge of AST, fasting insulin, and PNPLA3 genotype is significantly better than a score based on clinical or metabolic profiles alone in determining the risk of NASH.

Ketone body production is differentially altered in steatosis and non-alcoholic steatohepatitis in obese humans.

BACKGROUND & AIMS: Levels of ketone bodies have been reported to be both increased and decreased in individuals with non-alcoholic fatty liver disease. We investigated whether the metabolism of ketone bodies is different in simple steatosis and in non-alcoholic steatohepatitis (NASH). METHODS: Serum low molecular weight molecules including ketone bodies were measured using high-throughput proton (1H) nuclear magnetic resonance in 116 (76 categorized unequivocally to those with normal liver, simple steatosis or NASH) morbidly obese individuals [age 47.3 ± 8.7 (mean ± SD) years, body mass index 45.1 ± 6.1 kg/m(2) , 39 men and 77 women] with histological assessment of NASH and analysis of gene expression in the liver. Finally, we correlated ß-hydroxybutyrate (ß-OHB) levels with NASH predicting score in Metabolic Syndrome in Men Study (METSIM) population study (n = 8749 non-diabetic men). RESULTS: Levels of ketone bodies were lower in individuals with NASH compared to individuals with simple steatosis (P = 0.004 and P = 0.018 for ß-OHB and acetoacetate respectively). Lower levels of ß-OHB were associated with the NASH predicting score in the METSIM study (P = 0.001). Liver inflammation correlated with mRNA expression of genes regulating ketolysis in the liver (Spearman correlation 0.379-0.388, P < 0.0006 for ACAT1, ACSS2 and BDH1). CONCLUSION: Lower levels of ketone bodies in individuals with NASH compared to individuals with simple steatosis suggest a decrease in ketone body metabolism in NASH.

A population-based study on the prevalence of NASH using scores validated against liver histology.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease in Western countries. Diagnosis of NASH requires a liver biopsy. We estimated the prevalence of NASH non-invasively in a population-based study using scores validated against liver histology. METHODS: Clinical characteristics, PNPLA3 genotype at rs738409, and serum cytokeratin 18 fragments were measured in 296 consecutive bariatric surgery patients who underwent a liver biopsy to discover and validate a NASH score ('NASH score'). We also defined the cut-off for NASH for a previously validated NAFLD liver fat score to diagnose NASH in the same cohort ('NASH liver fat score'). Both scores were validated in an Italian cohort comprising of 380, mainly non-bariatric surgery patients, who had undergone a liver biopsy for NASH. The cut-offs were utilized in the Finnish population-based D2D-study involving 2849 subjects (age 45-74 years) to estimate the population prevalence of NASH. RESULTS: The final 'NASH Score' model included PNPLA3 genotype, AST and fasting insulin. It predicted NASH with an AUROC 0.774 (0.709, 0.839) in Finns and 0.759 (0.711, 0.807) in Italians (NS). The AUROCs for 'NASH liver fat score' were 0.734 (0.664, 0.805) and 0.737 (0.687, 0.787), respectively. Using 'NASH liver fat score' and 'NASH Score', the prevalences of NASH in the D2D study were 4.2% (95% CI: 3.4, 5.0) and 6.0% (5.0, 6.9%). Sensitivity analysis was performed by taking into account stochastic false-positivity and false-negativity rates in a Bayesian model. This analysis yielded population prevalences of NASH of 3.1% (95% stimulation limits 0.2-6.8%) using 'NASH liver fat score' and 3.6% (0.2-7.7%) using 'NASH Score'. CONCLUSIONS: The population prevalence of NASH in 45-74 year old Finnish subjects is ∼ 5%.

Genetic variation in PNPLA3 but not APOC3 influences liver fat in non-alcoholic fatty liver disease.

BACKGROUND AND AIM: A recent study in Indian subjects suggested common variants in apolipoprotein C3 (APOC3) (T-455C at rs2854116 and C-482T at rs2854117) to contribute to non-alcoholic fatty liver disease (NAFLD), plasma apoC3 and triglyceride concentrations. Our aim was to determine the contribution of genetic variation in APOC3 on liver fat content and plasma triglyceride and apoC3 concentrations in a larger European cohort. METHODS: A total of 417 Finnish individuals were genotyped for rs2854116 and rs2854117 in APOC3 and the known rs738409 in patatin-like phospholipase domain-containing protein 3 (PNPLA3) influencing liver fat. Plasma apoC3 concentration was measured enzymatically, and liver fat by proton magnetic resonance spectroscopy. RESULTS: APOC3 wild-type homozygotes and variant allele (T-455C or C-482T or both) carriers did not differ with regard to liver fat, apoC3 concentrations, triglyceride-, high density lipoprotein-, fasting plasma glucose, insulin-, alanine aminotransferase- and aspartate aminotransferase-concentrations, nor was there a difference in prevalence of NAFLD. In contrast, carriers of the PNPLA3 GG genotype at rs738409 had a 2.7-fold (median 11.3%) higher liver fat than those with the CC (median 4.2%) genotype. The PNPLA3 rs738409 was also an independent predictor of liver fat, together with age, gender, and body mass index. CONCLUSION: Genetic variants in PNPLA3 but not APOC3 contribute to the variance in liver fat content due to NAFLD.

Circulating triacylglycerol signatures in nonalcoholic fatty liver disease associated with the I148M variant in PNPLA3 and with obesity.

We examined whether relative concentrations of circulating triacylglycerols (TAGs) between carriers compared with noncarriers of PNPLA3(I148M) gene variant display deficiency of TAGs, which accumulate in the liver because of defective lipase activity. We also analyzed the effects of obesity-associated nonalcoholic fatty liver disease (NAFLD) independent of genotype, and of NAFLD due to either PNPLA3(I148M) gene variant or obesity on circulating TAGs. A total of 372 subjects were divided into groups based on PNPLA3 genotype or obesity. Absolute and relative deficiency of distinct circulating TAGs was observed in the PNPLA3(148MM/148MI) compared with the PNPLA3(148II) group. Obese and 'nonobese' groups had similar PNPLA3 genotypes, but the obese subjects were insulin-resistant. Liver fat was similarly increased in obese and PNPLA3(148MM/148MI) groups. Relative concentrations of TAGs in the obese subjects versus nonobese displayed multiple changes. These closely resembled those between obese subjects with NAFLD but without PNPLA3(I148M) versus those with the I148M variant and NAFLD. The etiology of NAFLD influences circulating TAG profiles. 'PNPLA3 NAFLD' is associated with a relative deficiency of TAGs, supporting the idea that the I148M variant impedes intrahepatocellular lipolysis rather than stimulates TAG synthesis. 'Obese NAFLD' is associated with multiple changes in TAGs, which can be attributed to obesity/insulin resistance rather than increased liver fat content per se.