**10. New diagnostic platforms in nafld**

NAFLD is a disease with wide spectrum: from steatosis through inflammation to fibrosis and finally cirrhosis and hepatocellular carcinoma, even in absence of cirrhosis [72]. The strongest predictor of fibrosis progression in NAFLD is steatohepatitis. The most important features are hepatocellular degeneration (ballooning) and inflammatory cell infiltration.

and insulin grow factor acid labile subunit, in a 3-panel model. These panels performed in the diagnosis of the diverse NAFLD stages get an area under the receiver operator curve

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Metabolomics. In the natural history of NAFLD the progression to hepatic fibrosis occurs only in 10 to 25% of cases, leading to cirrhosis, end-stage liver disease or hepatocellular car‐ cinoma. The strongest predictor of fibrotic progression, apart from pre-existing fibrosis, is steatohepatisis. A two-hit model has been proposed as an explanation for why some pa‐ tients progress to NASH. In a first step, because of insulin resistance, adipose tissue has en‐ hanced triglyceride lipolysis, which leads to increased serum free fatty acids, and impaired hepatic triglyceride export. In this model, hepaticsteatosis (hit 1) exposes the liver parenchy‐ ma to environmental and extracellular hepatic insults (hit 2), leading to inflammation, stea‐ tonecrosis and fibrosis. Impaired mitochondrial oxidation and lipid export may also

Leptin system is also implicated, and its receptor expression is related to fibrosis degree [85].

As it was explained in the introduction, irisin is a newly identified hormone. Irisin is pro‐ duced in muscle cells induced in exercise [86-88]. Irisin activates changes in adipose tissue, and make its change from white adipose tissue to brown adipose tissue, and this causes a significant increase in total body energy expenditure and resistance to obesity-linked insulin

Inflammation is considered to be the central clue for the progression of NAFLD, the origins and components are considered in this review [89]. Hepatocytes injured by toxic lipid mole‐ cules play a central role in the recruitment of innate immunity involving Toll-like receptors (TLR), Kuppfer cells, lymphocytes and neutrophils and possibly inflammasome. On this way, a study was carried to determine the lipidomic signature in NAFLD [90]. Using proteo‐ mic tools (mass spectrometry) the investigators found metabolites from nonenzymatic oxi‐ dation product of arachidonic acid and from impaired peroxisomal polyunsaturated fatty acid (PUFA). This study links to another, where investigators characterize metabolic profile to distinguish steatosis and NASH [91], they also found arachidonic acid, among other sub‐ stances, relation to NASH and fibrosis. Metabolomics analysis was performed to NAFLD patients showing a lower concentrations of glutathione, an antioxidant substance, in this

The key pro-inflammatory signalling pathways in NASH are nuclear factor-kappa B (NFkB) and c-Jun N-terminal kinase (JNK). It could be possible that inflammation in NASH could originate outside the liver. Gut microbiota, the related Kupffer/TLR response, in‐ flamed adipose tissue and circulating inflammatory cell can contribute or act as co-factors that triggers or maintain hepatic injury. In a study conducted in our centre to study the rela‐ tionship between endotoxemia and NAFLD, we found higher levels of LBP (Lipopolysac‐ charide-binding protein) in patients with NASH when compared to patients with simple steatosis [93]. The LBP increase correlates with the level of tumor necrosis factor alfa (TNFalfa) which is overexpressed in patient with NASH and significant fibrosis. [94] Detailed in‐

resistance. So this advance opens new pathogenic pathways in NAFLD.

(AUROC) ranging from 0,83 to 0,91 [83].

contribute to hepatic fat deposition.

group [92].

These new techniquesinclude genomics, metabolomics and proteomics.

Genomics. Gene expression studies provide an insight into possible mechanism of patho‐ genesis as well as potential biomarkers of disease. One method for studying gene expression is micro-arrays of DNA. A study using this test found 34 gens with different expression in NASH vs controls, these genes where implicated in lipid metabolism and extracellular ma‐ trix remodelling [73]. Another study compared gene expression in NASH-related cirrhosis with other causes of cirrhosis. In NASH cirrhosis group genes involved in anti-oxidant stress were underexpressed, along with genes involved in fatty and glucose metabolism [74]. In our centre we used micro-arrays to study gene expression in obese patients with NAFLD [75]. Obese patients with NASH without fibrosis show an overexpression of proin‐ flammatory and proapoptotic genes; and those with fibrosis show an overexpression of fi‐ brogenic genes, including the leptin receptor Ob-Rb.

