Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome (1) is one of the most common cause of chronic liver disease worldwide.The prevalence of NAFLD is expected to increase due the rising epidemic of the obesity and type II diabetes (diabesity) the most important components of the metabolic syndrome (2). NAFLD is a spectrum of disease ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), advanced fibrosis and even cirrhosis (3). While simple steatosis is rather a stable condition, NASH, defined by the presence of necroinflammatory activity with or without fibrosis, may have a progressive course to cirrhosis and its complications and is associated with a significant increase in overall and liver-related morbidity and mortality (4). Definite diagnosis of NASH relies on liver biopsy which still remains the gold standard for differentiation between NASH and simple steatosis. Liver biopsy gives information not only on the degree of steatosis, but also on severity of inflammatory activity and of fibrosis stages (5). Despite these advantages, liver biopsy is an invasive procedure, associated with various complications, may be subject to sampling errors, due to heterogenous distribution of the lesions (6) and is not a suitable tool for a disease with such a high prevalence.

Due to the potential progressive course of NASH and limited access to liver biopsy, several non-invasive biomarkers or panel of biomarkers for NASH have been developed. Some excellent reviews have been recently published on this subject (7-9).

From the histopathologic point of view steatosis as a prerequisite condition, necroinflammation, hepatocyte ballooning and apoptosis are the leading features of NASH (10). Apoptosis has a role both in experimental model of NASH as well as in human NASH (11) and correlates with fibrosis and necroinflammatory activity (12).

However, apoptosis is just one of the mechanism involved in cell death in liver injury, a process that associates other events: necrosis, programmed necrosis (necroptosis) and autophagy (13, 14). Dead hepatocites or dying, but viable liver epithelial cells, irrespective of mechanism promote liver inflammation and fibrogenesis, significant features that differentiate NASH from simple steatosis. A complex pathogenic process is involved: the clearance of apoptotic bodies through phagocytosis by Kupffer cells and hepatic stellate cells, enhanced production of proinflammatory and profibrogenic cytokines and chemokines (15). From the practical point of view, death or dying hepatocites release cytoskeletal proteins, mainly caspase-cleaved or uncleaved cytokeratin 18 (CK-18) and their assessment improves the detection of liver cell death (16).

From the pathophysiological point of view, insulin resistance (17), imbalance of adipocytokines, inflammation (18) apoptosis and necrosis (11) play a key role in development of NASH and, therefore, biochemical markers of these events may be useful for the differentiation of NASH from simple steatosis.

In the present study, we tried to test a panel of serological markers reflecting the main pathophysiological events involved in the development of NASH: insulin resistance, adipokines imbalance (namely adiponectin), inflammation, necrosis and apoptosis with the aim to improve diagnosis of NASH. We compared diagnostic performance of the newly proposed biomarker panel with previously described non-invasive tests for predicting NASH.


PATIENTS AND METHODS
Study population

The study involved 79 patients (56 men and 23 women) with biopsy proven NAFLD. Liver biopsy was performed for abnormal liver function tests lasted for at least 6 month and suspected NAFLD at grey scale ultrasonography.

The patients with other liver disease etiologies: hepatitis B or C, autoimmune liver disease, Wilson disease, hemochromatosis, 1-antitripsin deficiency, HIV infection, patients with a history of hepatotoxic or steatosis-inducing drugs or those with daily alcohol intake exceeding 10 g/day for women and 20 g/day for men were excluded. Patients with a history of an inflammatory disease, current infection, or history of cancer, as well as those receiving treatment with PPAR- agonists were also excluded.

The study was performed in accordance with the Declaration of Human Rights (Helsinki) and with its further revision and was approved by the local Ethics Committee. A written informed consent was obtained from each patients.

Clinical evaluation

The assessment included history and physical examination. Prevalence of diabetes, hypertension and hyperlipidemia was assessed by review of past medical history. Heigh, weight, and waist circumference were determined. Body mass index (BMI) was calculated (kg/m2). The diagnosis of metabolic syndrome (MS) and its components was based on the Consensus elaborated in 2009 (20).

Laboratory investigations

A blood sample was obtained after overnight fasting for the determination of plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamiltransferase (GGT), glucose, cholesterol, tryglicerides, HDL-cholesterol. All the determinations were made on an automatic analyser (Konelab 301-Thermo Electron Corp, Finland).

Other serum samples were obtained in the same day with the liver biopsy, centrifugated at 2500 g for 10 minutes and serum aliquots were stored at –80°C until further analyses.

Fasting insulin levels were measured by ELISA method (Mercodia ELISA, Sweden). The degree of insulin resistance was calculated according to the homeostasis model assessment for insulin resistance (HOMA-IR) by the formula: fasting insulin level (mIU/l) x fasting glucose level (mg/dl)/405 (20). HOMA-IR was calculated for all patients, taking into account that none had fasting glucose level >140 mg% or underwent treatment with insulin.

