Usefulness of combined validation of insulin growth factor 1 and serum Adiponectin level to anticipate the early stage of nonalcoholic Steatohepatitis

Background: Non-alcoholic fatty liver disease (NAFLD) is considered one of the most common causes of chronic liver disease globally. NAFLD prevalence is increasing in parallel with other manifestations of metabolic syndrome. Diagnosis of liver fibrosis depends on imaging and biopsy. Adiponectin and insulin growth factor 1 (IGF-1) are metabolic markers associated with liver dysfunction. Aim of the study: Was to determine the possibility of using adiponectin and IGF-I in the diagnosis of early-stage nonalcoholic steatohepatitis (NASH) and the validity of the results in older age groups. Methodology: Comparative cross-sectional study included 60 hepatic patients: 30cases with NASH compared to 30cases with simple steatosis. Both IGF-1 and serum adiponectin were assessed in serum via Enzyme-linked Immunosorbent assay (ELISA). Levels of both analytes were correlated with clinical condition, abdominal ultrasonography (US), immunological and virological data. Results: Adiponectin and IGF-1 levels were significantly lower in the NASH group; compared to the simple steatosis group (p < 0.01). Also, the significant negative correlation between both of them and liver enzymes (p <0.01) reveals that liver and parenchymal dysfunction is linked with lower serum level of IGF-I and adiponectin. Older patient’s subgroup analysis showed similar results. Conclusion : Insulin growth factor 1 and Serum adiponectin are less in patients with NASH compared to those with simple steatosis so they can possibly be used in early laboratory diagnosis of NASH. These results are also valid in older age patients.


Introduction
The most frequent form of chronic liver disease, nonalcoholic fatty liver disease (NAFLD), impacts 30% of Western populations.It is a metabolic disorder linked with metabolic syndrome, associated with excessive triglyceride accumulation in the hepatocytes [1], and is a frequent indication for liver transplantation [2].Histologically NAFLD is categorized into two groups; the first group is simple steatosis group, where there is hepatic steatosis with no hepatocellular damage's proof [3].However, in the second group, nonalcoholic steatohepatitis (NASH), there is hepatocyte injury (ballooning) associated with inflammation and steatosis, with or without fibrosis [4].
Diagnosis of hepatic steatosis is dependent on imaging and histology only, with the elimination of the reasons for secondary hepatic fat buildup as steatogenic therapy's utilization, hereditary syndromes, consumption of alcohol, or metabolic insulinresistance syndrome where most patients are suffering from frank obesity, hypertension, dyslipidemia with uncontrolled glucose level.Insulin resistance states are correlated with great levels of adipokines.Adipokines are cytokines released via adipose tissue as adiponectin, Tumor necrosis factor α (TNFα), leptin, Transforming growth factor-β (TGF-β), and resistin [4,5].
Routine liver biopsy is an invasive technique, and it is not preferable to diagnose NAFLD.Also, it has several limitations concerning cost, sampling variability, multiple complications, and inter-observer skills [6].Still, ultrasonography represents the first line in the diagnosis of NAFLD, among other imaging studies.It has many advantages concerning availability, safety, also its convenience, and relatively low cost [7].
Diagnosis of fatty liver by contrast-enhanced CT has limited sensitivity.However, MRI is useful to exclude fatty infiltration.It can detect the quantity of fatty infiltration across the whole liver [8].Although, liver enzymes and previous imaging tests (ultrasound, CT, and MRI) can't be reliable for assessing steatohepatitis and fibrosis degree in cases with NAFLD.Therefore, new non-invasive biomarkers are necessary to recognize steatohepatitis in patients with NAFLD [6].
High concentrations of adiponectin circulate in the serum [9], behaving as insulin in insulin-sensitive tissues (muscles & liver) by stimulation of adenosine monophosphateactivated protein kinase to increase oxidation of fatty acid and glucose utilization [10][11][12].Many previous studies investigate the role of adiponectin in decreasing insulin resistance and also its role in attenuating liver fibrosis and inflammation, as it can suppress inflammation by direct inhibition of hepatic TNF-α [13].Deficiency of adiponectin was associated with high aminotransferase levels and liver disease progression [14,15].
In 2010, Polyzos et al. detected level changes of adiponectin throughout the adipose tissue's expansion leading to metabolic syndrome progression and the concomitant progression of NAFL to NASH, leading to NASHcorrelated cirrhosis [16,17].Increased inflammatory cytokines in the liver will result in collagen deposition, liver injury, and finally fibrosis [14].
Insulin-like growth factor-1 (IGF-1) is a hormone that affects metabolism, growth, and development.The liver secretes large amounts of IGF-1 and its binding protein stimulated by growth hormone so, the metabolism of lipids, protein, and carbohydrates is affected by growth hormone through, IGF-1 which increases levels of circulating free fatty acids (FFA) and lipolysis [18].Increased level of IGF-I stimulates the uptake of peripheral glucose with decrease synthesis of hepatic glucose improving insulin sensitivity.Dal et al. and Takahashi Y detected a decrease in levels of IGF-1 and its binding proteins in chronic liver diseases, which was associated with GH resistance [19,20]

Study design and Setting:
This study was a comparative cross-sectional study that was conducted on 60 patients who attended the outpatient clinic of Ain Shams University Hospitals.Their ages ranged from thirty to seventy years, and they were divided into 2 groups: 30 cases with NASH in one group and 30 cases with simple steatosis in the other group.

