Changes in diet and lifestyle have resulted in a dramatic increase in the prevalence of obesity and metabolic syndrome in Western countries and many Asian countries. This has resulted in a significant increase in the incidence of non-alcoholic fatty liver disease (NAFLD), which is considered to be a hepatic manifestation of metabolic syndrome. NAFLD has become an important public health issue because of its high prevalence. NAFLD consists of two clinical entities: simple steatosis and non-alcoholic steatohepatitis (NASH). Currently, NAFLD is the most common cause of chronic liver disease in these countries. In this review, we summarize the current concepts relating to the characteristics and diagnosis of NAFLD/NASH.
Non-alcoholic fatty liver disease (NAFLD) is considered to be a hepatic manifestation of metabolic syndrome. NAFLD has become an important public health issue because of its high prevalence. NAFLD consists of two clinicopathological entities: simple steatosis, which generally follows a benign non-progressive clinical course, and non-alcoholic steatohepatitis (NASH), which may progress to cirrhosis and hepatocellular carcinoma. The diagnosis of NAFLD is based on the following three criteria: non-alcoholic, detection of steatosis either by imaging or by histology, and appropriate exclusion of other liver diseases. Alcoholic liver disease can occur when daily alcohol consumption exceeds 20 g in women or 30 g in men. Thus, non-alcoholic indicates lower levels of these alcohol consumptions. However, there is still no clear consensus regarding the threshold alcohol consumption for defining non-alcoholic liver disease. Then, there is the strong recommendation for a change in the nomenclature, such as use of the term metabolic fatty liver and metabolic steatohepatitis. NASH has emerged as a clinicopathological entity, and even now, a liver biopsy remains the gold standard for making a definitive diagnosis. However, liver biopsy has several drawbacks. In general practice, NAFLD is a convenient-to-use term for the diagnosis and management of these patients, and serum biomarkers that indicate the severity of fibrosis serve as clinically useful tools for the identification of NAFLD in patients with bridging fibrosis or cirrhosis. In the future, improved understanding of the pathogenesis of NASH and new technologies may contribute to the diagnostic process and provide reliable, non-invasive alternatives to liver biopsy.
Nomenclature of NAFLD/NASH
NAFLD is characterized by excessive accumulation of fat, or steatosis, in the liver in individuals with a history of a little or no alcohol consumption. While simple steatosis accounts for 80–90% cases of NAFLD, NASH accounts for the remaining 10–20%. Simple steatosis is mostly a benign non-progressive clinical entity, while NASH can progress to cirrhosis or even hepatocellular carcinoma (HCC). NASH is histologically characterized by hepatic steatosis associated with evidence of liver cell injury (ballooning degeneration) and inflammation, steatohepatitis, and varying degrees of fibrosis; these histological features are indistinguishable from those of alcoholic hepatitis. NASH has emerged as a distinct clinicopathological entity,[1-6] and even now, a liver biopsy still remains the “gold standard” for making a definitive diagnosis.
Traditionally, fatty disorders of the liver have been classified as alcoholic or non-alcoholic (Fig. 1). Primary NAFLD/NASH is associated with obesity, diabetes, or dyslipidemia, and the so-called insulin resistance or metabolic syndrome. Secondary NAFLD/NASH is rare and may be associated with many conditions such as polycystic ovary syndrome, endocrine diseases, sleep apnea, and pancreatoduodenal resection, etc. According to the practice guideline proposed by American Association for the Study of Liver Diseases, steatogenic medications are not included as a cause of NAFLD; however, historically, drug-induced fatty liver has been included under NAFLD. Therefore, the classification is still confusing. Using the term “non-alcoholic” to describe fatty liver disease associated with all other etiologies than alcohol consumption renders the condition heterogeneous. Then, there is the strong recommendation for a change in the nomenclature, such as use of the term metabolic fatty liver and metabolic steatohepatitis.[2, 5, 6] Thus, there is no consensus yet on the best way to classify fatty disorders of the liver.