Total serum adiponectin, and high sensitivity IL-6 (HS-IL-6) were determined by ELISA method (Quantikine human adiponectin and Quantikine HS-IL-6; R and D Systems, UK). The sensitivity of those assays was 0.079 ng/ml for adiponectin and 0.016 pg/ml for HS-IL-6. Adiponectin levels were calculated for the whole group and also separately for diabetics.

Total cytokeratin-18 (M65 antigen) was determined by commercially available Kit (M65 ELISA, Peviva AG, Sweden) with a sensitivity of 11 U/L, according to the manufacturer’s instructions. This method is based on the capture (M6) and detection (M5) of antibodies directed against two different epitopes of CK-18, independently of the cleavage status (21).

Morphopatological study

Liver biopsies were performed under ultrasonographic guidance and stained with hemotoxilin-eosin and Masson’s trichrome and were assessed by a senior hepatopathologist (AS) blinded to the clinical or biological characteristics of the patients. The biopsies were graded according to the NAFLD scoring system proposed by the National Institute of Diabetes and Digestive and Kidney Diseases NASH Clinical Research Network and reported as NAFLD activity score (NAS) (22). According to this scoring system NAS is the unweighted score of steatosis (0-3), lobular inflammation (0-3) and ballooning (0-2). The stage of fibrosis was assessed using a four-point scale: 0= no fibrosis; 1= mild/moderate zone 3 perisinusoidal fibrosis or portal/periportal fibrosis only; 2= perisinusoidal and portal/periportal fibrosis; 3= bridging fibrosis and 4= cirrhosis.

According to the NAS score patients were classified as not-NASH (NAS2), borderline NASH (NAS=3-4) and definite NASH (NAS5).

For further analysis we took into consideration two group of patients: not-NASH and definite NASH.

Statistical analysis

All factors were included in descriptive statistics. Values were presented as mean, standard deviation and median and percentiles (25th, 75th) for all continuous variables and frequencies for categorical factors. Univariate analysis was done in order to compare the patients with NASH to those with not-NASH. Student’s t test and Mann-Whitney test were used to compare continuous variables and Pearson’s chi-square test for categorical variables. Correlations between variables were established using Spearmen’s rank correlation. Variables that achieved statistical significance with univariate analysis were included in multivariate analysis in order to evaluate the independent factors associated with NASH. According to criteria common in medical studies, the boundary of statistical significance was accepted as p0.05.

Receiving operating characteristics (ROC) analysis was performed to assess the role of each studied serological marker in the diagnosis of NASH. The area under ROC curves (AUROCs) and corresponding 95% confidence intervals (95% CIs) were assessed. Cut-off values for each parameters were determined. The sensibility (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of the serum biomarkers in predicting definitive diagnosis of NASH were calculated using ROC analysis. De Long’s method was used for comparing AUROCs (23). The MedCalc® 9.3.9.0. software and SPSS software version 15.0 (SPSS Inc. Chicago, IL, USA) were used for the statisical analysis.


RESULTS
Histopathological data in patients with non-alcoholic fatty liver disease

The histological findings are shown in Table 1. The not-NASH group included 20 patients and NASH group (NAS5) 59 patients.

Table 1. Histological characteristics of the liver biopsies.

* -number (percent); †mean±standard deviation.

The patients with NASH had significantly higher degrees of steatosis and inflammation. None of the not-NASH patients had fibrosis.

Clinical, demographic and biochemical features

Individual characteristics of not-NASH and NASH patients are shown in Table 2. The NASH patients were older, had higher ALT, HOMA-IR, M65, HS-IL-6 levels, but lower adiponectin levels.

Table 2. Clinical, demographic and biochemical features.

* -number (percent); † -mean±standard deviation; ‡ -median (25-75 percentiles).

Predictive factors of nonalcoholic steatohepatitis versus not-nonalcoholic steatohepatitis

In univariate analysis, age, ALT and HOMA-IR were associated with NASH. The prevalence of metabolic syndrome was not different between these two groups (p=0.7).

Adiponectin, apoptosis and necrosis marker (M65), and HS-IL-6 were also significantly higher in patients with NASH vs. not-NASH patients.

Taking into account that patients with diabetes have lower serum levels of adiponectin we analyzed the overall group in comparison to the diabetes group and did not find any statistically significant difference (5736.2±4154.4 ng/ml vs. 4283.9±1937.9 ng/ml; p=0.222).

All factors significantly associated with NASH were taken into account for multivariate analysis (Table 3). Only adiponectin, IL-6 and CK-18 were independently associated with NASH.

Table 3. Predictor factors of NASH in multivariate analysis.

