Cytokeratin-18 Fragments Research Articles

Serum Cytokeratin-18 Fragment Levels Are Useful Biomarkers for Nonalcoholic Steatohepatitis in Children

Nonalcoholic steatohepatitis (NASH) is the most aggressive form of nonalcoholic fatty liver disease (NAFLD). Noninvasive methods to identify children with NASH are urgently needed. The aim of this study was to evaluate the use of plasma cytokeratin-18 (CK18) fragment levels, a marker of increased hepatocyte apoptosis, as a non-invasive biomarker for pediatric NASH. Consecutive children with biopsy-proven NAFLD were included and blood samples were collected at the time of the biopsy. The diagnosis of NASH was based on Brunt's criteria. Histological features were scored: steatosis (0-3), lobular inflammation (0-3), ballooning (0-2), and portal inflammation (0-2). NAFLD activity score was calculated (0-8) and fibrosis stage was scored (0-4). We measured plasma CK18 levels using the M30-Apoptosense enzyme-linked immunosorbent assay kit. A total of 201 subjects were included in the study. The mean age was 10.7±2.5 years and 37% were male. NASH was diagnosed in 140 patients with a mean NAFLD activity scoring of 4.4±1.3. CK18 levels were significantly higher in subjects with NASH compared with not NASH (322.1 U/l±104.8 vs. 164.2 U/l±62, respectively; P<0.001). The risk of having NASH on liver biopsy increased with increased CK18 levels (P<0.001). For every 10 U/l increase in CK18 levels, the likelihood of having NASH increased by 70% after adjusting for multiple confounders. The performance of CK18 level for the diagnosis of NASH was excellent with an area under the receiver operating characteristics curve of 0.933. CK18 is a promising non-invasive biomarker for NASH in children with fatty liver disease.

Evaluation of the diagnostic value of serum and tissue apoptotic cytokeratin-18 in patients with chronic hepatitis C

Background and study aimsHepatitis C virus (HCV) is considered the most common aetiology of chronic liver disease (CLD) in Egypt. The disease severity ranges from mild illness to cirrhosis and hepatocellular carcinoma. A role for apoptosis in liver damage caused by HCV chronic infection has been suggested. Cytokeratin 18 (CK-18) is the major intermediate filament protein in the liver and is a known caspase substrate in hepatocyte apoptosis. Therefore, we analysed the serum and tissue levels of CK-18 in patients with chronic HCV infection to evaluate its role in hepatocyte apoptosis. We also correlated CK-18 expression with the severity of hepatic pathology. Patients and methodsThis study examined 80 Egyptian patients with liver disease. There were 69 patients with chronic hepatitis C and 11 patients with hepatitis C-induced cirrhotic changes. Fifteen healthy controls were also included in the study. The levels of CK-18 fragment were quantified in paired serum and liver biopsy samples. ResultsThe serum and tissue CK-18 levels were reduced in chronic HCV patients compared to early cirrhosis patients. This result indicates that serum levels of CK-18 and the hepatic expression of CK-18 might play an important role in disease progression. The serum and tissue levels of CK-18 were significantly increased and directly correlated with inflammation severity, stage of fibrosis, and ALT levels in the chronic HCV group and the cirrhotic liver group. There was no significant difference in viral load between patient cohorts. ConclusionThe serum level and the hepatic expression of CK-18 are related to disease activity and are directly correlated with METAVIR scoring. This result suggests that serum CK-18 levels may be useful for monitoring disease activity in chronic HCV and liver cirrhosis patients.

Prediction of response to neoadjuvant chemotherapy in breast cancer patients by circulating apoptotic biomarkers nucleosomes, DNAse, cytokeratin-18 fragments and survivin

Biomarkers predicting response to neoadjuvant chemotherapy in locally confined breast cancer (LBC) are highly needed. We prospectively assessed serial blood levels of apoptotic biomarkers nucleosomes, DNAse activity, cytokeratin-18 fragments (M30) and survivin in 51 LBC patients and correlated them with response to neoadjuvant treatment and established tumor markers. As controls, we used 31 healthy subjects, 13 patients with benign diseases and 28 with metastatic breast cancer (MBC). Levels of nucleosomes and survivin were elevated in LBC and MBC while M30, CEA and CA 15-3 levels were only elevated in MBC. During neoadjuvant chemotherapy, LBC patients with no change of disease (N=13) had significantly higher pretherapeutic levels of nucleosomes than patients with remission (N=38). We conclude that apoptotic biomarkers bear valuable information for diagnosis and therapy response prediction in LBC patients.