Most recent genomic tests, such GWAS (Genome-wide association studies) provide a method for evaluating a large number of single nucleotide polymorphisms (SNP) with the same experi‐ ment. A study performed with a GWAS study found a SNP in farnesyldiphosfatasefarnesyl‐ transferase 1 (FDFT1) which was associated with different histological parameters (a SNP with portal inflammation and another different SNP with fibrosis stage) and the total NAFLD activ‐ ity score [76]. In an earlier GWAS study [77], an SNP in PNPA3 (adiponutrin/patatin-like phos‐ pholipase-3) was strongly associated with both hepatic fat content and hepatic inflammation. The prevalence of this mutation may explain the difference in susceptibility to NAFLD seen in different ethnicities [77]. A subsequent study [78], confirmed the relationship between this SNP and histological score, the no association with metabolic syndrome.

These studies are incredibly interesting and they could help the development of new nonin‐ vasive markers, nevertheless all of them share limitations, mainly concerning to sample size. It is easily understandable given the fact that they use expensive and complex tools [51].

Proteomics. Proteomic tools look specifically at protein expression patterns and profiles. There are several approaches to proteomic studies depending on the used tool. These tools are complex; they are based on diverse types of mass-spectrometry. For more detailed infor‐ mation refer to [79, 80]. The different proteomic platforms support the use of either liver tis‐ sue or blood. This platform allows identifying, quantifying and comparing proteins in the study groups of interest. That novel approach has been applied for the study of NAFLD [81-84]. These studies have found several proteins related to disease progression: alfa and beta-hemoglobin [84], lumican and FABP1 (fatty acid binding protein-1) [82]; and finally fi‐ brinogen beta-chain, retinol binding protein-4, serum amiloyd p-component, lumican, trans‐ grelin-2 and CD5-like antigen, in 6-panel model and complement component 7, transgrelin-2 and insulin grow factor acid labile subunit, in a 3-panel model. These panels performed in the diagnosis of the diverse NAFLD stages get an area under the receiver operator curve (AUROC) ranging from 0,83 to 0,91 [83].

**10. New diagnostic platforms in nafld**

178 Liver Biopsy – Indications, Procedures, Results

brogenic genes, including the leptin receptor Ob-Rb.

NAFLD is a disease with wide spectrum: from steatosis through inflammation to fibrosis and finally cirrhosis and hepatocellular carcinoma, even in absence of cirrhosis [72]. The strongest predictor of fibrosis progression in NAFLD is steatohepatitis. The most important features are hepatocellular degeneration (ballooning) and inflammatory cell infiltration.

Genomics. Gene expression studies provide an insight into possible mechanism of patho‐ genesis as well as potential biomarkers of disease. One method for studying gene expression is micro-arrays of DNA. A study using this test found 34 gens with different expression in NASH vs controls, these genes where implicated in lipid metabolism and extracellular ma‐ trix remodelling [73]. Another study compared gene expression in NASH-related cirrhosis with other causes of cirrhosis. In NASH cirrhosis group genes involved in anti-oxidant stress were underexpressed, along with genes involved in fatty and glucose metabolism [74]. In our centre we used micro-arrays to study gene expression in obese patients with NAFLD [75]. Obese patients with NASH without fibrosis show an overexpression of proin‐ flammatory and proapoptotic genes; and those with fibrosis show an overexpression of fi‐

Most recent genomic tests, such GWAS (Genome-wide association studies) provide a method for evaluating a large number of single nucleotide polymorphisms (SNP) with the same experi‐ ment. A study performed with a GWAS study found a SNP in farnesyldiphosfatasefarnesyl‐ transferase 1 (FDFT1) which was associated with different histological parameters (a SNP with portal inflammation and another different SNP with fibrosis stage) and the total NAFLD activ‐ ity score [76]. In an earlier GWAS study [77], an SNP in PNPA3 (adiponutrin/patatin-like phos‐ pholipase-3) was strongly associated with both hepatic fat content and hepatic inflammation. The prevalence of this mutation may explain the difference in susceptibility to NAFLD seen in different ethnicities [77]. A subsequent study [78], confirmed the relationship between this

These studies are incredibly interesting and they could help the development of new nonin‐ vasive markers, nevertheless all of them share limitations, mainly concerning to sample size. It is easily understandable given the fact that they use expensive and complex tools [51].