Diagnostic value of individual independent predictors of nonalcoholic steatohepatitis

The AUROCs, 95%CI, cut-off values and Se, Sp, PPV and NPV were established for each independent predictor (Table 4). The best performance was obtained for M65 antigen, followed by IL-6 and adiponectin.

Table 4. Diagnostic value of independent predictors for diagnosis of NASH (NAS5).

The correlation between independent predictors of nonalcoholic steatohepatitis and histological characteristics

Some correlations were found between serological markers and liver histologic characteristics in NASH patients. Adiponectin was inversely correlated with steatosis grade, but not with lobular inflammation or hepatocyte ballooning. M65 levels were correlated with steatosis, lobular inflammation and hepatocyte ballooning, while IL-6 was correlated with lobular inflammation, but not with hepatocyte ballooning or steatosis (Table 5). All independent predictors were correlated with NAS. HOMA-IR was positively correlated with steatosis (r=0.502; p=0.0001) and negatively with adiponectin (r=–0.271; p=0.038). We also found a negative correlation between adiponectin and CK-18 (r=–0.365; p=0.004) and a positive correlation between CK-18 and IL-6 (r =0.279; p=0.032).

Table 5. Correlations between serological markers and histologic characteristics for NASH patients (NAS5).

The combination of serological markers

Combining independent predictors of NASH at the cut-off values for each parameter we found an increased diagnostic performance. The best value was obtained for the triple combination: adiponectin, M65 and IL-6 with an AUROC of 0.903 (Table 6).

Table 6. Combination of two or three independent predictors for diagnosis of NASH (NAS5).

DISCUSSION
In an attempt to identify a novel pathophysiological-based non-invasive panel of serological biomarkers aimed to distinguish between NASH and not-NASH patients we have studied some markers involved in the pathogenetic events leading to NASH.

Our study showed that individual noninvasive markers: adiponectin, markers of inflammation and those of necrosis and apoptosis had similar diagnostic values to those reported by previous studies. It was not surprising that we did not find a statistically difference between HOMA-IR values in NASH compared to not-NASH patients, taking into account that insulin resistance is a “pathophysiological hallmark of NAFLD” (17). For NASH patients HOMA-IR was positively correlated with steatosis and negatively with adiponectin levels. Among the adipokines, adiponectin has been studied extensively. Adiponectin has anti-inflammatory, antisteatotic, antifibrogenic and antiapoptotic effects in the liver (24, 25, 26) and also a significant metabolic effect, improving hepatic insulin resistance (27).

Our study found that adiponectin levels were significantly decreased in NASH as compared to not-NASH patients. This difference was found even after the adjustment for potential confounders. Similar results were obtained when diabetic patients were removed from the analysis, similar to another study (28). Lower adiponectin levels were reported by other studies in NASH (29) and some of them found that hypoadiponectinemia was independently associated not only with the degree of steatosis, but also with the severity of liver histology: necroinflammation (30) or even fibrosis (30, 31). In our study adiponectin was negatively associated with insulin sensitivity and the grades of steatosis, but was not correlated with the severity of liver disease, as in other reports (27, 32).

A better AUROC of 0.765 for adiponectin as a single individual marker for NASH was obtained by Shimada et al. (33).

In NAFLD, the development of hepatic inflammation is the critical stage in the progression from simple steatosis to NASH (18). A study that investigated the liver expression of selected genes related to inflammation and immune response associated with obesity, steatosis and NASH of morbidly obese patients found 58 out of 222 genes upregulated in NASH patients. IL-6 gene was upregulated 5.9 fold in NASH patients as compared to patients having grade 3 steatosis (34).

IL-6 is a cytokine secreted by adipocytes, immune and endothelial cells as well as by hepatocytes. A long term exposure to IL-6 may sensitize the liver to apoptotic cell death (35). Our study found an increased serum level of IL-6 in NASH patients. The levels of IL-6 were correlated with total CK-18 levels (M65 antigen) and with lobular inflammation. Similar correlations between serum levels of IL-6 and histological substrate - the degree of inflammation and stage of fibrosis was reported in another study (36). The levels of IL-6 and sIL-6R were increased in patients with NASH compared with patients having simple steatosis (37). The role of IL-6 in the pathogenisis of NASH is also supported by a study that demonstrated that patients with NASH have a dysregulated cytokine metabolism at baseline and a significant decrease in IL-6 levels after lifestyle changes and vitamin E administration (38).

Hepatocyte apoptosis and Fas expression are leading features of human NASH (11). The accumulated knowledge of the pathways involved in hepatocyte apoptosis and necrosis led to the identification of several markers: the total levels of caspase-generated CK-18 fragments (M30 antigen), reflecting apoptosis; the total amount of uncleaved and caspase-cleaved CK-18 by M65 ELISA assay and M65 EpiDeath (M65ED) ELISA (39), reflecting total cell death, that is both apoptosis and necrosis and the difference between M65 and M30 antigens, reflecting necrosis (40).