The Association Between Hepatic Fat Content and Liver Injury in Obese Children and Adolescents

OBJECTIVENonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH) are highly prevalent in obese youth. Herein, we aimed to study the association between hepatic fat accumulation as assessed by magnetic resonance imaging and circulating levels of cytokeratin-18 (CK-18) fragments, a robust NASH biomarker, and to explore the impact on this association of ethnicity, insulin resistance, and single nucleotide polymorphisms (SNPs) associated with steatosis (rs738409 in the PNPLA3, rs1260326 in the GCKR) or NASH severity (rs2645424 in the FDFT1).RESEARCH DESIGN AND METHODSTwo-hundred twenty-nine obese youths (87 Caucasians, 61 African Americans, and 81 Hispanics; mean age, 12.8 ± 2.9 years; mean BMI, 31.4 ± 7.4) underwent magnetic resonance imaging, oral glucose tolerance test, and CK-18 levels measurement; 12 subjects underwent liver biopsy.RESULTSAfrican Americans showed lower CK-18 levels than Hispanics (P < 0.001) and Caucasians (P = 0.004). Hepatic fat content (HFF%) and whole body insulin sensitivity index (WBISI) modulated CK-18 levels in Caucasians and Hispanics (P = 0.02 and P = 0.011), but not in African Americans; in fact, CK-18 was associated with HFF% and WBISI in Caucasians (P = 0.0018 and P < 0.0001) and Hispanics (P < 0.0001 and P = 0.02), but not in African Americans (both P = 0.5). The PNPLA3 SNP showed association in Caucasians (P = 0.02) and Hispanics (P = 0.05), and FDFT1 SNP showed an association in Caucasians (P = 0.05) and Hispanics (P = 0.02), with the same trend in African Americans (P = 0.07).CONCLUSIONSAfrican Americans have lower levels of CK-18 than Caucasians and Hispanics irrespective of HFF% and insulin resistance. Moreover, SNPs in the PNPLA3 and FDFT1 may drive the individual predisposition to development of hepatic injury.

Hepatic reticuloendothelial system cell iron deposition is associated with increased apoptosis in nonalcoholic fatty liver disease

The aim of this study was to examine the relationship between the presence of hepatic iron deposition, apoptosis, histologic features, and serum markers of oxidative stress (OS) and cell death in nonalcoholic fatty liver disease (NAFLD). Clinical, biochemical, metabolic, and independent histopathologic assessment was conducted in 83 unselected patients with biopsy-proven NAFLD from a single center. Apoptosis and necrosis in serum was quantified using serum cytokeratin 18 (CK18) M30 and M65 enzyme-linked immunosorbent assays and in liver by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in situ. Serum malondialdehyde (MDA) and thioredoxin-1 (Trx1) levels were measured to evaluate OS. Presence of reticuloendothelial system (RES) cell iron in the liver was associated with nonalcoholic steatohepatitis (P < 0.05) and increased hepatic TUNEL staining (P = 0.02), as well as increased serum levels of apoptosis-specific (M30; P = 0.013) and total (M65; P = 0.006) CK18 fragments, higher MDA (P = 0.002) and lower antioxidant Trx1 levels (P = 0.012), compared to patients without stainable hepatic iron. NAFLD patients with a hepatocellular (HC) iron staining pattern also had increased serum MDA (P = 0.006), but not M30 CK18 levels or TUNEL staining, compared to subjects without stainable hepatic iron. Patients with iron deposition limited to hepatocytes had a lower proportion of apoptosis-specific M30 fragments relative to total M65 CK18 levels (37% versus ≤25%; P < 0.05). Presence of iron in liver RES cells is associated with NASH, increased apoptosis, and increased OS. HC iron deposition in NAFLD is also associated with OS and may promote hepatocyte necrosis in this disease.