Proteomics. Proteomic tools look specifically at protein expression patterns and profiles. There are several approaches to proteomic studies depending on the used tool. These tools are complex; they are based on diverse types of mass-spectrometry. For more detailed infor‐ mation refer to [79, 80]. The different proteomic platforms support the use of either liver tis‐ sue or blood. This platform allows identifying, quantifying and comparing proteins in the study groups of interest. That novel approach has been applied for the study of NAFLD [81-84]. These studies have found several proteins related to disease progression: alfa and beta-hemoglobin [84], lumican and FABP1 (fatty acid binding protein-1) [82]; and finally fi‐ brinogen beta-chain, retinol binding protein-4, serum amiloyd p-component, lumican, trans‐ grelin-2 and CD5-like antigen, in 6-panel model and complement component 7, transgrelin-2

These new techniquesinclude genomics, metabolomics and proteomics.

SNP and histological score, the no association with metabolic syndrome.

Metabolomics. In the natural history of NAFLD the progression to hepatic fibrosis occurs only in 10 to 25% of cases, leading to cirrhosis, end-stage liver disease or hepatocellular car‐ cinoma. The strongest predictor of fibrotic progression, apart from pre-existing fibrosis, is steatohepatisis. A two-hit model has been proposed as an explanation for why some pa‐ tients progress to NASH. In a first step, because of insulin resistance, adipose tissue has en‐ hanced triglyceride lipolysis, which leads to increased serum free fatty acids, and impaired hepatic triglyceride export. In this model, hepaticsteatosis (hit 1) exposes the liver parenchy‐ ma to environmental and extracellular hepatic insults (hit 2), leading to inflammation, stea‐ tonecrosis and fibrosis. Impaired mitochondrial oxidation and lipid export may also contribute to hepatic fat deposition.

Leptin system is also implicated, and its receptor expression is related to fibrosis degree [85].

As it was explained in the introduction, irisin is a newly identified hormone. Irisin is pro‐ duced in muscle cells induced in exercise [86-88]. Irisin activates changes in adipose tissue, and make its change from white adipose tissue to brown adipose tissue, and this causes a significant increase in total body energy expenditure and resistance to obesity-linked insulin resistance. So this advance opens new pathogenic pathways in NAFLD.

Inflammation is considered to be the central clue for the progression of NAFLD, the origins and components are considered in this review [89]. Hepatocytes injured by toxic lipid mole‐ cules play a central role in the recruitment of innate immunity involving Toll-like receptors (TLR), Kuppfer cells, lymphocytes and neutrophils and possibly inflammasome. On this way, a study was carried to determine the lipidomic signature in NAFLD [90]. Using proteo‐ mic tools (mass spectrometry) the investigators found metabolites from nonenzymatic oxi‐ dation product of arachidonic acid and from impaired peroxisomal polyunsaturated fatty acid (PUFA). This study links to another, where investigators characterize metabolic profile to distinguish steatosis and NASH [91], they also found arachidonic acid, among other sub‐ stances, relation to NASH and fibrosis. Metabolomics analysis was performed to NAFLD patients showing a lower concentrations of glutathione, an antioxidant substance, in this group [92].

The key pro-inflammatory signalling pathways in NASH are nuclear factor-kappa B (NFkB) and c-Jun N-terminal kinase (JNK). It could be possible that inflammation in NASH could originate outside the liver. Gut microbiota, the related Kupffer/TLR response, in‐ flamed adipose tissue and circulating inflammatory cell can contribute or act as co-factors that triggers or maintain hepatic injury. In a study conducted in our centre to study the rela‐ tionship between endotoxemia and NAFLD, we found higher levels of LBP (Lipopolysac‐ charide-binding protein) in patients with NASH when compared to patients with simple steatosis [93]. The LBP increase correlates with the level of tumor necrosis factor alfa (TNFalfa) which is overexpressed in patient with NASH and significant fibrosis. [94] Detailed in‐ formation in pathophysiology of NAFLD and NASH is not the aim of this paper, if you are interested refer to this review [89].

[6] Vos MB, Welsh J. Prevalence of suspected NAFLD is increasing among U.S. adoles‐ cents. Digestive Disease Week 2012; May 19-22, 2012; San Diego, California. Abstract

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