Clinical features of NAFLD/NASH
According to data from annual health checkups, 10–40% of Japanese adults have ultrasonography (US) -diagnosed NAFLD.[7-9] NASH is observed in 10–20% of cases of NAFLD, while the estimated prevalence of NASH is 1–8%. Age and gender differences in both the prevalence and severity of NAFLD/NASH are well known, which may just reflect the differences in the prevalence of obesity and metabolic syndrome in the general population[9-11] (Figs 2, 3). The prevalence of NAFLD increased with the severity of risk factors; it was 10–20% in non-obese individuals, around 50% in those with a body mass index (BMI) more than 25 kg/m2 but less than 30 kg/m2, and around 80% in those with a BMI over 30 kg/m.[2, 7, 9] The prevalence of NAFLD was around 50% in type 2 diabetes and around 50% in patients with dyslipidemia. The prevalence of NAFLD also shows ethnic differences; it is higher in Hispanics followed by white and lower in African Americans. Family members of subjects with NAFLD are also at increased risk because of genetic background.[13-15]
NAFLD patients are usually asymptomatic until the condition progresses to liver cirrhosis. Therefore, NAFLD is often detected based on the presence of hepatic steatosis on abdominal US during routine health checkups or clinical visits for other diseases among non-alcoholic individuals. Most patients with NAFLD have insulin resistance; obesity, diabetes, or dyslipidemia. While NAFLD could be the result of insulin resistance, a causal role of NAFLD in insulin resistance has also been reported. Thus, there could be a vicious cycle involving these diseases. NAFLD is no longer considered to be a primary liver disease but rather as a part of metabolic syndrome. Blood chemistry shows mild elevation of transaminases, and also other evidence for liver dysfunction in the cirrhotic stage.
The long-term prognoses of NAFLD, including histologically diagnosed simple steatosis, NASH and cirrhotic NASH have been reported from population-based studies as well as hospital-based cohort studies (Table 1).[17-28] According to these studies, the prognoses vary widely among these conditions.[17-28] Longitudinal histological studies have confirmed the benign clinical course of simple steatosis, although a few studies have reported the development of cirrhosis in some patients with “simple steatosis.” Progression to fibrosis in NASH appears to occur more frequently among patients whose baseline liver biopsies demonstrate greater necroinflammatory changes.
|Author||Diagnosis||n||Average F/U (years)|
|Overall n (%)||Liver-related/overall (%)|
|Adams et al.||NAFLD**||420||7.6||21 (5%)|| |
< 0 >
|Ekstedt et al.||NAFLD***||129||13.7||10 (7.8%)|| |
< 0 >
|Rafiq et al.||NAFLD***||131||18.5||NR|| |
< 0 >
|Söderberg et al.||NAFLD***||118||21||9 (7.6%)|| |
< 0 >
|Sørensen et al.||NAFLD**||1800||6.2||0|| |
< 0 >
|Teli et al.||Simple*** Steatosis||40||9.6||0|| |
< 0 >
|Dam-Larsen et al.||Simple*** Steatosis||170||20.4||2 (1.2%)|| |
< 0 >
|Evans et al.||NASH***||26||8.7||1 (4%)|| |
< 0 >
|Hui et al.||Cirrhotic-NAFLD***||23||7.0||100%|| |
< 0 >
|Sanyal et al.||Cirrhotic-NAFLD***||152||10||100%|| |
< 3 >
|Yatsuji et al.||Cirrhotic-NAFLD***||68||3.4||100%|| |
< 14 >
|Söderberg et al.||Cirrhotic-NAFLD***||9||21||100%|| |
< 0 >
|Ascha et al.||Cirrhotic-NAFLD**||195||3.2||100%|| |
< 0 >
It has been reported that as compared with individuals in the general population, those with NAFLD show a lower-than-expected survival with a standardized mortality ratio of 1.34 to 1.69 because of increases in the risk of cardiovascular diseases and liver-related death.[17, 18, 20, 23] The most common causes of death in patients with NAFLD are cardiovascular disease and malignancy, followed by liver-related death. However, overall, NAFLD appears to be slowly progressive, with liver-related morbidity and mortality occurring only in a minority of subjects. The reported risk factors for the development of cirrhosis are older age, presence of diabetes, and morbid obesity.