Sterol Regulatory Element-Binding Factor 2 (SREBF-2) Predicts 7-Year NAFLD Incidence and Severity of Liver Disease and Lipoprotein and Glucose Dysmetabolism

We prospectively assessed the impact of a sterol regulatory element-binding factor-2 (SREBF-2) polymorphism on the risk of developing nonalcoholic fatty liver disease (NAFLD) and on liver histology and lipoprotein and glucose metabolism in biopsy-proven NAFLD. In a population-based study, we followed 175 nonobese, nondiabetic participants without NAFLD or metabolic syndrome at baseline, characterized for the SREBF-2 rs133291 C/T polymorphism, dietary habits, physical activity, adipokines, C-reactive protein (CRP), and endothelial adhesion molecules. A comparable cohort of NAFLD patients underwent liver biopsy, an oral glucose tolerance test with minimal model analysis to yield glucose homeostasis parameters, and an oral fat tolerance test with measurement of plasma lipoproteins, adipokines, and cytokeratin-18 fragments. After 7 years, 27% of subjects developed NAFLD and 5% developed diabetes. SREBF-2 predicted incident NAFLD and diabetes and CRP and endothelial adhesion molecule changes. In biopsy-proven NAFLD patients, SREBF-2 predicted nonalcoholic steatohepatitis (odds ratio 2.92 [95% CI 2.08–4.18], P = 0.002) and the severity of tissue insulin resistance, β-cell dysfunction, and oral fat intolerance (characterized by higher postprandial lipemia, cholesterol enrichment of triglyceride-rich lipoproteins and oxidized LDLs, HDL cholesterol fall, adipokine imbalance, and postprandial apoptosis activation). An SREBF-2 polymorphism predisposes individuals to NAFLD and associated cardiometabolic abnormalities and affects liver histology and glucose and lipid metabolism in biopsy-proven NAFLD.

Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury

Hepatocyte cell death via apoptosis and necrosis are major hallmarks of ethanol-induced liver injury. However, inhibition of apoptosis is not sufficient to prevent ethanol-induced hepatocyte injury or inflammation. Because receptor-interacting protein kinase (RIP) 3-mediated necroptosis, a nonapoptotic cell death pathway, is implicated in a variety of pathological conditions, we tested the hypothesis that ethanol-induced liver injury is RIP3-dependent and RIP1-independent. Increased expression of RIP3 was detected in livers of mice after chronic ethanol feeding, as well as in liver biopsies from patients with alcoholic liver disease. Chronic ethanol feeding failed to induce RIP3 in the livers of cytochrome P450 2E1 (CYP2E1)-deficient mice, indicating CYP2E1-mediated ethanol metabolism is critical for RIP3 expression in response to ethanol feeding. Mice lacking RIP3 were protected from ethanol-induced steatosis, hepatocyte injury, and expression of proinflammatory cytokines. In contrast, RIP1 expression in mouse liver remained unchanged following ethanol feeding, and inhibition of RIP1 kinase by necrostatin-1 did not attenuate ethanol-induced hepatocyte injury. Ethanol-induced apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive nuclei and accumulation of cytokeratin-18 fragments in the liver, was independent of RIP3. CYP2E1-dependent RIP3 expression induces hepatocyte necroptosis during ethanol feeding. Ethanol-induced hepatocyte injury is RIP3-dependent, but independent of RIP1 kinase activity; intervention of this pathway could be targeted as a potential therapeutic strategy.

