Non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is the pathological accumulation of fat in the liver in the absence of alcohol intake. Described initially only 30 years ago⁽Reference Adler and Schaffner^1, Reference Ludwig, Viggiano and McGill²⁾, it is now the leading cause of liver disease in developed countries, with an estimated prevalence of 20–35% in the general population. The occurrence of NAFLD is strongly linked to obesity, insulin resistance and other components of the metabolic syndrome. From a nutrition and public health perspective the two major concerns are the rising incidence of NAFLD in children and the convincing evidence that NAFLD is an independent risk factor for CVD.

The term NAFLD encompasses a spectrum of histologically-defined liver disorders. Disease can progress from macrovesicular lipid accumulation in the hepatocytes (termed steatosis) to non-alcoholic steatohepatitis (NASH; steatosis in the presence of inflammatory infiltrate possibly with some fibrosis) to outright fibrosis, cirrhosis and even hepatocellular carcinoma. A combination of environmental and genetic factors determines individual risk of NAFLD development and progression, with a clear role for nutrition as a modifiable environmental risk factor. Pathogenesis of NAFLD was initially envisaged as a ‘two-hit process’⁽Reference Day and James³⁾ with fat accumulation in hepatocytes viewed as the primary insult and increased oxidative stress leading to inflammation being the second ‘hit’ in the progression to NASH and fibrosis. However, at the cellular level mechanisms influencing disease progression are clearly multi-factorial and dependent on numerous genetic and environmental interactions. Future analysis and modelling of NAFLD progression must take account of this complexity in a systems biology fashion.

Non-alcoholic fatty liver: disease development

The pathogenesis of NAFLD begins with the accumulation of lipid in the liver. As only a minority of patients with hepatic steatosis progress to the necroinflammatory steatohepatitis and develop fibrosis, it was originally conceptualised that a second ‘hit’ is required to induce cellular events (e.g. oxidative stress) leading to inflammation, cell death and fibrosis⁽Reference Day and James³⁾. As with all complex diseases, it is now recognised that the phenotypic expression of NAFLD depends on a myriad of genetic, behavioural and environmental interactions⁽Reference Charlton⁴¹⁾.

While hepatic steatosis can be viewed simplistically as an imbalance between the processes of lipid accumulation and lipid disposal, the cellular and molecular regulation of hepatic metabolism is intricate and has been the subject of a recent extensive review⁽Reference Musso, Gambino and Cassader⁴²⁾. Fat in the liver is acquired from the diet via lipoproteins, by de novo lipogenesis or circulating NEFA, and is removed through VLDL secretion or by β-oxidation. Derangements in each of these pathways have been associated with NAFLD. Traditionally, it was considered that lipid accumulation in the liver occurs in the context of obesity and peripheral insulin resistance, with lipid coming from an elevated plasma NEFA pool caused by increased activity of hormone-sensitive lipase and increased lipolysis from engorged adipocytes. Multiple lines of evidence suggest that this explanation is likely to be too naïve; indeed, it has been postulated that systemic insulin resistance might be secondary to NAFLD⁽Reference Parekh and Anania⁴³⁾.

Stable-isotope research in patients with NAFLD has shown that although 60% of the TAG in the liver comes from the peripheral NEFA pool, elevations in fasting de novo lipogenesis also contribute substantially⁽Reference Donnelly, Smith and Schwarzenberg⁴⁴⁾. Additional work has observed both an increase in adipose lipolysis and increased VLDL-TAG secretion from the liver in patients with NAFLD⁽Reference Fabbrini, Mohammed and Magkos⁴⁵⁾. However, not only is the increase in VLDL-TAG secretion insufficient to account for the increase in hepatic TAG content in these patients, but the VLDL-TAG is derived predominantly from ‘non-systemic’ NEFA, either from de novo lipogenesis or lipolysis of visceral fat. This finding is interesting in the light of very recent proton magnetic resonance spectroscopy and MRI data showing that hepatic lipid content is directly correlated with visceral fat levels⁽Reference Guerrero, Vega and Grundy⁴⁶⁾. The study highlights ethnic differences in the manifestations of insulin resistance; although Hispanics and African-Americans have similar frequencies of insulin resistance, African-Americans are less likely to have NAFLD, hypertriacylglycerolaemia and elevated visceral fat depots. These data, as well as data showing NAFLD in non-obese individuals with normal glucose and lipid levels⁽Reference Bacon, Farahvash and Janney⁴⁷⁾, suggest that NAFLD can and does occur without the backdrop of insulin resistance.