We conducted a comparative analysis of the natural history of 68 patients with biopsy-proven cirrhotic NASH and 69 age- and sex-matched patients with liver cirrhosis associated with hepatitis C virus infection (LC-C). The mean age of the patients with cirrhotic NASH was 62.7 years. Patients with cirrhotic NASH showed a similar survival rate to that of the patients with LC-C (75.2% and 73.8%, respectively), although the rate of development of HCC was lower (5-year HCC development rate: 11.3% for cirrhotic NASH vs 30.5% for LC-C). The leading cause of death in patients with cirrhotic NASH was HCC, followed by liver failure (Fig. 4). All previous studies have confirmed that patients with cirrhotic NASH exhibit a similar clinical course to those with LC-C, and the reported rates of development of HCC in these patients were similar to our data (around 10% at 5 years).[20, 25-28]
Characteristics of HCC in NAFLD/NASH
Concerning the risk factors for the development of HCC, we identified advanced fibrosis, older age, histological low-grade inflammation, and low aspartate aminotransferase (AST) levels as the risk factors for presence of HCC. It is well known that when NASH progresses to the end stage, the necroinflammatory changes and serum transaminase levels gradually decline; therefore, presence of low-grade inflammation and a low serum AST level may indicate the end stage of cirrhotic NASH. Accordingly, the risk factors for the development of HCC in patients with NASH are the features of end-stage NASH and older age.
We compared the clinical features of 34 NASH-related HCC (NASH-HCC) patients and 56 age-, sex-, and treatment-matched patients with hepatitis C virus infection-related HCC (HCV-HCC). As expected, there was a significantly higher prevalence of obesity, diabetes, and dyslipidemia in the NASH-HCC group. Serum transaminases were significantly higher in the HCV-HCC group, while the serum gamma-glutamyl transferase level was significantly higher in the patients with NASH-HCC. The 5-year survival rate was 55.2%, and the 5-year recurrence rate after curative treatment was 69.8% in patients with NASH-HCC. The survival and recurrence rates were similar in the two groups. HCC in NASH may also be of multicentric origin, similar to the case of HCC associated with viral hepatitis.
According to previous studies, 10–75% of all NASH-related HCCs occur in patients with non-cirrhotic NASH. The high incidence of HCC arising from non-cirrhotic NASH may be partly due to the fact that the diagnosis of NASH is based on histology, and liver tissue can only be obtained by liver biopsy or surgery in patients with preserved liver function. Moreover, end-stage cirrhotic NASH cannot be diagnosed with any confidence because of its “burned out” histology. These points may introduce significant bias. Further studies are required to clarify the true incidence of HCC arising from non-cirrhotic NASH.
How is NAFLD/NASH diagnosed?
The diagnosis of NAFLD is based on the presence of the following three criteria: non-alcoholic, detection of steatosis either by imaging or by histology, and appropriate exclusion of other liver diseases.[1-6] NASH is diagnosed based on the presence of steatohepatitis on liver biopsy. Given the lack of surrogate markers yet for the diagnosis of NAFLD, it is important to exclude other liver diseases such as alcoholic liver diseases, viral hepatitis, autoimmune liver diseases, and metabolic or hereditary liver diseases. However, the prevalence of NAFLD is extremely high, NAFLD is often complicated by other liver diseases such as viral hepatitis, etc., and NAFLD exacerbates liver damage and reduces the response to treatments. Epidemiological studies have shown that alcoholic liver disease can occur when the daily alcohol consumption exceeds 20 g in women and 30 g in men. Then, NAFLD is diagnosed when the alcohol consumption is lower than the aforementioned in the respective sexes. Serum transaminases are not helpful for the diagnosis of steatosis because 50–80% of patients with hepatic steatosis have normal transaminase levels. In stage 3 fibrosis, fibrosis markers such as hyaluronic acid, etc., are elevated, and in the cirrhotic stage, reduction of the platelet count and evidence of liver dysfunction such as elevation of the serum bilirubin and ammonia, etc., are noted.
