Causes of secondary fat accumulation.
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder of our times. The spectrum of this disease goes from steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. NAFLD can appear in the context of many conditions. Probably, NAFLD could be a component of metabolic syndrome, with its complete phenotypic expression: insulin resistance, obesity, type 2 diabetes, hypertension, hypercholesterolemia, and hypertriglyceridemia.
The pathogenesis involves insulin resistance, hepatic fat deposition, increased oxidant stress, apoptosis, inflammation and fibrosis. At present day, a new hormone has been discovered. Muscle cells products this new hormone, called irisin. Irisin can induce changes in adipose tissue.
Diagnosis of NAFLD cannot be performed with a single test and it should be one of exclusion, as well.
Nowadays, there is not a single therapeutic intervention. The focus of management should be treatment of the risk factors for NASH (insulin resistance, obesity…). Principal methods used for weight management are dietary modifications and life style changes. Then, pharmacotherapy may include insulin sensitizers, cholesterol-lowering agents, anti-obesity and anti-oxidant agents. Morbid obese patients may benefit from surgical weight loss, reducing the progression of NASH.
NAFLD definition  requires that there is evidence of hepatic steatosis, either by imaging or by histology and there are no causes for secondary hepatic fat accumulation (Table 1). NAFLD is usually associated with metabolic risk factors such as metabolic syndrome, obesity, diabetes mellitus, and dyslipidaemia.
|Excessive alcohol consumption.|
Hepatitis C (genotype 3)
Medication (amiodarone, methotrexate, tamoxifen, corticosteroids)
Medications (valproate, anti-retroviral medicines)
Acutte fatty liver of pregnancy
Inborn errors of metabolism (LCAT deficiency, cholesterol ester storage disease, Wolman disease)
NAFLD includes a constellation of histological findings that goes from steatosis, to necroinflammation, called NASH and progression to advanced fibrosis and cirrhosis.
NAFLD is becoming the leading cause of liver disease. One of the causes is the increasing of obesity.
The prevalence of NAFLD is increasing. Recent studies presented in the Digestive Diseases Week 2012 summarizes this increased prevalence over the last 20 years [5, 6]. Investigators report an increasing in obesity. This increase is followed by a rising in steatosis and NASH, the presence of steatosis among obese people has increased from 23% in the 80s, 43% in the 90s and finally to 60% nowadays . Even in non-obese patients, the prevalence of steatosis increased from 12%, to 27% and 36%, respectively .
In children/adolescents, over the last 20 years, obesity has increased from 11% to 21%, suspected NAFLD from 4% to 10, and the prevalence of altered aminotransferases among obese adolescents has increased from 17% to 37% .
4. NAFLD and liver biopsy
Liver biopsy remains the gold standard for characterizing liver histology in patients with NAFLD. However, it is expensive and carries some morbidity and very rare mortality risk. Thus, it should be performed in those who would benefit the most from diagnostic, therapeutic guidance, and prognostic perspectives.
The last guideline for NAFLD management recommends liver biopsy : in patients who are at risk to have steatohepatitis and advance fibrosis; theses patients could be identified by the presence of metabolic syndrome and NAFLD fibrosis score; and a liver biopsy should be considered in patients in whom other etiologies are suspected and cannot be excluded without a liver biopsy.
Liver biopsy allows confirming the diagnosis, evaluation and semiquantitation of necroinflammatory lesions and fibrosis.
On the other hand, liver biopsy suffers from challenges. An adequate biopsy represents only 1/50000-1/65000 of the organ. Sampled area should be carefully chosen and sample length must be enough, a least 15mm. This size can reduce sample error. Finally, experienced pathologist is important to haver a greater yield of findings.
5. Histology of NAFLD
NAFLD represents a histopathologic spectrum ranging from steatosis alone, to necroinflammation, summarized as NASH; and progression to advanced fibrosis and cirrhosis.
The histologic characterization of NAFLD and NASH may include description of steatosis and cell injury in addition to inflammation and fibrosis. Kleiner and Brunt  propose categorizing the histologic changes when studying NAFLD as follows in table 2.
The main histological characteristic of NAFLD is the accumulation of fat in the form of triglyicerides within hepatocytes, lesion termed steatosis (Figure 1 and 2); this term is defined by the guideline  as NAFL – non-alcoholic fatty liver, where the risk of progression to cirrhosis and liver failure is minimal. The presence of >5% steatoic hepatocytes in a liver biopsy is accepted as the minimum criterion for thehistological diagnosis of NAFLD .
|No significant evidence of fatty liver disease.||Insufficientsteatosis for diagnosis of steatosis, without other changes (ballooning, fibrosis) that would suggest steatohepatitis.|
Steatosis with inflammation.
