**1. Introduction**

In the 1990s, nonalcoholic steatohepatitis (NASH) was considered to be a relatively uncommon disease, only occurring in obese women with type 2 diabetes. Things changed quickly after a study came up in 1996 describing NASH the second most commonly occurring liver disease in patients after acute and chronic viral hepatitis infection [1]. Even more important for the field of NASH was a review in 1998 highlighting that fibrosis and cirrhosis are complications associated with NASH in more than 15–50% of all cases [2]. In the same article, Day and James postulated the two-hit hypothesis according to which the pathogenesis of NASH is initiated by a first hit predominantly caused by accumulating lipids in hepatocytes leading to apoptosis of these cells and excessive oxidative stress. Steatosis then sensitizes the liver to develop advanced NASH by a potential second hit driven by infiltrating immune cells, which release inflammatory mediators such as cytokines. This shift to an inflammatory milieu can finally lead to fibrotic tissue remodeling which can easily progress to end stage liver disease such as cirrhosis and the development of hepatocellular carcinoma (HCC). Today, this hypothesis is considered to be inadequate to explain the multiple and complex disease drivers to nonalcoholic fatty liver disease (NAFLD). Lately the multiple-hit hypothesis is better framing the variable circumstances implicating disease development and progression [3]. However, studies show that disease progression

is not always linear and it is not fully clear who is more likely to progress to more advanced stages [4]. Considered as the hepatic manifestation of metabolic syndrome, NAFLD is in most cases associated with type 2 diabetes and dyslipidemia (Figure 1) [5].

**Figure 1.** Risk factors, symptoms, diagnosis, and treatment of nonalcoholic liver disease. Predisposition and disease progression in NAFLD/NASH is influenced by comorbidities [6], genetic determinants [7], and environmental factors including drugs and toxins [8]. The range of resulting symptoms and complication of NAFLD/NASH can vary from very mild to life-threating. Diagnosis and monitoring of disease can be done by blood tests [9], liver function tests [10] and imaging [11]. However, a liver biopsy should be performed based on an individualized decision and is still the gold standard in scoring and grading of steatosis, inflammation, and fibrosis [12]. Although an ultimate therapy is still missing, beneficial effects on NAFLD/NASH progression are energy restriction, lifestyle changes, improved diets, and elevated physical activity [13]. In addition, in biopsy-proven NASH and fibrosis, medication with antiglycemic drugs, insulin sensitizers, synbiotics, or compounds interfering with fat metabolism or preventing oxidative stress have favorable effects on disease outcome [13,14]. Surgical procedures, including bariatric surgery, to treat obesity and liver transplant after liver failure are extreme forms in NAFLD/NASH treatment [15].

Taking the direct association with insulin resistance and obesity into account, NAFLD is a global health burden with rising impact [16,17]. It is known that almost 25% of the global population is affected by NAFLD and/or its complications, making it the most common chronic and progressive liver disease especially in industrialized countries [18]. Models even predict a prevalence of NAFLD in adults of more than 30% of the total population with around 20% being diagnosed as NASH. Looking at these developments, calculations show that the incidence of HCC and NASH-related liver transplantation will be doubled until 2030 [19–21].

Up-to-date weight loss and lifestyle changes are still the only possible ways to overcome NAFLD. Regrettably, very few patients successfully achieve long term weight reduction. Therefore, there is an urgent need for the development of a pharmacological treatment for patients with NASH and fibrosis. NASH and fibrosis typically develop asymptomatic till they progress to end-stage liver disease at which liver transplantation is the only cure available. At present, first guidelines consent to the use of pioglitazone, vitamin E and pentoxifylline in patients with NASH to reduce steatosis, but they still have many potential side-effects or have low efficiency [22–25]. Other agents such as obeticholic acid,

cenicriviroc, elafibranor and selonsertib are actually in clinical trial phase III (Figure 2) [26]. Although these various clinical trials show promising results to target NAFLD and NASH therapeutically, there is no approved pharmacological treatment available yet [27–31].

**Figure 2.** Established compounds and drug candidates evaluated for the treatment of NAFLD/NASH in clinical trial phase III. Pioglitazone (PPARγ agonist), vitamin E (antioxidant), pentoxifylline (anti-tumor-necrosis factor-α (TNF-α) agent), Obeticholic acid (farnesoid X receptor antagonist), cenicriviroc (CCR2/CCR5 inhibitor), elafibranor (PPARα/δ agonist), and selonsertib (ASK1 inhibitor) have different molecular targets. The different biological activities of the drugs point to the complexity of NAFLD/NASH, having a large variety of potential therapeutic drug targets. The depicted structures were generated with the open source molecule viewer Jmol using data deposited in the PubChem compound database with CIDs: 4829, 14985, 4740, 447715, 11285792, 9864881, and 71245288). For more details about the biological activity of each drug refer to [32].