Abdominal US is currently the most common method employed for qualitative assessment of hepatic steatosis because it is non-invasive, widely available, cheap, and provides useful information. Presence of hepatic steatosis on abdominal US is usually defined based on the presence of at least two of the following findings: increased hepatorenal contrast, liver brightness, deep attenuation, and vascular blurring (Fig. 5). However, the diagnosis by US has several limitations; it is subjective, operator-dependent, shows poor sensitivity for the detection of mild steatosis, and is a poor tool for quantifying the steatosis. Both computed tomography (CT) and magnetic resonance imaging (MRI) seem to be more objective and more sensitive techniques for the quantification of steatosis, but MRI is still less widely available and much more expensive. For the diagnosis of steatosis by CT, the liver-to-spleen attenuation ratio is measured, and the diagnosis of steatosis is made when the ratio is less than 0.9. Of course, CT also has limitations with respect to the diagnosis of steatosis, including poor sensitivity for the detection of mild steatosis, X-ray exposure of the patients, and unavailability for patients with hemosiderosis. Unfortunately, none of these imaging modalities is useful for the diagnosis of NASH.
Concerning interference with the detection of steatosis by advanced fibrosis, the decrease in the detection sensitivity is marked for both US and CT. The sensitivity of US and CT for advanced fibrosis is also decreased markedly in patients with severe steatosis and obese patients, being more marked for US. An awareness of these disadvantages of the common imaging modalities would be useful for a more precise diagnosis of hepatic steatosis and fibrosis in patients with NAFLD.
The principal histological features of NASH are as follows: presence of macrovesicular steatosis, ballooning degeneration of the hepatocytes, and mixed lobular inflammation. These characteristic pathological features with Mallory hyaline and pericellular fibrosis are predominantly seen around the central veins (zone 3) (Fig. 6). Atypical features have been reported in pediatric cases and morbidly obese cases, such as more periportal steatosis (zone 1), little or no ballooning or Mallory hyaline, and more portal-based chronic inflammation and fibrosis.
Three important pathological classifications have been proposed for NAFLD: Matteoni's classification, Brunt's classification, and the NALFD activity score (NAS).[35-37]
In 1999, Matteoni et al. described a classification system that served to distinguish between NASH and non-NASH. They divided 132 NAFLD patients into four categories: type 1, steatosis alone; type 2, steatosis with lobular inflammation only; type 3, steatosis with hepatocellular ballooning; and type 4, type 3 plus either Mallory–Denk bodies or fibrosis. They confirmed the benign clinical course of patients with type 1 or 2 NAFLD and the progressive clinical course of patients who had either type 3 or 4 NAFLD. As a result of these differences, these authors defined type 1 and type 2 histological forms of NAFLD as “non-NASH,” and type 3 and type 4 as NASH. However, this classification did not include an assessment of the severity or pattern of NASH, such as the degree of steatosis, inflammation, location of these changes (i.e. lobular or portal), or the degree of fibrosis.
In the same year as Matteoni's classification system was published, Brunt et al. proposed a semiquantitative grading and staging system for NASH. This classification was applicable to only NASH and not to the entire spectrum of NAFLD.
In 2005, the NASH Clinical Research Network Pathology Committee developed and validated a histological scoring system based on Brunt's classification, NAS, as a semiquantitative instrument by which to judge treatment responses or disease progression in clinical studies. The NAS system addresses the full spectrum of NAFLD and is applicable to both adult and pediatric NAFLD patients. The score is determined as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3), and ballooning degeneration (0–2). A score of ≥ 5 correlated with the diagnosis of NASH made independently by an experienced pathologist without using the score; likewise, scores of less than 3 were correlated with “not NASH,” and scores of 3 or 4 were regarded as borderline. In regard to fibrosis, stage 1 referred to perisinusoidal fibrosis in zone 3 (perivenular area: delicate [1A] and dense [1B]), and detection of portal fibrosis without perisinusoidal fibrosis was defined as 1C. Stage 2 was characterized by perisinusoidal and portal/periportal fibrosis. Stage 3 was defined as bridging fibrosis and stage 4 as cirrhosis. Although the authors reminded us that the NAS system was never intended to be used for the diagnosis of NASH, NAS has frequently been used as a surrogate method for establishing the diagnosis of NASH. Then, they assessed the relation between NASH diagnosed by NAS and pathological diagnosis of steatohepatitis (in this case, NASH) and found that the definitive diagnosis of NASH was not always correlated with threshold values of the NAS. They concluded that clinical pathologists should be encouraged not to use NAS as a categorical approach for the diagnosis of NASH.