Steatosis with nonspecific fibrosis
|Steatosis without specific changes to suggest a form of steatohepatitis. This category may include spotty lobular inflammation and/or mild degrees of fibrosis of uncertain significance.|
- zone 3 borderline steatohepatitis
|Form of steatoshepatitis most common in adults; defined as a zone 3 centered injury pattern that includes steatosis, inflammation, ballooning injury, (often with Mallory-Denk bodies) with or without fibrosis. Borderline steatohepatitsis has some, but not all ofthe features that would allow a diagnosis of steatohepatitis.|
|Zone 1, borderline pattern||Form of steatohepatitis that occurs mainly in young children, characterized by zone 1-centered (portal inflammation, portal-based fibrosis, zone 1 steatosis, ballooning injury in zone 1 if present).|
|Cryptogenetic fibrosis/ cirrhosis||Presence of fibrosis (usually advanced) or cirrhosis, with little ton o steatosis and no changes (ballooning, Mallory-Denk bodies) that would suggest borderline or definite steatohepatitis. Other explanations for fibrosis (besides steatohepatitis) should be considered.|
Steatosis in NAFLD is usually macrovesicular, which refers to hepatocytes with single large intracytoplasmatic fat droplet or smaller well defined droplets displacing the nucleus to the cell periphery. This macrovesicularsteatosis is usually present in a zone 3 or panacinar distribution; it differs from zone 1 steatosis that is a common distribution in chronic hepatitis C. Azonal steatosis is most often seen in biopsies with advanced fibrosis .
The extent of steatosis can be evaluated and classified semi-quantitative. The most reproducible method follows the acinararchiqueture dividing the liver parenchyma in thirds and assessing percentage involvement bay steatoic hepatocytes  – table 3.
|Mild||0 – 33%|
|Moderate||33 – 66%|
NASH, under this concept is the histology pattern of NAFLD, which is at risk of developing advance fibrosis. The minimal criteria for the histopathological diagnosis of adult NASH include steatosis, hepatocyte injury, usually in form of ballooning, and lobular inflammation, typically localized in acinar zone 3 [10, 11].
The key feature for the diagnosis of NASH is the ballooning injury (Figures 3 and 4), and it is considered a marker of apoptosis . This type of cell injury is characterized by a cell that becomes enlarged and the cytoplasm becomes irregularly clumped with optically clear, nonvesiculated areas. Ballooned cells are seen most frequently in zone 3 near the hepatic veins, and lose this localization, becoming portal inflammation more prominent when the disease progresses and in severe cases. Immunostaining of hepatocyte keratins 8 and 18 might help to identify ballooned hepatocytes . Ballooned degeneration is difficult to diagnose even by trained pathologist, for that reason it can show significant inter-observer variation .
Ballooning degeneration is associated with an increased liver-related mortality .
Mallory-Denk Bodies (MDB), also known as Mallory bodies, are eosinophilic, ropey cytoplasmatic inclusion bodies in the hepatocyte of patients with chronic liver disease. This type of lesion contains abnormal cytokeratin 8 and 18 filaments that have been ubiquinated.
Mallory bodies have an importance in disease progression and it is suggested a possible prognostic role in steatohepatitis . In a recent study, the presence of MBD was significantly associated with liver-related mortality .
Both ballooning degeneration and MDB can trigger the development of apoptosis. Apoptotic (acidophil) bodies are common in NASH. They can be identified as rounded, eosinophilic cytoplasmic fragments, which appear to be free within the sinusoids or surrounded by Kupffer or other inflammatory cells. Apoptosis has been validated as an accurate marker for diagnosis of NASH based on immunochemistry in liver tissue .
Inflammatory infiltrates (Figure 5) can be seen in the hepatic acini/lobules or the portal tract. Lobular inflammation is usually mild, consists of a mixed inflammatory cell infiltrate, composed of lymphocytes, some eosinophils, and a few neutrophils. Polymorphs can be observed around ballooned hepatocytes that are called “satellitosis” (Figure 6). Kuppfer cells aggregates as lobular microgranulomas and lipogranulomas may appear 
Hematoxylin-eosin stain.Portal chronic mononuclear cell inflammation in adult NASH is common and mild. When portal inflammation is greater than lobular other aetiologies should be ruled out, such as chronic hepatitis C . On the other side, a greater portal inflammation than lobular inflammation can be seen in successfully treated patients . In a large database of liver biopsies from the NASH Clinical Research Network, including adults and children, portal chronic inflammation was associated with clinical and histologic features of severity and advance disease .
