**3. Diagnosis of steatohepatitis**

A key element in the diagnosis of NAFLD is the differentiation of NASH from NAFL and the staging of the liver fibrosis, given that patients with NASH and advanced fibrosis are those at the greatest risk of developing hepatic complications and cardiovascular disease [54–56].

#### **3.1. Liver biopsy**

As it was mentioned earlier, the chosen method to evaluate the grade of histological lesion is still the liver biopsy. However, liver biopsy has well-known limitations and cannot be proposed for all patients, given the high prevalence of NAFLD worldwide. Liver biopsy is invasive and is not without complications. Besides, there are other drawbacks: (1) sampling error, since a typical liver biopsy samples only 1/50,000 of all liver tissues, and histological lesions of NASH are unevenly distributed throughout the liver parenchyma [57]; (2) inter- and intra-observer variability, as observed by Gawrieh et al. although there was a high agreement ratio in the assessment of steatosis grading and fibrosis staging between pathologists, the agreement was suboptimal for lobular inflammation and hepatocellular ballooning [58]; and (3) the existence of different criteria for the definition of NASH. The Non-alcoholic Steatohepatitis Clinical Research Network (NASH-CRN) proposed the system termed the NAS scoring system in order to classify NAFLD according to severities of fatty change, inflammation and hepatocellular ballooning [3]. NAS is markedly reproducible and is useful for assessing therapeutic effects in Clinical trials, but it is incapable of diagnosing NASH in patients with burned-out NASH, in whom fatty changes and inflammatory cell infiltration resolving in fibrosis have progressed [59].

Given these limitations, non-invasive methods have been developed for the diagnosis of NASH and fibrosis as a first option to examine NAFLD patients and to help determine which require a liver biopsy. The ideal test should be economical, reproducible and capable of diagnosing the whole spectrum of lesions, including within NAFLD, and even reflecting the changes produced on initiating specific treatment. Nowadays, we do not have a test available that has these characteristics, so these non-invasive methods are based on diverse complementary approaches: clinical factors, genetics, serological markers, image tests and transient elastography [60, 61].

#### **3.2. Risk factors associated with non-alcoholic steatohepatitis and progressive disease**

according to the grade of activity (inflammation and hepatocyte injury) and the stage of fibrosis, the best predictors of the disease progression [53, 54]. The advantages and, especially, the

From what has been mentioned so far, we can specify a series of characteristics that indicate a patient with NAFLD: (1) radiological evidence of steatosis or CAP >248 dB/m ± Abnormal liver blood test, (2) the presence of insulin resistance or another component of the metabolic syndrome, (3) consumption of alcohol of <30 g/d in men and <20 g/d in women and (4) exclusion from other causes of chronic liver disease (viral hepatitis, cholestatic diseases, autoimmune hepatitis, hemochromatosis, α1 antitrypsin deficiency, Wilson's disease, drug-induced liver injury and celiac disease) [24, 25]. Once the initial diagnosis of NAFLD has been made, our next step is to evaluate the stage of disease and the necessity of carrying out a liver biopsy.

A key element in the diagnosis of NAFLD is the differentiation of NASH from NAFL and the staging of the liver fibrosis, given that patients with NASH and advanced fibrosis are those at the greatest risk of developing hepatic complications and cardiovascular disease [54–56].

As it was mentioned earlier, the chosen method to evaluate the grade of histological lesion is still the liver biopsy. However, liver biopsy has well-known limitations and cannot be proposed for all patients, given the high prevalence of NAFLD worldwide. Liver biopsy is invasive and is not without complications. Besides, there are other drawbacks: (1) sampling error, since a typical liver biopsy samples only 1/50,000 of all liver tissues, and histological lesions of NASH are unevenly distributed throughout the liver parenchyma [57]; (2) inter- and intra-observer variability, as observed by Gawrieh et al. although there was a high agreement ratio in the assessment of steatosis grading and fibrosis staging between pathologists, the agreement was suboptimal for lobular inflammation and hepatocellular ballooning [58]; and (3) the existence of different criteria for the definition of NASH. The Non-alcoholic Steatohepatitis Clinical Research Network (NASH-CRN) proposed the system termed the NAS scoring system in order to classify NAFLD according to severities of fatty change, inflammation and hepatocellular ballooning [3]. NAS is markedly reproducible and is useful for assessing therapeutic effects in Clinical trials, but it is incapable of diagnosing NASH in patients with burned-out NASH, in whom fatty changes and inflammatory cell infiltration

Given these limitations, non-invasive methods have been developed for the diagnosis of NASH and fibrosis as a first option to examine NAFLD patients and to help determine which require a liver biopsy. The ideal test should be economical, reproducible and capable of diagnosing the whole spectrum of lesions, including within NAFLD, and even reflecting the

drawbacks of the liver biopsy are dealt with later.