Once steatosis is established multiple mechanisms contribute to lipid-induced cellular injury⁽Reference Browning and Horton⁴⁸⁾. Increased mitochondrial β-oxidation of NEFA leads to an increase in reactive oxygen species. Mitochondrial defects have been observed in patients with NAFLD and impairment of the respiratory chain also produces reactive oxygen species and leads to extramitochondrial NEFA oxidation in the peroxisomes and microsomes, producing further reactive oxygen species. The overall increase in oxidative stress leads to lipid peroxidation, DNA and protein damage and ultimately cell death. In addition, the aldehyde by-products of lipid peroxidation increase production of pro-inflammatory cytokines and recruit inflammatory cells into the liver. Inflammation and the activation of hepatic stellate cells lead to collagen production and the initiation of fibrosis⁽Reference Browning and Horton⁴⁸⁾.

Last, the pathogenesis of NAFLD is also mediated by transcription factors such as the sterol regulatory element-binding protein-1, the carbohydrate responsive element-binding protein and the PPAR, which are reviewed elsewhere⁽Reference Anderson and Borlak⁴⁹⁾, and influenced by circulating cytokines and adipokines. The levels of all these molecules will depend on both genetic polymorphisms and the presence or absence of disease (e.g. obesity or immune), adding a further level of complexity to the pathogenesis of NAFLD. A review of clinical studies has shown low levels of the anti-inflammatory adiponectin and high levels of the pro-inflammatory resistin, TNFα and IL-6 cytokines in patients with NAFLD⁽Reference Tsochatzis, Papatheodoridis and Archimandritis⁵⁰⁾. Genetic polymorphisms in these cytokines have also been investigated in patients with NAFLD, as described later.

Non-alcoholic fatty liver disease: progression and associated mortality

The prognosis of patients with NAFLD depends on the histological stage of the disease. Simple steatosis may have a relatively benign course with no increased risk of mortality, whereas NASH may progress quite rapidly and is associated with markedly increased risk of mortality⁽Reference Dam-Larsen, Franzmann and Andersen^51–Reference Ekstedt, Franzen and Mathiesen⁵⁴⁾. Progression in NAFLD is difficult to assess, requiring years of follow-up and repeat biopsies that are prone to sampling errors⁽Reference Ratziu, Charlotte and Heurtier^17, Reference Ratziu, Bugianesi and Dixon⁵⁵⁾. The longest prospective study of the natural history of NAFLD using repeat biopsies had a mean follow-up of 13·7 years and involved 129 patients⁽Reference Ekstedt, Franzen and Mathiesen⁵⁴⁾. It was reported that 47% of patients presenting with steatosis progressed to NASH and 25–50% of patients presenting with NASH developed advanced fibrosis or cirrhosis in 8–13 years (Fig. 1). Although the results underscore the fact that patients with steatosis can and do develop steatohepatitis, mortality was not found to be changed in the group presenting with steatosis, whereas survival was reported to be significantly lower in patients that had presented with NASH (70% v. 80% in the reference population, P=0·01). Patients with NAFLD-associated cirrhosis have been shown to have a very poor 10-year prognosis, with 50% needing a liver transplant, 20% dying from a liver-related cause and 7% developing hepatocellular carcinoma (Fig. 1)⁽Reference Sanyal, Banas and Sargeant⁵⁶⁾. It is of great concern that a very recent study on the natural history of NAFLD in children has demonstrated that paediatric NAFLD is also progressive and can result in end-stage liver disease and death⁽Reference Feldstein, Charatcharoenwitthaya and Treeprasertsuk⁵⁷⁾. In the cohort of sixty-six children followed over a mean of 6·4 years, two children underwent liver transplantation and two children died, while observed serial biopsies showed progression in 80% of patients.

Fig. 1. Progression of non-alcoholic fatty liver disease. Of the patients presenting with steatosis 47% progress to steatohepatitis and 25–50% of patients presenting with steatohepatitis develop fibrosis or cirrhosis within 8–13 years⁽Reference Ekstedt, Franzen and Mathiesen⁵⁴⁾. The 10-year prognosis for patients with NAFLD-related cirrhosis is very poor with 50% requiring a liver transplant, 20% dying of a liver-related cause and 7% developing hepatocellular carcinoma⁽Reference Sanyal, Banas and Sargeant⁵⁶⁾. – – –▸, Potential for reversal.