Paper alert

Paper alert

Nonalcoholic fatty liver disease in Asia: A story of growth

Ten years ago, few if any researchers in Asia showed interest in nonalcoholic fatty liver disease (NAFLD). Today, NAFLD is increasingly recognized as a major chronic liver disease not only in Western countries but also in Asia. Its importance is exemplified by its high prevalence, disease progression, and association with major medical disorders. In Asia, 15-30% of the general adult population suffers from NAFLD. In patients with diabetes and metabolic syndrome, the reported prevalence is typically over 50%. Patients with the active form of NAFLD, namely steatohepatitis (NASH), may have fibrosis progression and eventually develop cirrhosis. Patients with NASH-related cirrhosis have similar mortality to those with other causes of cirrhosis, and they have a high risk of developing hepatocellular carcinoma up to 2-3% per year. In addition, NAFLD patients have a high prevalence of cardiovascular disease and colorectal neoplasm. One major challenge for practicing clinicians is how to identify patients with significant liver disease among many who are found to have NAFLD. While liver biopsy is traditionally considered the gold standard for disease staging, it is invasive and unpleasant, and is an impractical tool for a disease that affects a quarter of the general population. To this end, new developments in transient elastography and biomarkers such as cytokeratin-18 fragments can help exclude significant liver fibrosis and NASH, respectively. This article summarizes a young researcher's journey through this exciting area of research and what he has learned from amazing people all around the world.

Development of an Accurate Index for Predicting Outcomes of Patients With Acute Liver Failure

Patients with acute liver failure (ALF) have high mortality and frequently require liver transplantation (LT); few reliable prognostic markers are available. Levels of M30, a cleavage product of cytokeratin-18 caspase, are significantly increased in serum samples from patients with ALF who die or undergo LT. We developed a prognostic index for ALF based on level of M30 and commonly measured clinical variables (called the Acute Liver Failure Study Group [ALFSG] index) and compared its accuracy with that of the King's College criteria (KCC) and Model for End Stage Liver Disease (MELD). We also validated our model in an independent group of patients with ALF. Serum levels of M30 and M65 antigen (the total cytokeratin-18 fragment, a marker of apoptosis and necrosis) were measured on 3 of the first 4 days following admission of 250 patients with ALF. Logistic regression was used to determine whether the following factors, measured on day 1, were associated with LT or death: age, etiology; coma grade; international normalized ratio (INR); serum pH; body mass index; levels of creatinine, bilirubin, phosphorus, arterial ammonia, and lactate; and log(10) M30 and log(10) M65. The area under the receiver operating characteristic (AUROC) was calculated for the ALFSG and other indices. Coma grade, INR, levels of bilirubin and phosphorus, and log(10) M30 value at study entry most accurately identified patients who would require LT or die. The ALFSG index identified these patients with 85.6% sensitivity and 64.7% specificity. Based on comparison of AUROC values, the ALFSG Index (AUROC, 0.822) better identified patients most likely to require LT or die than the KCC (AUROC, 0.654) or MELD (AUROC, 0.704) (P = .0002 and P = .0010, respectively). We validated these findings in a separate group of 250 patients with ALF. The ALFSG index, a combination of clinical markers and measurements of the apoptosis biomarker M30, better predicts outcomes of patients with ALF than the KCC or MELD.

Serum cytokeratin 18 as a biomarker for gastric cancer

Cytokeratin 18 (CK18) fragments are released into circulation during epithelial cell death. M30 (reflects caspase-cleaved CK18 fragment) and M65 (reflects total CK18 fragment) enzyme-linked immunosorbent assay (ELISA) detect circulating CK18 fragments released during caspase-dependent or total cell death, respectively; thus, CK18 has the potential of being a biomarker for epithelial cancers. In the present study, we investigated the serum levels of M30 and M65 in patients with gastric cancer, determined correlation of these levels with clinical features, and evaluated the usefulness of these enzymes as diagnostic and prognostic markers. We enrolled 54 gastric cancer patients and 12 healthy volunteers in this study. We measured the serum levels of M30 and M65 by quantitative ELISA. The levels of M30 and M65 in gastric cancer patients were significantly higher than those in healthy volunteers (P=0.001, P<0.001). The enzyme levels were elevated with the progress of gastric cancer. The sensitivity and specificity of M30 as a diagnostic marker were 67.5 and 90.9%, respectively, and those of M65 were 70.1 and 90.9%, respectively. The serum levels of M30 and M65 in patient with early gastric cancer were elevated in 38.1 and 66.7%, respectively. Further, increased serum level of M65 is an independent indicator of poor prognosis (P=0.036). The serum levels of M30 and M65 may be useful biomarkers for gastric cancer as diagnostic markers that can reflect the extent of cancer. Moreover, M65 levels can be used as a prognostic indicator.