**2.3. Initial diagnosis of NAFLD**

8 Liver Research and Clinical Management

**3. Diagnosis of steatohepatitis**

resolving in fibrosis have progressed [59].

**3.1. Liver biopsy**

The best predictor of the evolution of NAFLD is the presence of necroinflammation and fibrosis in liver biopsy; however, there are more and more studies reporting no insignificant rates of progression of simple steatosis [5, 6, 62]. A first study that analyzed patients with NAFLD and paired biopsies demonstrated that even patients with simple steatosis can progress to NASH and advanced fibrosis, especially in the presence of metabolic risk factors [6]. Therefore, there is a series of non-modifiable and modifiable factors in patients associated with a greater risk of development of NASH and more progressive disease.

Various transversal studies have demonstrated that the disease is more severe in older patients, although this phenomenon could be due to the sum of pathogenic factors and a greater duration of the liver disease itself and the associated diseases [8, 63, 64]. In fact, the longitudinal studies have not managed to demonstrate that age is a factor that aggravates the disease per se [65]. The association between sex and fibrosis progression is controversial; two transversal studies show that men and post-menopause women have a greater risk of fibrosis in comparison with pre-menopause women; moreover, precocious menopause is associated with a greater risk of fibrosis [66–69]. Other non-modifiable factors are genetic; dozens of genes with multiple polymorphisms associated with NAFLD have been discovered thanks to genome-wide association studies (*GWAS*), but the number of strongly validated genes in large independent cohorts is limited to two, *patatin-like phospholipase domain containing 3* (PNPLA3) and *transmembrane 6 superfamily member 2* (TM6SF2) [70]. The presence of the single nucleotide polymorphisms (SNPs) rs738409 and rs58542926 of the genes PNPLA3 and TM6SF2, respectively, has been associated with a greater risk of NAFLD, as well as a more severe disease [71–76]. Recently, an SNP of IL28b (also implicated in the response to interferon in chronic hepatitis C patients (VHC)) has been associated with an increment in fibrosis in NAFLD patients [77]. Moreover, in a controlcase study carried out by our working group, we have observed that the presence of the variants rs1421085 and rs1558902 of the fat mass and obesity-associated (FTO) gene confer a high risk of liver inflammation particularly in patients of normal weight with NAFLD (unpublished).

On the other hand, NAFLD tends to be more severe in patients with various factors of the metabolic syndrome, particularly DM2 and obesity. In fact, the reduction in weight and good glycemic control are associated with an improvement in inflammation and liver fibrosis [11, 78, 79]. However, it is known that NASH can also be present in slim subjects although it is unknown whether the natural history of the disease in these slim subjects is similar to that present in obese subjects. As for arterial hypertension, it is arguable whether its treatment improves the histology of NASH [5, 80]. Another factor of the metabolic syndrome, frequent in NAFLD patients, is dyslipidemia, fundamentally in the form of hypertriglyceridemia and atherogenic dyslipidemia [64, 81]; but moreover, a recent study has related the very low-density lipoprotein (VLDL) profile with the NAFLD severity, observing that a decrease in small VLDL particle concentration is associated with more advanced fibrosis [82]. Vitamin D deficiency is also frequent among NAFLD patients, and its levels have been correlated negatively with the severity of steatosis, inflammation and fibrosis [83, 84].