Vascular alterations in NAFLD. Recent paper has focused the study of NASH in microvessels of the liver . This work has found an intraacinar branch of the hepatic artery in the perivenular region in active steatohepatitis. This finding is important because it can lead to confusion for a portal tract resulting in an equivocal diagnosis. Likewise, the presence of this vessel correlates with higher stage of fibrosis.
Fibrosis in adult NASH usually starts in acinar zone 3 and has characteristic “chicken wire” pattern due to deposition of collagen an other extracellular matrix fibres along the sinusoids of zone 3 and around the hepatocytes (Figure 7 and 8). Portal fibrosis has been reported in cases of morbid obesity-related NASH and in pediatric NASH. Fibrosis predicts clinical outcomes in NASH . there was noter from this study that the progression of the fibrosis is accompanied of steatosis reduction. Approximately 37% to 41% of patients with NAFLD have fibrosis progression over 3 to 10 years [22, 23]. The higher rates of fibrosis progression were related to: body mass index, diabetes and low initial fibrosis . When periportal fibrosis was not present, there was a 100% of negative predictive value in predicting liver-related outcomes . Steatosis, inflammation, ballooning and Mallory hyaline were not associated with liver-related mortality after adjusting for the presence of fibrosis . The inclusion of fibrosis explains why the recent classifications for NASH used by Younossi  and Matteoni , independently correlated with liver-related mortality. This observation shows the importance of fibrosis in NAFLD, patients with NASH and fibrosis portends a higher risk of death .
Other histological lesions that may be seen in NASH include megamitochondria, glycogenated nuclei and iron deposition.
Megamitochondria (giant mitochondria) are round or needle-shaped, eosinophilic, intracytoplasmatic inclusions more commonly observed in hepatocytes with microvesicularsteatosis. This abnormal mitochondria is a result of injury from lipid peroxidation or represent an adaptive change . Glycogenated nuclei are vacuolated nuclei usually observed in periportal hepatocytes. Their presence is more frequent in non-alcoholic etiology and it is rare in alcoholic injury .
Finally, hepatic siderosis might be seen in NAFLD. One study of 293 liver biopsies (34,5% of patients with NAFLD) investigates the relationship between iron deposition and NAFLD . Stainable hepatic iron described three histological patterns: hepatocellular pattern, reticuloendothelial system cell – RES - (mainly Kupffer cell) pattern and mixed. RES pattern was associated with advanced fibrosis and higher histological features of portal inflammation, ballooning and definite NASH .
6. Histologic scoring systems
NAFLD histologic criteria requires an accumulation of more than 5% of fat deposition, mainly in form of triglycerides. NAFLD was first described by Ludwig and colleagues , and since then several systems for grading and staging NAFLD have been proposed.
In 1999, Matteoni and colleagues characterized histologic subtypes that correlate with clinical outcomes  – table 4. In 2005, NASH Clinical Research Network developed NAFLD activity score (NAS) . This score comprises four features evaluated semi-quantitatively: steatosis, lobular inflammation, hepatocellular ballooning and fibrosis. Fibrosis was classified separately – table 5. When NAS is >5 sensitivity and specificity for definite NASH were 0,75 and 0,83, respectively. Finally, a recent classification for NAFLD has been proposed by Younossi and colleagues . This classification includes the evaluation of these histologic features: steatosis with centrilobular ballooning, and/or Mallory-Denk bodies of fibrosis – see table 6.
|Type 1||Simple steatosis alone||No NASH|
|Type 2||Statosis + lobular inflammation only||No NASH|
|Type 3||Steatosis + hepatocellular ballooning||NASH without fibrosis|
|Type 4||Steatosis, ballooning, Mallory bodies or fibrosis||NASH with fibrosis|
|NASH requires a score of ≥ 4 with at least 1 point o ballooning injury.|
|Perisinusoidal zone 3|
|Persinusoidal and portal/periportal||2|
|Simple steatosis alone||No NASH|
|Steatosis + lobular inflammation only||No NASH|
|Steatosis with centrilobular ballooning and/or Mallory-Denk bodies||NASH|
|Any steatosis with centrilobularpericellular/perisinusoidal or briding fibrosis||NASH|
The most important difference between NAS and subtype classifications is that the latters include fibrosis and this provides a better prediction of liver-related mortality in patients with NAFLD .