Nonalcoholic steatohepatitis versus steatosis: Adipose tissue insulin resistance and dysfunctional response to fat ingestion predict liver injury and altered glucose and lipoprotein metabolism

Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH). Though liver-related risk seems confined to NASH, it is currently unclear whether NASH has a higher risk of cardiovascular disease (CVD) and diabetes than SS as a result of the coexistence of obesity and other cardiometabolic confounders. Adipose tissue is an emerging modulator of liver disease in NAFLD and of cardiometabolic disease in the general population. We evaluated in SS and NASH (1) glucose homeostasis and cardiovascular risk profile and (2) the effect of adipose tissue dysfunction, assessed in fasting conditions and postprandially, on liver injury, glucose and lipoprotein metabolism, and markers of early atherosclerosis. Forty nonobese, nondiabetic, normolipidemic biopsy-proven NAFLD patients (20 with SS and 20 with NASH) and 40 healthy subjects, matched for overall/abdominal adiposity and metabolic syndrome, underwent an oral fat load test, with measurement of plasma triglyceride-rich lipoproteins, oxidized low-density lipoproteins, adipokines, and cytokeratin-18 fragments, and an oral glucose tolerance test with minimal model analysis to yield glucose homeostasis parameters. Circulating endothelial adhesion molecules were measured, and adipose tissue insulin resistance (adipose IR) index and visceral adiposity index were calculated. Despite similar fasting values, compared to SS, NASH showed a more atherogenic postprandial lipoprotein profile, an altered adipokine response (i.e., higher resistin increase and an adiponectin fall), and hepatocyte apoptosis activation after fat ingestion. Adipose IR index, endothelial adhesion molecules, and hepatic insulin resistance progressively increased across NAFLD stages. NASH, but not SS, showed an impaired pancreatic β-cell function. On multiple regression analysis, adipose IR index and postprandial adiponectin independently predicted liver histology and altered cardiometabolic parameters. Adipose tissue dysfunction, including a maladaptive adipokine response to fat ingestion, modulates liver injury and cardiometabolic risk in NAFLD.

Short-term exercise reduces markers of hepatocyte apoptosis in nonalcoholic fatty liver disease

Increased hepatocyte apoptosis is a hallmark of nonalcoholic fatty liver disease (NAFLD) and contributes to the profibrogenic state responsible for the progression to nonalcoholic steatohepatitis (NASH). Strategies aimed at reducing apoptosis may result in better outcomes for individuals with NAFLD. We therefore examined the effect of a short-term exercise program on markers of apoptosis-plasma cytokeratin 18 (CK18) fragments, alanine aminotransferase (ALT), aspartate aminotransferase (AST), soluble Fas (sFas), and sFas ligand (sFasL)-in 13 obese individuals with NAFLD [body mass index 35.2 ± 1.2 kg/m(2), >5% intrahepatic lipid (IHL) assessed by (1)H-MR spectroscopy]. Exercise consisted of treadmill walking for 60 min/day on 7 consecutive days at ∼85% of maximal heart rate. Additionally, subjects underwent an oral glucose tolerance test and a maximal oxygen consumption (Vo(2max)) test before and after the exercise intervention. The Matsuda index was used to assess insulin sensitivity. We observed significant decreases in CK18 fragments (558.4 ± 106.8 vs. 323.4 ± 72.5 U/l, P < 0.01) and ALT (30.2 ± 5.1 vs. 24.3 ± 4.8 U/l, P < 0.05), and an increase in whole body fat oxidation (49.3 ± 6.1 vs. 69.4 ± 7.1 mg/min, P < 0.05), while decreases in circulating sFasL approached statistical significance (66.5 ± 6.0 vs. 63.0 ± 5.7 pg/ml, P = 0.06), as did the relationship between percent change in circulating CK18 fragments and ALT (r = 0.55, P = 0.05). We also observed a significant correlation between changes in fat oxidation and circulating sFasL (rho = -0.65, P < 0.05). There was no change in IHL following the intervention (18.2 ± 2.5 vs. 17.5 ± 2.1%, NS). We conclude that short-term exercise reduces a circulatory marker of hepatocyte apoptosis in obese individuals with NAFLD and propose that changes in the proapoptotic environment may be mediated through improved insulin sensitivity and increased oxidative capacity.