Multiple risk factors have been associated with NAFLD progression including older age⁽Reference Garcia-Monzon, Martin-Perez and Iacono^23, Reference Ratziu, Giral and Charlotte^24, Reference Angulo, Keach and Batts⁵⁸⁾, BMI⁽Reference Ratziu, Giral and Charlotte^24, Reference Fassio, Alvarez and Dominguez^53, Reference Angulo, Keach and Batts⁵⁸⁾, insulin resistance⁽Reference Ekstedt, Franzen and Mathiesen⁵⁴⁾, diabetes⁽Reference Adams, Sanderson and Lindor^59, Reference Rafiq, Bai and Fang⁶⁰⁾, metabolic syndrome⁽Reference Marchesini, Bugianesi and Forlani⁶¹⁾ and histological NASH on diagnosis⁽Reference Matteoni, Younossi and Gramlich^52, Reference Ekstedt, Franzen and Mathiesen^54, Reference Gramlich, Kleiner and McCullough⁶²⁾. A current systematic review of ten studies has examined risk factors for progression from NASH to advanced fibrosis⁽Reference Argo, Northup and Al-Osaimi⁶³⁾. Using multivariate analysis the review has found that only age and the presence of inflammation on initial biopsy are independent predictors of progression in 221 patients over a 5·3-year follow-up. The well-documented limitations associated with systematic reviews⁽Reference Crowther and Cook⁶⁴⁾, particularly the choice and heterogeneity of included studies in this case, may explain why obesity, BMI and diabetes were not found to be significant predictors in this analysis.

Data relating to the mortality risks associated with NAFLD are somewhat conflicting. The first population-based cohort study that examined NAFLD and mortality has determined survival among 420 patients with NAFLD, identified through imaging or biopsy, after a mean follow-up of 7·6 years⁽Reference Adams, Lymp and St Sauver⁶⁵⁾. In agreement with case series from specialist clinics⁽Reference Matteoni, Younossi and Gramlich^52, Reference Ekstedt, Franzen and Mathiesen^54, Reference Rafiq, Bai and Fang⁶⁰⁾ NAFLD was found to be associated with lower survival and increased risk of mortality from liver disease. After 10 years of follow-up (n 161) survival was reported to be 77% v. 87% in the general population. Subsequently, three independent groups have analysed data from the US Third National Health and Nutrition Examination Survey for mortality risk among suspected cases of NAFLD based on elevated ALT levels⁽Reference Ong, Pitts and Younossi^66–Reference Ruhl and Everhart⁶⁸⁾. While two groups have found NAFLD to be associated with an increase in overall mortality⁽Reference Ong, Pitts and Younossi^66, Reference Dunn, Xu and Wingard⁶⁷⁾, one of these two groups has found NAFLD to be associated with a very large increase in liver-associated mortality⁽Reference Ong, Pitts and Younossi⁶⁶⁾, whereas the other group has reported a striking link between NAFLD among 45–54 year olds and CVD mortality⁽Reference Dunn, Xu and Wingard⁶⁷⁾. Conversely, the third group has reported no association between elevated ALT levels and all-cause or CVD mortality but an association with liver-related mortality⁽Reference Ruhl and Everhart⁶⁸⁾. The differences may be attributable to variations in study design, patient selection strategy and statistical analysis. While these studies have the advantage of very large sample sizes from a representative sample of the general population, diagnosis of NAFLD is based on elevated ALT levels, which do not differentiate between steatosis and steatohepatitis and, as previously mentioned, are likely to severely underestimate NAFLD incidence and therefore the magnitude of mortality risk. However, the data overall show NAFLD is far from a benign diagnosis.

Genetic and environmental factors associated with non-alcoholic fatty liver disease development and progression

The development of NAFLD is strongly linked to obesity and insulin resistance. However, as there are obese individuals and individuals with diabetes who do not have NAFLD, and since NAFLD can occur in normal-weight individuals with normal glucose and lipid levels⁽Reference Bacon, Farahvash and Janney⁴⁷⁾, there are obviously multiple genetic and environmental factors determining NAFLD development and progression (Fig. 2).