Inclusion criteria:
NAFLD patients are diagnosed by evidence of excessive hepatic fat accumulation in the liver parenchyma by abdominal sonar with no other causes of steatosis nor history of significant alcohol consumption [48].
NAFLD was subdivided into 2 groups: the first group included NASH patients who were diagnosed by abdominal ultrasonography and elevated liver enzymes.The second group included patients with simple steatosis diagnosed by abdominal ultrasonography with normal liver enzymes [48].

Exclusion criteria:
Causes of elevated liver enzymes other than NALFD i.e., history of alcohol consumption (>20 g day for women and >30 g/day for men), history of use of medications known to precipitate steatohepatitis (e.g., valproate, amiodarone, or prednisone), viral hepatitis (hepatitis B, or C), metabolic causes of steatohepatitis, e.g., Hemochromatosis and Wilson disease and autoimmune liver disease.[48].

Statistical Analysis:
For statistical analysis, MedCalc ver.20 (MedCalc, Ostend, Belgium) was utilized.Non-numerical data were presented as frequency and percentage, while nonparametric numerical data were expressed as Inter-quartile range (IQR) and Median.For comparing between various groups of nonnumerical factors, the Chi-square test was utilized.In the case of non-parametric numerical records, the Mann-Whitney U assay was utilized for contrasting between two groups.Spearman's rank correlation coefficient (r) was utilized to validate the correlation degree between adiponectin and IGF-1 levels and basic clinical, radiological, laboratory, and hepatic markers variables.Forward logistic regression analysis was utilized to detect the independent parameters that correlate with NASH occurrence.The diagnostic performance of IGF-1 and adiponectin levels were validated in aspects of their diagnostic specificity and sensitivity using the ROC Curve (receiver operating characteristic) to find out the best cut-off value.
There was a highly significant positive correlation between Adiponectin level and IGF-1 (p = 0.0002), but there was no significant correlation with age even among older subgroup.However, a highly significant negative correlation was detected between adiponectin level and other parameters as ALT, AST, direct and total bilirubin (p < 0.01).Also, a highly significant negative correlation was detected between IGF-1 and the same hepatic parameters (ALT, AST, total, and direct bilirubin) (p < 0.01) (Table 3).

Discussion
This

Conclusion
Insulin growth factor 1 and Serum adiponectin can be used in early diagnosis of NASH (rather than simple steatosis) these results can also be applied to older age groups.
There might be a protective or even a therapeutic role for IGF-1 and adiponectin versus the progression of NASH; this role needs to be further investigated.

List of abbreviation:
 NAFLD) Non-alcoholic fatty liver disease  (IGF-1) insulin growth factor 1  (NASH) nonalcoholic steatohepatitis  (TNFα) Tumor Necrosis Factor Alpha  (TGF-β) Transforming growth factor beta  (CT) computed tomography  (MRI) magnetic resonance imaging  (GH) growth hormone  (HBSAg) hepatitis b surface antigen  (HCV Ab) hepatitis c virus antibody  (ANA) antinuclear antibody  (ASMA) anti-smooth muscle antibody  (IIF) Indirect immunofluorescence  (BMI) body mass index  (US) ultrasound  (ALT) alanine aminotransferase  (AST) aspartate aminotransferase  (ELISA) Enzyme-linked Immunosorbent assay comparative cross-sectional study was done on 60 NFALD patients to correlate severity of fatty liver and levels of serum adiponectin and IGF 1.The average age of our patients was 42 to 44, which is most likely the result of a sedentary lifestyle without exercise and a nutritious diet.Many prior authors as Jamali et al. and Pandey et al. showed the same observation concerning the relatively young age of patients [27, 28].On the other hand, our study showed similar results and correlations in older patients.This implies that the same statistical conclusions can be generalized to those participants.

Table ( 1
): Comparison between the 2 groups as per basic clinical data.Comparison between the 2 groups as per hepatic markers utilizing Mann-Whitney's U test.Correlation analysis of adiponectin and IGF-1 with clinical and laboratory criteria.

Table ( 4
): Roc-curve of hepatic markers to anticipate patients with NASH.