With the aim of improving the diagnostic value of the biomarkers, predictive models have been developed that combine some of these serum biomarkers with analytical parameters and clinical variables, but they have not been adequately validated, so up to now, they are not

Diagnosis and Characterization of Non-Alcoholic Fatty Liver Disease

http://dx.doi.org/10.5772/intechopen.72668

11

Emerging fields in the search for non-invasive biomarkers of NAFLD are proteomics, metabo-

Proteomics provides essential information about the biologically active entity named protein. Thanks to proteomic analysis, key changes in serum protein expression levels have been demonstrated between control subjects and patients with different stages of fatty liver [108]. In the last years, studies about the use of metabolomic to discover biomarkers of progression of NAFLD have received great interest, and not only in this liver disease [109–111]. In fact, a Spanish group has developed the so-called OWL Liver Test that consists in the determination

**Model Variables Sensitivity (%) Specificity (%)**

HAIR score Hypertension, ALT, insulin 80 89

NASHTest Age, gender, weight, 88 50

NASH score PNPLA3 genotype, insulin, 75 74

Nice model Ck18, ALT, metabolic 84 86

NAFLD Diabetes, gender, BMI, 91 47

OxNASH Age, BMI, AST, 13- 81 –

Hydroxyoctadecadienoic

acid, linoleic acid

**Table 2.** Predictive models for non-alcoholic steatohepatitis.

recommendable in clinical practice [102–107] (**Table 2**).

resistance

height, cholesterol, triglycerides, α2 macroglobulin, apolipoprotein A1, haptoglobin, GGT, ALT,

AST, bilirubin

AST

diagnostic triglycerides, M30, M65-

panel M30

syndrome

*3.3.1. Emerging fields*

lomics and epigenetics.

Another possible factor associated with NAFLD progression is the alcohol consumption, a controversial aspect as despite there being a limit above which the consumption of alcohol would define alcoholic steatohepatitis (≥ 60 g/d in women and ≥80 g/d in men), it is not clear that we are confronting a pathology different to NASH given that the pathogeny of these entities presents a great similarity. Moreover, the quantification of alcohol consumption is quite subjective, imprecise, habitually underestimated and not contrasted with objective determinations through biomarkers. At present, there is no agreement on the impact of light-moderate consumption of alcohol on NAFLD given that the literature available about this topic shows contradictory results relate to NAFLD progression [85, 86]. Nevertheless, it seems that all the relevant studies are in favor of a possible benefit from the moderate alcohol consumption, defined as the consumption of up to one drink a day for women and two drinks a day for men [87]. While the consumption of large doses of alcohol leads to the development of insulin resistance and to the infiltration of macrophages into the adipose tissue [88], moderate consumption has been associated with an improvement in the sensitivity to insulin and high concentrations of adiponectin [89–91]. Various studies suggest a significant association between the moderate consumption of alcohol and the less histological severity of NAFLD [92, 93]. As for the development of HCC, only one prospective study exists that evaluates the consumption of alcohol with the risk of HCC in NAFLD, finding a greater risk of this tumor with moderate use of alcohol; however, this study is carried out in patients with cirrhosis due to established NASH, without evaluating the impact of alcohol on patients with a less severe disease [94].

#### **3.3. Non-invasive diagnosis of non-alcoholic steatohepatitis**

There is still no available image test in clinical practice capable of differentiating NAFL from NASH, so various biomarkers have been evaluated to predict the existence of NASH, which are related to pathogenesis pathways of the disease (apoptosis/cellular death, inflammation and oxidative stress).

The most studied serum biomarker associated with the presence of NASH is cytokeratin 18 fragments (CK18-F), a product of the degradation resulting from the apoptosis of hepatocytes mediated by caspase 3 [95], which is measured using enzyme-linked immunosorbent assay (ELISA). Various studies have demonstrated a significant increase in CK18-F in NASH patients in comparison with NAFL patients, and a positive correlation with fibrosis and the histological components of NASH [96, 97]. However, the sensitivity and specificity of this test are quite low, around 60% [98]. Oxidized low-density lipoprotein (LDL), thiobarbituric acid reactive substances (TBARS) and malonaldehyde have been used as markers of oxidative stress, but the results are contradictory [99, 100]. Among the markers of inflammation studied include leptin, protein C reactive, interleukin 6, hyaluronic acid, adiponectin and tumor necrosis factor α (TNFα). All of them have been evaluated in short series or pilot studies in heterogeneous groups of patients with contradictory results [101].

With the aim of improving the diagnostic value of the biomarkers, predictive models have been developed that combine some of these serum biomarkers with analytical parameters and clinical variables, but they have not been adequately validated, so up to now, they are not recommendable in clinical practice [102–107] (**Table 2**).