Fig. 2. Genetic and environmental factors associated with non-alcoholic fatty liver disease (NAFLD) development and progression. —▸, Comorbidities (obesity, insulin resistance, hyperlipidaemia and inflammation) in addition to genetic and environmental factors influence the development and progression of NAFLD. Genetic (–··–▸) and environmental (– –▸) factors also influence the development of comorbid conditions.

Initial evidence for a genetic component to NAFLD comes from familial clustering studies⁽Reference Struben, Hespenheide and Caldwell^69, Reference Willner, Waters and Patil⁷⁰⁾ and the ethnic variation in NAFLD prevalence⁽Reference Browning, Szczepaniak and Dobbins⁷⁾. Various genetic single-nucleotide polymorphisms have been investigated in NAFLD including single-nucleotide polymorphisms in the adiponectin⁽Reference Musso, Gambino and De Michieli^71, Reference Tokushige, Hashimoto and Noto⁷²⁾, IL-6⁽Reference Carulli, Canedi and Rondinella⁷³⁾, TNFα⁽Reference Tokushige, Takakura and Tsuchiya-Matsushita⁷⁴⁾ and apoE⁽Reference Sazci, Akpinar and Aygun⁷⁵⁾ genes. ‘Candidate gene’ studies in small cohorts have yielded somewhat conflicting results in different populations. The first genome-wide association scan conducted in 2008 in a large multiethnic population targeting NAFLD has identified the patatin-like phospholipase domain-containing protein 3 (also known as adiponutrin) gene as being strongly associated with hepatic TAG content⁽Reference Romeo, Kozlitina and Xing⁷⁶⁾. Allelic variants of the patatin-like phospholipase domain-containing protein 3 gene have been found to be associated with high and low amounts of hepatic fat in Hispanics and African-Americans respectively, and to be correlated with the high and low prevalence of NALFD in these populations. Remarkably, patatin-like phospholipase domain-containing protein 3 has also been independently identified in a separate population-based genome-wide scan as one of two genetic loci influencing plasma levels of ALT⁽Reference Yuan, Waterworth and Perry⁷⁷⁾. Undoubtedly, as genome sequencing continues to get cheaper and faster, studies of this type will reveal more genes that are causally related to the pathogenesis of NAFLD.

Nutrition and physical activity are important environmental factors that determine risk in NAFLD. Excess food intake and lack of exercise contribute to weight gain, which has been shown to contribute to the progression of liver fibrosis in patients with NAFLD⁽Reference Ekstedt, Franzen and Mathiesen⁵⁴⁾. Specific dietary factors may also play either protective or antagonistic roles in the development and progression of NAFLD. Nutrient intakes in individuals with NAFLD have been assessed in Italian⁽Reference Musso, Gambino and De Michieli⁷⁸⁾, Japanese⁽Reference Toshimitsu, Matsuura and Ohkubo⁷⁹⁾, Israeli⁽Reference Zelber-Sagi, Nitzan-Kaluski and Goldsmith⁸⁰⁾ and US⁽Reference Kim, Kallman and Bai⁸¹⁾ populations; no UK cohort has been assessed to date. An increased consumption of meat and soft drinks and low consumption of fish were found to be associated with NAFLD cases compared with controls. Unsurprisingly, low intakes of PUFA and high intakes of saturated fat and cholesterol were also shown to be associated with NAFLD. Data from two randomised controlled trials in patients with NAFLD have shown beneficial effects from 6 months of dietary supplementation with n-3 PUFA compared with dietary advice alone⁽Reference Spadaro, Magliocco and Spampinato^82, Reference Zhu, Liu and Chen⁸³⁾. Reductions in fatty liver observed by ultrasonography as well as improvements in serum ALT and TAG levels were found to be greater in the patients supplemented with n-3 PUFA. In both trials the patients receiving n-3 PUFA were found to be more likely to have complete fatty liver regression, suggesting a beneficial therapeutic effect for n-3 PUFA supplementation in NAFLD. Other studies have shown higher-carbohydrate and lower-fat diets to be associated with more progressive disease⁽Reference Solga, Alkhuraishe and Clark^84, Reference Kang, Greenson and Omo⁸⁵⁾. Conversely, very recent animal data have shown that in both mice⁽Reference Bruce, Cagampang and Argenton⁸⁶⁾ and non-human primates⁽Reference McCurdy, Bishop and Williams⁸⁷⁾ exposure to a maternal high-fat diet leads to a disturbing development and progression of NAFLD in the offspring.