Younossi et al. assessed the ability to predict the long-term liver-related mortality based on the pathological characteristics. The study cohort consisted of 209 patients with biopsy-proven NAFLD who were followed up for at least 5 years. The results of their multivariate analysis identified only fibrosis as an independent predictor of liver-related mortality. According to the findings of this study, assessment of the severity of hepatic fibrosis is essential for determining the prognosis in patients with NASH.
Indication of liver biopsy
NASH has emerged as a distinct clinicopathological concept, and even now, biopsy evaluation is considered the “gold standard” for a definitive diagnosis. However, liver biopsy has several drawbacks; it is an expensive and invasive procedure and is fraught with the possibility of sampling error and variability in pathologist interpretation. Moreover, given the extremely high prevalence of NAFLD, a liver biopsy would be poorly suited as a diagnostic test for NASH. Accordingly, at present, liver biopsy may only be considered in NAFLD patients who are considered to be at an increased risk of developing NASH with advancing fibrosis or are suspected to have coexisting other chronic liver diseases.[5, 6] In general practice, NAFLD is a convenient-to-use term for the diagnosis and management of these patients, and serum biomarkers that indicate the severity of fibrosis serve as clinically useful tools for the identification of NAFLD in patients with bridging fibrosis or cirrhosis.
Non-invasive assessment of NASH and advanced fibrosis in NAFLD
Recently, several biochemical markers and imaging modalities have been reported for predicting NASH and the severity of hepatic fibrosis.[41-47] An ideal biomarker should be simple to measure, accurate, reproducible, inexpensive, and readily available. In general, while most of the biomakers and scoring systems show similar accuracy for the detection of advanced fibrosis, their accuracy is weak for the diagnosis of mild fibrosis. The NAFLD Fibrosis Score is a widely validated scoring system for predicting the severity of fibrosis that is based on six readily assessable clinical variables (age, BMI, hyperglycemia, platelet count, albumin, AST/alanine aminotransferase ratio).
Several imaging techniques have also been advocated as non-invasive diagnostic tests for NASH. US-based transient elastography or FibroScan has shown promising results for assessment of the severity of liver fibrosis and degree of steatosis. However, these modalities are expensive and not widely available.
Pathogenesis of NASH
The development of NASH is thought to initiate from basal steatosis as the first hit, followed by a “second hit” that is capable of inducing necroinflammation; this hypothesis is the so-called “two-hit theory.”[48, 49] The second hit can include oxidative stress, especially that arising from mitochondrial stress, insulin resistance, inflammatory cytokines, etc. Autophagy may also play an important role in the pathogenesis of NASH. Recently, a new concept to explain the pathogenesis of NASH was reported by Tilg and Moschen, namely, the “multi-parallel hit” hypothesis. This hypothesis, based on reports that endoplasmic reticulum stress and cytokine-mediated stress can induce steatosis as well as necroinflammation, suggests that multiple hits act together in the development of NASH. Steatosis should therefore be considered as a part of the liver's early “adaptive” response to stress rather than as the first hit in disease progression.
I have summarized the characteristics and diagnosis of NAFLD/NASH. There is still no clear consensus regarding the threshold alcohol consumption for defining “non-alcoholic” liver disease. In the future, a change in the nomenclature of NAFLD/NASH might be needed because there are so many obese people who drink much alcohol and show the histological features of steatohepatitis. Liver biopsy currently remains the gold standard for the diagnosis of NASH. In the future, improved understanding of the pathogenesis of NASH and new technologies may contribute to the diagnostic process and provide reliable non-invasive alternatives to liver biopsy.