On the basis of different imaging techniques and the combination of clinical factors the presence of NASH in an individual can be strongly suspected. However, the gold standard to diagnose NASH is still an invasive liver biopsy [4]. This is not only less accepted and harmful to the patient, but also often leading to late diagnoses at the point of end-stage liver disease due to the fact that NAFLD is in most cases asymptomatic prior to the transition to NASH. In addition, not all researchers agree with the view that there exists a necessity of differentiating by histology the so-called simply or benign fatty liver from NASH. This is due to the fact that NAFLD may be less benign than it is currently thought to be and that there exists a high degree of heterogeneity of NAFLD [33].

Twenty to thirty percent of all NAFLD patients progress to NASH-fibrosis. Therefore, there is an urgent need to find reliable noninvasive biomarkers and screening techniques to diagnose NAFLD and NASH and monitor patients at an earlier time point at which lifestyle changes and potential newly developed drugs can be used purposefully.

In addition, other less common conditions can cause similar clinical and histological phenotypes like NAFLD and NASH [34]. There is common sense that the primary causes for the development of NAFLD are obesity, type II diabetes, dyslipidemia, insulin resistance and some genetic disorders [34]. However, there are other less common conditions in which NAFLD is the consequence of secondary causes, including specific disorders of lipid metabolism (abetalipoproteinemia, hypobetalipoproteinemia, familial combined hyperlipidemia, glycogen storage disease, Weber–Christian syndrome, lipodystrophy), total parenteral nutrition, hepatitis C infection, severe surgical weight loss, medications (amiodarone, tamoxifen, methotrexate, corticosteroids, highly active antiretroviral therapy), starvation, Wilson's disease, environmental toxicity, and celiac disease [34].

Some other conditions associated with metabolic syndrome that can lead to NAFLD are the obstructive sleep apnea syndrome (OSAS), polycystic ovary syndrome (PCOS), and non-obese NAFLD. OSAS is a common sleep disorder, which is associated with chronic intermittent hypoxia and increased proinflammatory cytokine production [35]. OSAS and obesity often coexist and the chronic hypoxia induces hyperglycemia, insulin resistance, and hepatic lipid peroxidation, which are hallmarks of the metabolic syndrome [35]. Similarly, PCOS is a frequent endocrine disease in women associated with a number of metabolic consequences, including obesity, dyslipidemia, insulin resistance, type 2 diabetes, and low-grade inflammation [36]. Therefore, it is not surprising that PCOS patients are prone to develop NAFLD. Interestingly, NAFLD can also occur in non-obese individuals. Although these patients have a normal body mass index (BMI), they have metabolic abnormalities similar to those characteristically associated with obesity [37]. Consequently, these normal weight or lean individuals displaying obesity-related features are called metabolically obese but normal weight (MONW) patients [37]. Although the pathways and pathophysiological mechanisms driving NAFLD and NASH in non-obese persons are not completely understood, it is speculated that major risk factors are dysfunctional fat, decreased muscle mass, genetic factors, different patterns of the gut microbiota, and epigenetic changes occurring early in life [38].

To avoid diagnostic pitfalls in the distinction of NAFLD provoked by traditional risk factors of metabolic disease and secondary causes of NAFLD, it is essential that clinicians comprehensively evaluate the patient, because both conditions vary considerably and will require different therapeutic regimens [34,39].

This review summarizes the current screening methods used to complement the overall appearance of NAFLD/NASH and provides an outlook on potential upcoming candidates to replace the need of taking liver biopsies.

### **2. Blood and Serum Tests**

Liver biopsy is still the standard procedure to diagnose NASH. Besides the risk of complications during surgery, it involves a lot of bias because only a little specimen of the liver is taken, which not always represent the actual status of the entire liver [40]. This runs the risk to underestimate disease severity in many cases [41]. Finding reliable biomarkers which can be measured with less or even noninvasive techniques is therefore of urgent need.

However, NAFLD and NASH are complex multi factorial diseases and therefore no single surrogate marker is likely to be omniscient to predict clinical outcome or benefits of a therapy. Despite the fact that all biomarkers and scores have their limitations, interest is increasing rapidly in the use of these markers to predict information about progression and outcome of the disease. Therefore, respective surrogate biomarker and scores offered by the market should be used with much care and limited to situations where it has been demonstrated robust ability in disease management. In addition, there is an urgent need to improve standardization in the usage of these operations. On the other site, it is obvious that the surrogate markers can be extremely helpful when handled correctly. This was very recently demonstrated in a study using telemedicine-based comprehensive, continuous care intervention (CCl) together with carbohydrate restriction-induced ketosis and behavior changes. The respective study showed that a NAFLD liver fat score (i.e., N-LFS) was reduced in the CCl group, whereas it was not changed in a group of patients receiving usual care [42]. This exemplarily demonstrates that surrogate markers can provide good measurement for the efficacy of a specific therapy.

Here we will summarize blood and serum biomarkers, which are already available and discuss their benefits and shortcomings in the diagnosis and management of NASH and NAFLD.