In view of the role of oxidative stress in NAFLD pathogenesis several studies have examined antioxidant supplementation as a therapeutic intervention. A recent Cochrane review of six very small and extremely diverse clinical trials has found no evidence either to recommend or advise against the use of antioxidant supplements in patients with NAFLD⁽Reference Lirussi, Azzalini and Orando⁸⁸⁾. However, data from pilot studies⁽Reference Lavine^89, Reference Kugelmas, Hill and Vivian⁹⁰⁾ and one double-blind randomised placebo-controlled trial⁽Reference Harrison, Torgerson and Hayashi⁹¹⁾ have shown sufficient positive effects of vitamin E supplementation in patients with NAFLD for further investigation in two ongoing multicentre trials. The Pioglitazone versus Vitamin E versus Placebo for the Treatment of Non-diabetic Patients with Non-alcoholic Steatohepatitis Trial⁽Reference Chalasani, Sanyal and Kowdley⁹²⁾ in adults and the Treatment of Non-alcoholic Fatty Liver Disease in Children Trial⁽Reference Lavine, Schwimmer and Molleston⁹³⁾ both involve 96 weeks of treatment and will measure improvements in histology as well as ALT levels. While the former trial has three arms and includes a placebo group, the latter trial in children will compare metformin treatment with vitamin E supplementation. Data from these trials, whether negative or positive, should end the debate on the potential efficacy of vitamin E supplementation in NAFLD.

Small intestinal bacterial overgrowth may be an additional environmental factor involved in NAFLD pathogenesis, and dietary supplements such as probiotics could have a beneficial effect. Evidence from animal studies shows that small intestinal bacterial overgrowth increases gut permeability leading to portal endotoxaemia and increased circulating inflammatory cytokines, both of which have been implicated in the progression of NAFLD⁽Reference Brun, Castagliuolo and Di Leo⁹⁴⁾. Several studies have reported an association between small intestinal bacterial overgrowth and the progression of NAFLD⁽Reference Wigg, Roberts-Thomson and Dymock^95–Reference Sajjad, Mottershead and Syn⁹⁷⁾ and in one small human study treatment with antibiotics has been shown to result in an increase in fasting insulin levels⁽Reference Sajjad, Mottershead and Syn⁹⁷⁾. Limited evidence from animal models and one preliminary human study suggest that supplementation with the probiotic VSL#3® (a combination of nine strains of various bifidobacteria, lactobacillus and Streptococcus thermophilus; VSL Pharmaceuticals, Inc., Gaithersburg, MD, USA) may possibly be beneficial in NAFLD, but data are mixed⁽Reference Loguercio, Federico and Tuccillo^98–Reference Velayudham, Dolganiuc and Ellis¹⁰⁰⁾. More research in this area is warranted.

Non-alcoholic fatty liver disease, the metabolic syndrome and CVD risk

The metabolic syndrome was originally defined as a cluster of CVD risk factors with insulin resistance as the underlying pathogenic factor⁽Reference Reaven¹⁰¹⁾. The third report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) defines metabolic syndrome as being any three or more of five variables: increased waist circumference; hypertriacylglycerolaemia; hypertension; low HDL-cholesterol; hyperglycaemia⁽¹⁰²⁾. Metabolic syndrome is highly prevalent among patients with NAFLD in multiple populations and is strongly associated with progressive disease⁽Reference Marchesini, Bugianesi and Forlani^61, Reference Clark, Brancati and Diehl^103–Reference Kim, Choi and Shin¹⁰⁵⁾. Furthermore, a Japanese prospective study (n 3147) has demonstrated that patients with metabolic syndrome, as defined by Adult Treatment Panel III⁽¹⁰²⁾, are four to eleven times (men and women respectively) more likely to have developed NAFLD on follow-up⁽Reference Hamaguchi, Kojima and Takeda¹⁰⁶⁾. Likewise, children with metabolic syndrome, as defined by Adult Treatment Panel III⁽¹⁰²⁾, have been shown to be five times more likely to have NAFLD⁽Reference Schwimmer, Pardee and Lavine³⁸⁾. This strong relationship between NAFLD and the metabolic syndrome has led to the description of NAFLD as the hepatic component of the metabolic syndrome.