7. NAFLD in special populations
In some cases in a study with a few number of patients treated with an insulin sensitizer, histologic evaluation of post-treatment liver biopsy showed that increased portal inflammation is a feature related to resolution of NASH, and it is associated to a change in the quality of zone 3 perisinusoidal fibrosis from dense to delicate .
|The Adult Treatment Panel III clinical definition of the metabolic syndrome:|
- Requires the presence of three or more of the following features:
|Waist circumference greater than 102 cm in men or greater than 88 cm in women.|
Triglyceride level 150mg/dl or greater.
High-density lipoprotein (HDL) cholesterol level less than 40 mg/dl in men and less than 50 mg/dl in women.
Systolic blood pressure 130 mmHg or greater or diastolic pressure 85 mmHg or greater.
Fasting plasma glucose level 110 mg/dl or greater.
In the near future, we will have to get used to new types of treatment, for example, “metabolic surgery”, which might be performed to non-morbid obese patients with diabetes, and to the new changes in hepatic parenchyma following endoscopic procedures performed to treat obesity.
Portal fibrosis is common in pediatric NAFLD and may evolve to periportal fibrosis and bridging fibrosis in some patients, whereas progression to cirrhosis is observed in rare cases .
It is not clear, that Type 2 NASH, described as pediatric NASH, is an entity by itself or it is another stage of the spectrum of NASH which could be a predictor of those who have a severe disease .
8. Imaging tecnology in NAFLD
Ultrasonography (US), computed tomography (CT), and magnetic resonance (MR) can identify liver steatosis but not steatohepatitis, nevertheless they provide anatomical and morphological information. The sensitivity of these imaging methods is optimal for steatosis over 33%. When advance liver disease, radiology techniques can provide indirect signs of cirrhosis, such as portal hypertension, or may be useful for the screening and diagnosis of hepatocellular carcinoma (HCC). Imaging technique may help to differentiate diffuse from focal form of steatosis. Hepatic fatty infiltration can present as focal steatosis (a focal area of steatosis in an otherwise normal liver) or as focal fatty sparing (fatty change with sparing of certain areas) .
|Liver echotexture||Liver parenchyma is homogeneous and no difference in contrast between liver and kidney||Slight increase in echo pattern||Intermediate||Gross discrepancy of the increased hepatic to renal cortical echogenicity|
|Echo penetration an visibility of diaphragm||Liver structure is clearly defined from the surface to diaphragm. The outline of the diaphragm is clearly visualized.||Mild attenuation of sound beam through the liver||Intermediate||Marked attenuation of sound beam through the liver, the diaphragm is not visualized.|
|Clarity of liver blood vessel structure||Vessel wall and lumen of vessel can clearly visualized||Slight decrease definition of portal venule walls||Intermediate||Only the main portal walls can be visualized with absence of all smaller portal venule walls|
Magnetic Resonance can detect steatosis by exploiting the difference of resonance frequencies between water and fat proton signals. The sensitivity and specificity of MRI in detecting as low as 5% of liver fat infiltration are 85% and 100%, respectively . The detection of the fatty liver can be seen in “white/bright” when applying in-phase T1 images and “black” when applying out-of-phase images, compared to the signal intensity of the spleen and paraespinal muscles. Another technic of MR imaging with fat saturation may quantify more accurately liver fat infiltration, especially in patients who have fibrosis.
MR spectroscopy can reliably quantify even minimal steatosis, as low as 0,5% . In has been based on the ubiquitous protons hydrogen and phosphorus , and more than 5% of fat content on MR spectroscopy indicates presence of steatosis . Its routine application is limited by cost and lack of availability, and it remains a research tool.
(60-95% / 84-100%)
|Noninvasive, widely aviable, low cost, repetition,|
Useful for screening
Qualitative assessment of steatosis.
Only accurate when moderate-to-severe fat infiltration.
(50-86% / 75-87%)
|Noninvasive, semiquantitative assessment of fat content.|
Detects focal or diffuse infiltration.
Iron overload, copper and fibrous tissue could be confounding factors.