Non-invasive diagnosis of non-alcoholic steatohepatitis by combined serum biomarkers

The diagnosis of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is limited by the need for liver biopsy. We aimed at testing the accuracy of cytokeratin-18 fragment (CK-18), adipocyte fatty acid binding protein (AFABP) and fibroblast growth factor 21 (FGF21) for the diagnosis of NAFLD and NASH. 146 patients with biopsy-proven NAFLD and 74 age- and gender-matched healthy controls were included. Serum CK-18, AFABP and FGF21 levels were determined by enzyme-linked immunosorbent assay. Serum CK-18, AFABP, and FGF21 increased in a stepwise fashion in control subjects (median 103 U/L, 15.4 ng/ml, and 104 pg/ml), patients with non-NASH NAFLD (263 U/L, 18.9 ng/ml, and 249 pg/ml) and NASH (418 U/L, 19.4 ng/ml, and 354 pg/ml) (p<0.001, 0.060, and 0.016, respectively). The area under receiver-operating characteristics curve to diagnose NAFLD and NASH was 0.91 and 0.70 for CK-18, 0.66 and 0.59 for AFABP, and 0.84 and 0.62 for FGF21. At cut-offs of 203 and 670 U/L, CK-18 had 71% negative predictive value (NPV) and 77% positive predictive value (PPV) to exclude and diagnose NASH. A 2-step approach measuring CK-18 followed by FGF21 further improved the NPV to 74% and PPV to 82%. In a validation cohort of 51 patients with paired liver biopsies, the NPV and PPV of the 2-step approach were 67% and 78%, respectively. CK-18 is the most accurate biomarker for NAFLD and NASH. A two-step approach using CK-18 and FGF21 further improves the accuracy in diagnosing NASH.

Clinical Applications for Biomarkers of Acute and Chronic Graft-versus-Host Disease

Acute and chronic graft-versus-host disease (aGVHD, cGVHD) are serious complications of allogeneic hematopoietic cell transplantation. The complex pathophysiology of these disease processes is associated with immune system activation, the release of cytokines and chemokines, and alterations in cell populations. The blood levels of specific protein and cellular levels in patients with GVHD have correlated with the development, diagnosis, and prognosis of GVHD. Here, we review the most promising biomarkers for aGVHD and cGVHD with clinical relevance. The utility of GVHD biomarkers in clinical care of allogeneic hematopoietic cell transplantation recipients needs to be proven through clinical trials, and potential approaches to trial design are discussed.

Prospective Biopsy-Controlled Evaluation of Cell Death Biomarkers for Prediction of Liver Fibrosis and Nonalcoholic Steatohepatitis

Fibrosis and steatosis are major histopathological alterations in chronic liver diseases. Despite various shortcomings, disease severity is generally determined by liver biopsy, emphasizing the need for simple noninvasive methods for assessing disease activity. Because hepatocyte cell death is considered a crucial pathogenic factor, we prospectively evaluated the utility of serum biomarkers of cell death to predict different stages of fibrosis and steatosis in 121 patients with chronic liver disease. We compared the M30 enzyme-linked immunosorbent assay (ELISA), which detects a caspase-cleaved cytokeratin-18 (CK-18) fragment and thereby apoptotic cell death, with the M65 ELISA, which detects both caspase-cleaved and uncleaved CK-18 and thereby overall cell death. Both biomarkers significantly discriminated patients with different fibrosis stages from healthy controls. However, whereas both markers differentiated low or moderate from advanced fibrosis, only the M65 antigen could discriminate even lower stages of fibrosis. The M65 assay also performed better in distinguishing low (≤10%) and higher (>10%) grades of steatosis. In a subgroup of patients, we evaluated the biomarkers for their power to predict nonalcoholic steatohepatitis (NASH). Importantly, both markers accurately differentiated healthy controls or simple steatosis from NASH. However, only serum levels of M65 antigen could differentiate simple steatosis from healthy controls. Cell death biomarkers are potentially useful to predict fibrosis, steatosis, or NASH. Compared with the widely used apoptosis marker M30, the M65 assay had a better diagnostic performance and even differentiated between lower fibrosis stages as well as between healthy individuals and patients with simple steatosis.