Indeed, in the last 5 years NAFLD has emerged as an independent risk factor for CVD. Several studies have observed increased carotid intima-media thickness and carotid plaques in patients with NAFLD, including children⁽Reference Pacifico, Cantisani and Ricci^39, Reference Pacifico, Cantisani and Ricci^39, Reference Brea, Mosquera and Martin^107–Reference Kim, Kim and Huh¹¹¹⁾. Although there has been some conflicting work⁽Reference McKimmie, Daniel and Carr¹¹²⁾, a systematic review has found that carotid plaques are two to three times more likely to be observed in patients with NAFLD and intima-media thickness is strongly associated with NAFLD⁽Reference Sookoian and Pirola¹¹³⁾. Furthermore, multiple population studies have observed increased CVD events in patients with NAFLD diagnosed by ultrasonography and have demonstrated that the increased risk for CVD in NAFLD is independent of conventional risk factors and components of the metabolic syndrome⁽Reference Targher, Bertolini and Rodella^114–Reference Choi, Kim and Kim¹¹⁶⁾. These data add to population data that have shown that elevated liver enzymes are also associated with increased risk for CVD⁽Reference Dunn, Xu and Wingard^67, Reference Schindhelm, Dekker and Nijpels¹¹⁷⁾.

As the treatment of NAFLD involves correcting the same metabolic factors as those involved in CVD, it is prudent that all patients with NAFLD be evaluated for early atherosclerosis. Likewise, patients presenting with the metabolic syndrome or a high Framingham risk score⁽Reference Wilson, D'Agostino and Levy¹¹⁸⁾ should be evaluated for the presence of NAFLD. Children identified with NAFLD or the metabolic syndrome should be strongly counselled to avoid smoking, increase their physical activity and improve their nutrition in order to prevent the development of CVD.

The potential of systems biology

Systems biology is the study of a biological system (cell, tissue, organism) viewed as an integrated and interacting network of genes, proteins and biochemical reactions. This methodology combines computational modelling with experimental biology in order to discover emergent properties that arise from studying a system as a whole; it is these properties that ultimately determine how a system is controlled or regulated. A systems approach begins by constructing a predictive model from which a hypothesis is formed and then tested experimentally by perturbing the system. Experimental techniques are generally the global approaches of genomics, proteomics and metabolomics. Data obtained from these multiple approaches are integrated and compared with the original model, which is refined in an iterative process. In the context of human health it is believed that systems biology is essential to future drug discovery and, indeed, to a future of personalised, predictive and preventative medicine⁽Reference Hood, Heath and Phelps¹¹⁹⁾. In this vision drug targets would be rational and identified through the analysis of normal and disease-perturbed networks, while an individual could have their genome sequenced and in combination with extensive blood analyses would be able to obtain a ‘probabilistic future health history’. Although systems biology is in its infancy and this vision still seems far off, global approaches have been applied to NAFLD and have produced data that will be relevant to the future creation of an integrated ‘systems’ model of NAFLD.

Proteomic technologies are still evolving and the analysis of a complete proteome, in the manner of DNA microarray analysis of complete genomes, has yet to be realised. However, as disease biomarkers and drug targets are most likely to be proteins, proteomics is viewed as central to systems biology and key to predictive medicine⁽Reference Weston and Hood¹²⁰⁾. In the context of NAFLD proteomic techniques have been used to analyse adipose⁽Reference Calvert, Collantes and Elariny¹²¹⁾, liver⁽Reference Charlton, Viker and Krishnan¹²²⁾ and most recently serum⁽Reference Bell, Theodorakis and Vuppalanchi¹²³⁾ samples in patients with NAFLD. Ultimately, it is most likely that a serum diagnostic or prognostic tool for NAFLD would comprise a panel of proteins, and this research highlights the power of systems methodologies for identifying candidate protein biomarkers. Importantly, a ‘potential biomarker panel’ comprising six proteins has been demonstrated to have greater diagnostic utility than ALT to differentiate between patient groups⁽Reference Bell, Theodorakis and Vuppalanchi¹²³⁾. It is also noteworthy that two independent studies⁽Reference Charlton, Viker and Krishnan^122, Reference Bell, Theodorakis and Vuppalanchi¹²³⁾ have identified lumican, a protein that activates transforming growth factor β and smooth muscle actin, to be elevated in both liver and serum from patients with NASH. These studies have given insight into NAFLD pathogenesis and have identified candidate disease biomarkers worthy of prospective validation.