Not sensitive for mild-to-moderate amounts of steatosis.
|Magnetic resonance imaging|
(85% / 100%)
|Noninvasive, semiquantitative, and no radiation exposure.|
Detects focal or diffuse infiltration.
|Limitation in patients with iron overload.|
Not suitable for patients with claustrophobia or implantable devices.
|MR spectroscopy||Noninvasive, reproducible, accurate quantification.||High cost not widely available. Long time taking images.|
US, CT and MR are insensitive in differentiating hepatic steatosis from NASH, and they cannot be used to stage fibrosis [43, 48]. But in the near future, a novel method based on MRI imaging and a new software will be able to stage fibrosis and to distinguish NASH from no-NASH. Professor Romero-Gomez conducts this study and it will be soon published.
Table 9 summarizes advantages and disadvantages of these radiologic methods.
9. Non-invasive assesment in NAFLD
Liver biopsy remains a useful tool to confirm the diagnosis and exclude other disease or helps to discover concomitant chronic liver disease. It provides prognostic information by staging and grading this disease. At present non-invasive diagnostic markers could provide a new tool for differentiating fatty liver from NASH as well as for grading /staging NAFLD.
The investigation of these new diagnostic methods comes from the well known drawbacks of liver biopsy. These include sampling error, inadequate biopsy size, variability in pathologist interpretation, cost and associated morbidity (complications 0,3%, mortality rate 0,01%).
An ideal non-invasive test should be simple, reproducible, readily available, less expensive than liver biopsy, able to predict the full spectrum of liver fibrosis stages, and reflect changes occurring with therapy .
Some reviews provide an overview of the role of non-invasive test in NAFLD [48, 50, 51]. We will try to present many of these scores through a table (number 10) to summarize their characteristics. AUROC is a numerical data that assess the performance of a scoring system. AUROC value greater than 0,8 indicate good diagnostic performance. The closer the value to 1, the better performing the scoring system.
|NONINVASIVE DIAGNOSIS OF STEATOSIS|
|SteatoTest ||6 components of FibroTest-ActiTest, BMI, cholesterol, triglycerides, glucose||0,3|
|Fatty liver Index (FLI) ||BMI, waist circumference, triglycerides, GGT||<30|
|NONINVASIVE ASSESSMENT IN NASH|
|Palekar ||HA >55 mcg/l, age >50 years, female gender, AST >45 UI/ml, AAR >80.||≥3||0,763||73,7||65,7||68,2||71,4|
|CK-18 ||CK-18 plasma (apoptosis marker)||250 U/l||0,83||75||81|
|oxNASH ||Detection of lipid peroxidation products by chromatography-mass spectrometric||0,74-0,83||63-81||84-97|
|SHIMADA ||Serum adiponectin, type 4 collagen 7s level, HOMA-IR||94||74||94||74|
|NASH Diagnostics *||CK-18, cleaved CK-18, adiponectin, resistin||0,3825||0,73||71,4||72,7||83,3||57,1|
|NashTest ||Age, gender, BMI, triglycerides, cholesterol, alfa-2macroglobulin, GGT, haptoglobin, apolipoprotein-A1, total bilirubin||0,79||29||98||91||71|
|NONINVASIVE MANAGEMENT OF FIBROSIS IN NAFLD|
|NAFLD fibrosis score ||Age, BMI, IFG/diabetes, AAR, platelet, albumin||≤1,455|
|Pediatric NAFLD fibrosis index ||Age, waist circumference and triglycerides.||≥9: rule in liver fibrosis.||0,85||98,5||44,5|
|<3:rule out fibrosis||75,4**||75**|
|**Pre-test probability: 69%|
|BARD score ||BMI ≥28 =1, AAR ≥0,8 =2, diabetes=1,||≥2: advanced fibrosis||0,81||96|
|FIB-4 ||Age, AST, platelet, ALT||<1,3|
|APRI ||AST, platelet||0,98||Advanced fibrosis: 0,85||75||86||54||93|
|ELF ||HA, TIMP1, P3NP.||0,3576||For severe fibrosis 0,90.||80||90||71||94|
|-0,1068||For moderate fibrosis 0,82||70||80||70||80|
|ELF: -0,2070.||No fibrosis 0,76||61||80||81||79|
|BAAT ||Age ≥50years, BMI ≥28kg/m2 =1, triglycerides ≥7mmol/L=1, ALT ≥2N||0-1, for septal fibrosis||0,84||100||47||100|
|Fibrotest ||0,30||F0-1 VS F2-4: 075-085.|
F0-2 VS F3-4: 0,81-0,92
|Fibrometer ||Glucose, AST, platelets, ferritin, ALT, weight, age.||<0,611||F0-1 vs F2-4: 0,936-0,952|
|ULTRASOUND BASED TECHNIQUES IN FIBROSIS DETECTION IN NAFLD|
|Fibroscan ||Transient elastography||9,9 kPa|
|F ≥3: 0,99|
|ARFI ||4,24 kPa||F0-2 vsF3-4: 0,90||90||90|
|ARFI ||1,77 m/sec|
|F ≥3: 0,973|
|MAGNETIC RESONANCE IMAGING BASED TECHNIQUES|
|MR elastography||Discriminating NASH from simple steatosis (SS)||2,74 kPa||SS vs NASH: 0,93||94||73||85||89|
NASH no fibrosis.