Elevated serum CK18 levels in chronic hepatitis C patients are associated with advanced fibrosis but not steatosis

Cytokeratin-18 (CK-18) is a major intermediate filament protein in liver cells. The M30 fragment of CK-18 has been identified as a useful marker of apoptosis associated with fibrosis and steatosis in nonalcoholic steatohepatitis (NASH). We sought to assess the relationship of this marker and steatosis in a cohort of adult patients with chronic hepatitis C. The study cohort included sera from 267 treatment-naïve chronic hepatitis C (CHC) patients and 100 healthy controls with normal alanine aminotransferase (ALT). Biopsies from CHC patients were assessed for METAVIR fibrosis stage, Histology Activity Index (HAI) inflammation score and steatosis grade by expert histopathologists. The M30 fragment of CK-18 was quantified by ELISA. Wilcoxon Rank Sum, Spearman Correlation and Linear Regression tests were performed for statistical analysis. Median CK-18 levels were higher in CHC patients compared to controls (411 vs 196 U/L, P < 0.0001). Fibrosis stage was associated with increasing serum CK-18 levels (P = 0.015) and CK-18 levels were higher for F2-F4 vs F0-F1 (500 vs 344 U/L; P = 0.001). There was no association between CK-18 and increasing steatosis grade 1, 2 or 3 (460.7 vs 416.8 vs 508.3 U/L; P = 0.35) and presence or absence of steatosis (445.3 vs 365.8 U/L; P = 0.075). Fibrosis stage was independently associated with serum M30 in a multivariable linear regression model (P = 0.03). CK-18 levels were higher in CHC compared to healthy controls and associated with hepatic fibrosis. There was no difference in CK-18 M30 levels between CHC patients with and without steatosis. Although apoptosis may still contribute to hepatitis C virus (HCV)-mediated steatosis, our results suggest that serum CK-18 will not be a clinically useful test for identifying significant steatosis in CHC.

Effect of lectin-like oxidized LDL receptor-1 polymorphism on liver disease, glucose homeostasis, and postprandial lipoprotein metabolism in nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) affects 3-5% of the general adult population and predisposes to cirrhosis, cardiovascular disease (CVD), and diabetes through unclear mechanisms. Lectin-like oxidized LDL receptor-1 (LOX-1) has been connected to CVD risk in the general population and to insulin resistance and hepatic fibrogenesis in experimental models. The objective was to assess the effect of the common functional LOX-1 IVS4-14 A→G polymorphism on liver disease, adipokines, oxidative stress, lipoprotein metabolism, and glucose homeostasis in NASH. Forty nonobese, nondiabetic, normolipidemic biopsy-proven NASH patients and 40 age-, sex-, BMI-, and LOX-1 IVS4-14 A→G polymorphism--matched healthy control subjects underwent an oral-fat-load test (OFT), with measurement of plasma triglyceride-rich lipoprotein (TRLP) subfractions, oxidized LDL, total antioxidant status (TAS), adipokines (resistin and adiponectin), and cytokeratin-18 fragments (marker of hepatocyte apoptosis). The subjects also underwent an oral-glucose-tolerance test (OGTT), with minimal model analysis to yield variables of glucose homeostasis. The LOX-1 polymorphism was independently associated with liver histology (G allele carriers had more severe liver disease); during the OFT, the G allele was associated with small TRLP accumulation, lower TAS, adipokine imbalance (higher resistin and lower adiponectin), and increased cytokeratin-18 fragments. The G allele was also independently associated with insulin resistance, impaired pancreatic β cell function, and incretin effect during the OGTT. In NASH, the LOX-1 polymorphism is associated with liver disease severity and may predispose to CVD through modulation of postprandial small TRLPs and adipokine balance and to diabetes by affecting both insulin secretion and insulin sensitivity.

