Simple steatosis and non-alcoholic steatohepatitis (Part Two)

Cytokines

The burst of numerous cytokines such as TNF-α, TGF-β, IL-8, and IL-10 is a cellular action of oxidative stress. Free radicals and cytokines increase the liver's sensitivity to TNF-α. The burst of TNF-α can be induced, among other factors, by enteral endotoxins. TNF-α is also increased in leptin deficiency and the increase in its concentration further aggravates insulin resistance. Leptin deficiency appears during rapid weight loss. Cytokines induce and maintain the process of inflammation and fibrogenesis. Some of these results are still hypothetical and were previously formulated in relation to the pathogenesis of ASH. They also seem to be related to the pathogenesis of NASH.

Genetic factors

Even those who regularly drink alcohol do not always develop hepatic insufficiency, only 10% of them develop cirrhosis. Similarly, not only healthy women but also elegant men can develop NASH. This suggests that genetic factors have a partial responsibility in these entities. The genetic basis for NASH remains unclear. For example, genes may regulate mechanisms that initiate the process which ultimately leads to NASH, the intensity of the second strike, or the burst of different cytokines.

Candidate genes are under investigation, but to date, only the association with C282Y HFE (the hemochromatosis gene) has been studied. The results were negative, the same as in patients with alcoholic liver disease. In these studies, there are only coincidental positive associations for genes that code for glutathione S-transferase M1 and for genes involved in the synthesis of type 1 collagen and that code in vitro for CYP2E1*5 of the CYP2E1 gene. Neither for NASH nor for alcoholic liver diseases is there any association with the HFE gene or HLA genotype.

Genetic factors underline that NASH and ASH require further intensive research. In summary, genes influence the extent of oxidative stress, code for immunoregulation, influence the regulation of apoptosis, and affect the degree of steatosis.

Clinical data and course

Diagnosing NASH is difficult because it is a diagnosis of exclusion and heavily depends on the patient's reliability regarding alcohol consumption.

As with all chronic liver diseases, the clinical picture in NASH is nonspecific and may consist only of fatigue, abdominal complaints, and signs of bloating. In fact, most patients in the late stages are asymptomatic. In many cases, the discovery of high serum transaminases leads to further diagnostic procedures. As a rule, the liver may be soft but could be enlarged.

In ultrasonography, the early stages of NASH have been called “bright liver”. As the disease progresses, palpation may detect increased firmness and ultrasonography may find fibrosis, cirrhosis, liver with an irregular surface, regeneration nodules, and signs of portal hypertension.

Laboratory diagnosis is not always helpful. In 90% of patients, the level of transaminases is under 100 U/l. The AST/ALT ratio is less than 1. In some cases, γ-GT and alkaline phosphatase may have a slight increase.

But even in cases of alcoholic liver damage, hyperbilirubinemia, cholestasis, a decrease in serum albumins, and an increase in mean corpuscular volume develop in very late stages. These changes are even less characteristic for NASH. Hypertriglyceridemia and in 30% of cases hypercholesterolemia are common. Thus, laboratory data can suggest a suspected diagnosis, but they should never be considered definitive.

Serum and hepatic iron may increase together in patients with NASH. Serum transferrin saturation and ferritin are pathological in 10-60% of patients, however, no patient has genetic and histological data for hemochromatosis. Therefore, routine searches for hemochromatosis are not necessary.

The accumulation of iron in the hepatic tissue and in serum is explained by the death of inflamed cells leading to the intracellular release of iron and its deposition in the hepatic tissue.

The exclusion criteria used in suspected NASH cases are:

1. alcohol consumption of 30-40 gr. per day
2. serological viral markers for hepatitis B, D, or C
3. evidence for autoimmune liver disease
4. HFE genotype or histological evidence for hemochromatosis with pathological elevation of iron metabolism markers.

Setting an upper limit for non-significant alcohol consumption is problematic. Alcohol consumption of less than 20 gr per day for women and less than 30gr for men has been considered insignificant, however, other authors set a limit of less than 40 gr per week.

Other diagnostic aspects include:

1. body mass index (BMI = body weight in kg/height in m2) over 25, i.e., obesity
2. type 2 diabetes mellitus
3. hyperlipidemia
4. history of intestinal surgical procedure for weight reduction in massive obesity.
5. Rapid weight loss may be a factor leading to NASH as do other situations in which there is an increase in the distribution of free fatty acids.

Histology

In suspected cases of NASH, liver biopsy is a necessary next step. The biopsy proves evidence only in inflamed or fibrotic fatty liver but does not prove etiology. Typical histological findings in NASH are identical to those in ASH: fatty degeneration, lobular hepatitis, ballooning, cellular necrosis, Mallory bodies, apoptosis, periportal infiltrate, pericellular fibrosis in a network form (chicken wire fibrosis), and complete cirrhosis.

Only fatty deposits and portal and/or lobular inflammation (neutrophil granulocytes and mononuclear cells) do not confirm the diagnosis of NASH.

Differential diagnosis includes chronic hepatitis C, which can easily be confused with NASH, autoimmune hepatitis treated with steroids, early stages of Wilson's disease, and drug-induced hepatitis. The most difficult problem for the pathologist occurs in cases of autoimmune hepatitis or chronic hepatitis C in which the liver cirrhosis is already developed.

Three stages of NASH have been histologically identified: 1- the inactive or non-progressive form has only minor signs of inflammation. 2- the subacute or subfulminant stage of NASH is rare and cannot be differentiated from ASH. It is usually seen after surgical procedures for weight reduction. It is potentially fatal. 3- The chronic stage is the form with slow progression and the most common form that better corresponds to the clinical picture of NASH. Within these three stages, the severity of the disease can be graded on a scale from 1 to 4. The criteria for these grades are based on qualitative and quantitative histological changes present in NASH.

Evidence of mild inflammation with moderate increase in transaminases is not reliable from a diagnostic perspective and also presents minor clinical and prognostic significance. Therefore, the following questions can be posed: when is diagnostic biopsy justified and can pathologists routinely differentiate NASH from other conditions or at least assess the severity of the disease?

Three studies involving a total of 369 patients have shown that fibrosis, as an important marker documenting the progression of hepatic disease, was found only in 4% of patients under 45 years old with NASH, but significantly more often in older patients (p<0.001). In patients with normal weight (BMI<25) under 45 years old, fibrosis is practically not observed. From these findings, it is concluded that biopsy for the diagnosis of NASH (or FL) in patients under 40 years old is very unlikely to provide any significant information, therefore, it is unnecessary.

Liver biopsies are mainly necessary for stages of NASH in patients over 40-45 years old and for therapeutic monitoring. Biopsy is not necessary in young people with normal weight, in non-diabetic patients with low transaminases.

The diagnosis of NASH is supported by histological findings (identical to those of ASH), non-specific laboratory findings, significant alcohol consumption of less than 30 gr/day with irregular intervals (which corresponds to 2-3 glasses of wine) and the exclusion of other liver diseases. The best definition of NASH is:

NASH = ASH – ALCOHOL.