Most of the studies investigated the plasma CK-18 fragments levels that reflect the magnitude of apoptotic process and were predictive of NASH (12, 41).

The AUROC for the diagnosis of NASH was 0.83 and the levels of CK-18 fragments were correlated with the presence of all NASH features: steatosis, lobular inflammation and fibrosis (40). The value of this test was confirmed in obese patients (36, 40, 42) and pediatric populations (43).

In our study we determined the soluble form of total extracellular cytokeratin-18 (M65 antigen) and found a cut-off value of 340 U/l, with an AUROC of 0.791 for predicting NASH. M65 was correlated with steatosis, lobular inflammation and hepatocyte ballooning.

These results are in concordance with other studies that reported the same favourable predictive values in patients with NASH, both for M30 or M65 serum levels, even better for total CK-18 (M65) with an AUROC of 0.814 vs. caspase generated CK-18 fragments, with an AUROC of 0.711 (40). M65 levels could be used as a rapid screening test to differentiate NASH from simple steatosis in patients with NAFLD at a cut-off value of 300 U/l with a PPV of 81% and NPV of 85% (44). In a recent meta-analysis a pooled AUROC for CK-18 cleaved fragments was 0.82, with 78% sensitivity and 87% specificity (8).

Another diagnostic biomarker panel containing total CK-18 level, serum adiponectin and serum resistin gave an AUROC of 0.908, with a sensitivity of 95.9 and specificity of 70.2 (40). Based on the better knowledge on apoptosis, a panel of noninvasive markers for the diagnosis of NASH including CK-18 fragments and sFas levels with an AUROC of 0.930 was proposed. This model correctly classified 88% of the patients (45).

In a very recent study, the assessment of total CK-18 levels by M65 or M65ED ELISA as single markers performed better in distinguishing between NASH and NAFLD, when compared to the M30 assay, with an AUROC of 0.93 (CI 95%, 0.8–1.0), respectively 0.92 (CI 95% 0.79–1.0) and with a sensitivity of 100% and specificity of 80%. By multiple regression analysis, the total CK-18 levels assessed by M65 ELISA was found to have a predictive value for NASH independently of ALT levels (46). These findings undoubtedly will simplify the diagnosis of NASH. Furthermore, the assessment of total CK-18 was found to discriminate between insignificant fibrosis F0–F1 and significant fibrosis F2 (46).

It is worth mentioning that CK-18 antigens are not specific for NASH and their levels increase in other chronic liver diseases involving apoptosis: alcoholic hepatitis, viral hepatitis, hepatocellular carcinoma and other malignancies, cholangitis, cholestatic liver disease (47).

In the last part of the study we analyzed the possibility to increase the diagnostic value of studied markers by different combinations between them. By combining two individual markers studied in this investigation we obtained a better predictive value for NASH in increasing order: M65 and IL-6; adiponectin and M65 and adiponectin and IL-6, with an AUROC of 0.841 for the last combination.

The combination of all three markers gave a predictive value for NASH with an AUROC of 0.903, Sp=85.7% (PPV=94.2%) and Se=84.5% (NPV=66.7%). To our knowledge, this is the first pathophysiological-based panel of biomarkers that combines adiponectin, a marker of inflammation (IL-6) and a marker of necrosis and apoptosis (total CK-18) to distinguish NASH from simple steatosis.

Besides the panels already mentioned, there are some other combinations of biomarkers reflecting pathophysiological events in NASH. The combination of adiponectin/leptin ratio and HOMA-IR yielded an AUROC of 0.82 (48), while metabolic syndrome, ALT and CK -18 fragments levels, an AUROC of 0.88 (49).

From the point of view of predictive value for NASH, our combination comes on the fourth place after Tamimi et al. (45), Joka et al. (46) and Younossi et al. (40).

Our study had several strengths. We studied a well characterized biopsy proven cohort of patients with NASH. The parameters resulted from multivariate analysis had a strong pathophysiological support for the diagnosis of NASH. To our knowledge this is the first panel of biomarkers that combines a marker of inflammation (IL-6), a marker of necrosis and apoptosis (M65) and adiponectin.

At the same time we are aware of the study limitations: the relatively small sample of patients, the cross-sectional fashion of the study and the possibility of the sampling errors and intra-observer variability with regards to liver biopsy. The main limitation is the lack of the validation group.

In conclusion, our data support the concept that the combined investigation of markers that reflect the main pathophysiological pathways involved in the development of NASH could be proposed as predictor of this disease.

Conflict of interests: None declared.


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