APRI and FIB-4 have been evaluated in obese children and they might be useful in this special population . Pediatric NAFLD scores is a noninvasive model evaluated in obese children, and it may help clinicians to predict liver fibrosis but external validation is needed .
In the future, new serologic markers, such as CD36, will help to differentiate more accurately between NAFLD stages, we would be able to distinguish simple steatosis from NASH.
Although clinical and laboratory models may be useful in identifying a group of patients at a low risk of advance fibrosis and liver biopsy might be avoided, they are not enough for staging and prognostic purposes if patients are at risk of advance fibrosis . NAFLD Practice Guideline of 2012 recommends NAFLD fibrosis score to identify patients with higher likelihood of having bridging fibrosis and/or cirrhosis .
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 . The strongest predictor of fibrosis progression in NAFLD is steatohepatitis. The most important features are hepatocellular degeneration (ballooning) and inflammatory cell infiltration.
These new techniquesinclude genomics, metabolomics and proteomics.
Genomics. Gene expression studies provide an insight into possible mechanism of pathogenesis 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 matrix remodelling . 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 . In our centre we used micro-arrays to study gene expression in obese patients with NAFLD . Obese patients with NASH without fibrosis show an overexpression of proinflammatory and proapoptotic genes; and those with fibrosis show an overexpression of fibrogenic 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 experiment. A study performed with a GWAS study found a SNP in farnesyldiphosfatasefarnesyltransferase 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 activity score . In an earlier GWAS study , an SNP in PNPA3 (adiponutrin/patatin-like phospholipase-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 . A subsequent study , 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 noninvasive 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 .
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 information refer to [79, 80]. The different proteomic platforms support the use of either liver tissue 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 , lumican and FABP1 (fatty acid binding protein-1) ; and finally fibrinogen beta-chain, retinol binding protein-4, serum amiloyd p-component, lumican, transgrelin-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 .
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 carcinoma. 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 patients progress to NASH. In a first step, because of insulin resistance, adipose tissue has enhanced triglyceride lipolysis, which leads to increased serum free fatty acids, and impaired hepatic triglyceride export. In this model, hepaticsteatosis (hit 1) exposes the liver parenchyma to environmental and extracellular hepatic insults (hit 2), leading to inflammation, steatonecrosis 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 .
As it was explained in the introduction, irisin is a newly identified hormone. Irisin is produced 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 . Hepatocytes injured by toxic lipid molecules 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 . Using proteomic tools (mass spectrometry) the investigators found metabolites from nonenzymatic oxidation 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 , they also found arachidonic acid, among other substances, relation to NASH and fibrosis. Metabolomics analysis was performed to NAFLD patients showing a lower concentrations of glutathione, an antioxidant substance, in this group .
The key pro-inflammatory signalling pathways in NASH are nuclear factor-kappa B (NF-kB) 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, inflamed 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 relationship between endotoxemia and NAFLD, we found higher levels of LBP (Lipopolysaccharide-binding protein) in patients with NASH when compared to patients with simple steatosis . The LBP increase correlates with the level of tumor necrosis factor alfa (TNF-alfa) which is overexpressed in patient with NASH and significant fibrosis.  Detailed information in pathophysiology of NAFLD and NASH is not the aim of this paper, if you are interested refer to this review .
NAFLD is an emerging problem. The study of pathology is ever evolving which is allowing the development of new therapeutic targets, and the emergence of new diagnostic techniques allow better identification of patients who will benefit from new treatments.