Growth hormone, dehydroepiandrosterone and adiponectin levels in non‐alcoholic steatohepatitis: an endocrine signature for advanced fibrosis in obese patients

Liver-related clinical consequences of non-alcoholic fatty liver disease (NAFLD) are seen only in the minority of patients with advanced fibrosis. The aim of our study was to generate insight into a potential endocrine basis of steatohepatitis with advanced fibrosis in NAFLD. Biopsy and blood samples were prospectively collected from patients with medically complicated obesity. Patients were categorized, according to liver histology, into: (i) normal, (ii) simple steatosis (SS), (iii) non-alcoholic steatohepatitis (NASH) with fibrosis stage (FS) 0-1 and (iv) NASH with FS ≥ 2. A broad panel of potential biomarkers included DHEA-S, growth hormone (GH), homeostasis model assessment-insulin resistance (HOMA-IR), leptin, resistin, adiponectin and cytokeratin 18 (CK-18) fragments. We studied 160 patients (mean BMI 46.8 ± 8.2 kg/m(2) ). Liver biopsies demonstrated normal histology in 10%, SS in 45%, NASH with FS 0-1 in 37.5% and NASH with FS ≥ 2 in 7.5%. C-reactive protein, IL-6, GH, CK-18, adiponectin, HOMA-IR and quantitative insulin sensitivity check index (QUICKI) were significantly associated with NASH in univariate analysis, but overall predictivity of these parameters was low (AUC ROC = 0.62-0.68). In contrast, all patients with NASH with FS ≥ 2 had insulin resistance, as measured by QUICKI, and GH levels <0.45 ng/ml and all but one patient with NASH FS 2-3 had low DHEA levels (<123 μg/dl). Low serum levels of GH and DHEA are very common in patients with NASH with more advanced fibrosis. Other biomarkers, including CK-18 fragment levels, have predictivity characteristics that would be of low clinical utility for distinguishing patients with normal histology or SS from those with NASH. These findings demonstrate an endocrine profile associated with advanced fibrosis.

Serum markers for necroinflammatory activity in patients with chronic viral hepatitis and normal or mildly elevated aminotransferase levels

Reports on the usefulness of serum markers for predicting liver necroinflammation are limited. The aim of this study was to determine the serum markers that predict significant inflammation in patients with chronic hepatitis B (CHB) and C (CHC) and normal or mildly elevated serum aminotransferase levels. Two hundred twenty-seven patients with CHB or CHC with normal or mildly elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (≤60 IU/L) were enrolled in this study. Significant inflammation was defined as inflammatory grade ≥3 activities using the Batt-Ludwig scoring system. The correlation between liver histology and serum markers of liver inflammation was analysed. Forty-eight (21.1%) and eight patients (3.5%) had grade 3 and 4 inflammation respectively. Univariate analysis revealed that age, platelet coun, and AST, ALT, γ-glutamyl transpeptidase, alkaline phosphatase, hyaluronic acid, haptoglobin, apolipoprotein A1 and procollagen III N-terminal peptide levels were significantly different between the patients with and without significant inflammation. There were no significant differences in the cytokeratin-18 fragment levels between the two groups. On the basis of multivariate analysis, the AST and apolipoprotein A1 levels and stage of fibrosis were highly predictive of significant inflammation. Using AST and apolipoprotein cut-off values ≥44 IU/L and ≤100 ng/ml, respectively, the presence of significant inflammation was predicted with high specificity (96.5%) and with a negative predictive value of 76.3%. The AST and apolipoprotein A1 levels were shown to be independent predictors of significant inflammatory activities in patients with CHB and CHC and normal or mildly elevated aminotransferase levels.