#### *3.3.1. Emerging fields*

observing that a decrease in small VLDL particle concentration is associated with more advanced fibrosis [82]. Vitamin D deficiency is also frequent among NAFLD patients, and its levels have been correlated negatively with the severity of steatosis, inflammation and

Another possible factor associated with NAFLD progression is the alcohol consumption, a controversial aspect as despite there being a limit above which the consumption of alcohol would define alcoholic steatohepatitis (≥ 60 g/d in women and ≥80 g/d in men), it is not clear that we are confronting a pathology different to NASH given that the pathogeny of these entities presents a great similarity. Moreover, the quantification of alcohol consumption is quite subjective, imprecise, habitually underestimated and not contrasted with objective determinations through biomarkers. At present, there is no agreement on the impact of light-moderate consumption of alcohol on NAFLD given that the literature available about this topic shows contradictory results relate to NAFLD progression [85, 86]. Nevertheless, it seems that all the relevant studies are in favor of a possible benefit from the moderate alcohol consumption, defined as the consumption of up to one drink a day for women and two drinks a day for men [87]. While the consumption of large doses of alcohol leads to the development of insulin resistance and to the infiltration of macrophages into the adipose tissue [88], moderate consumption has been associated with an improvement in the sensitivity to insulin and high concentrations of adiponectin [89–91]. Various studies suggest a significant association between the moderate consumption of alcohol and the less histological severity of NAFLD [92, 93]. As for the development of HCC, only one prospective study exists that evaluates the consumption of alcohol with the risk of HCC in NAFLD, finding a greater risk of this tumor with moderate use of alcohol; however, this study is carried out in patients with cirrhosis due to established NASH, without

evaluating the impact of alcohol on patients with a less severe disease [94].

There is still no available image test in clinical practice capable of differentiating NAFL from NASH, so various biomarkers have been evaluated to predict the existence of NASH, which are related to pathogenesis pathways of the disease (apoptosis/cellular death, inflammation

The most studied serum biomarker associated with the presence of NASH is cytokeratin 18 fragments (CK18-F), a product of the degradation resulting from the apoptosis of hepatocytes mediated by caspase 3 [95], which is measured using enzyme-linked immunosorbent assay (ELISA). Various studies have demonstrated a significant increase in CK18-F in NASH patients in comparison with NAFL patients, and a positive correlation with fibrosis and the histological components of NASH [96, 97]. However, the sensitivity and specificity of this test are quite low, around 60% [98]. Oxidized low-density lipoprotein (LDL), thiobarbituric acid reactive substances (TBARS) and malonaldehyde have been used as markers of oxidative stress, but the results are contradictory [99, 100]. Among the markers of inflammation studied include leptin, protein C reactive, interleukin 6, hyaluronic acid, adiponectin and tumor necrosis factor α (TNFα). All of them have been evaluated in short series or pilot studies in

**3.3. Non-invasive diagnosis of non-alcoholic steatohepatitis**

heterogeneous groups of patients with contradictory results [101].

fibrosis [83, 84].

10 Liver Research and Clinical Management

and oxidative stress).

Emerging fields in the search for non-invasive biomarkers of NAFLD are proteomics, metabolomics and epigenetics.

Proteomics provides essential information about the biologically active entity named protein. Thanks to proteomic analysis, key changes in serum protein expression levels have been demonstrated between control subjects and patients with different stages of fatty liver [108].

In the last years, studies about the use of metabolomic to discover biomarkers of progression of NAFLD have received great interest, and not only in this liver disease [109–111]. In fact, a Spanish group has developed the so-called OWL Liver Test that consists in the determination


**Table 2.** Predictive models for non-alcoholic steatohepatitis.

of more than 500 serum metabolites through liquid chromatography coupled with mass spectrometry (LC–MS) in NAFLD patients obtaining a metabolomic profile that enables the differentiation between NAFL and NASH with good specificity and sensitivity [112]. Moreover, the same group thanks to the study of metabolomic profiles at the serum level observed two different subtypes of NAFLD according to the involvement of the methionine metabolism, subtype M and subtype no M, distinguishing those patients that could benefit from therapy with SAMe (S-adenosyl methionine) [113].

Recently, studies in rodents suggest that epigenetic events, inheritable events not caused by changes in DNA sequence, may influence susceptibility to NASH. The three most commonly described epigenetic mechanisms are DNA (CpG) methylation, post-translational histone modifications and microRNAs (miRNAs). Several miRNAs have been identified in serum/ plasma of NAFLD patients that show diagnostic potential for distinguishing NAFL from NASH and advanced fibrosis [114].
