In this study we have shown that::1) Sub groups of patients with HBeAg negative chronic hepatitis B who had extensive fibrosis and inflammation had also high serum hyaluronate level. The differences were statistically significant comparing to normal values and to the patients with milder liver involvement. 2) Serum hyaluronate had more significant correlation with severity of liver fibrosis and inflammation amongst the other variables. 3) In the regression analysis serum hyaluronate had best predictive value for liver fibrosis and inflammation in patients with chronic hepatitis B. 4) At cut-off point of 126.4 ngm/ml it could differentiates extensive fibrosis from milder ones with sensitivity of 90.9% and specificity of 98.1%. At the same cut-off point, it could differentiate extensive inflammation from milder ones with sensitivity of 63.6% and specificity of 92.6%.
Liver biopsy is the method of choice in evaluating fibrosis and inflammation in patients with parenchymal liver disease [5, 17], but it has several limitations which include false negative result of 24% especially in macro nodular cirrhosis [6, 7], post biopsy pain and discomfort , high cost , and death rate of 0.015% . In the past decade, several investigators focused on developing non-invasive test for liver fibrosis [16, 17, 22]. None of them proved to be perfect. Guechot et al showed that hyaluronate had better correlation to the degrees of fibrosis than PIIIP in chronic liver disease . In the study by Oberti et al, serum hyaluronate level was considered the most sensitive test for screening in viral hepatitis B, and C . Imbert-Bismuth et al suggested that, by fibro test 50% of liver biopsies could be avoided in patient with chronic hepatitis C . However fibro test was reanalysed in treated hepatitis C, but its complex equation will limit its usefulness in clinical practice . Thus, due to expense and complexity of Fibrotest-Actitest, it could not be utilized on routine basis in developing countries. Myers et al used Fibrotest and Actitest to decriminate.mild from extensive fibrosis and inflammation in patients with chronic hepatitis B . In Spite of the interesting results, it is difficult to compare it with our data. However, hyaluronic acid may have a lower sensitivity for minimal fibrosis (as in chronic hepatitis C) as well as an absence of independent assessment of both fibrosis and activity as given by FibroTest-Actitest. We were looking for a direct marker of fibrogenesis to be cheap, reproducible and simple. The price of hyaluronic acid test is about 10 US Dollars in Iran as well as in Europe. In this regard serum hyaluronate appears to be an appropriate choice.
Hyaluronate is a polysaccharide with molecular weight ranging from 4 × 103 to 8 × 106 Daltons. It forms constituent of extracellular matrix in all connective tissues [27–29]. It is mainly produced by mesenchymal cells and cleared by hepatic sinusoidal endothelial cells through a high affinity receptor (Kid = 6 × 10-11 M) with a maximum capacity of 104 molecules/cells [30–33] It has short half life and increases by age .
Alcohol, viruses, auto immune diseases, and inborn errors of metabolism could increase production of hyaluronate by activating hepatic stellate cells and decrease clearance by hepatic sinusoidal capilarization . Sinusoidal capilarization could be associated with shunting of blood which is an additional factor for increase of serum hyaluronate in this condition . It was shown that serum hyaluronate increase in alcoholic liver disease [5, 20, 35], primary billiary cirrhosis  and in patients with hepatitis C [12, 36]. In addition, it could be increased in rheumatoid disease due to overproduction by synovial cells [34, 38]. It also increases in renal failure because of disturbed clearance of low molecular weight hyaluronate by the kidneys .
Aetiology of liver damage in our patients was only HBV infection. None of them had received any alcohol in their life time. None of them had any evidence for renal failure or any other disease which could explain their liver disease except HBV. Bloods were taken in complete physical rest and fasting state in order to rule out other interfering factors like eating and physical activity. Our normal level was 20.4 ± 15.5 ngm/ml which is in agreement with manufacturer levels (28.5 ± 24) and what we reported by the other investigators with the same age groups [15, 16, 20, 40].
The proposed cut-off points in different studies were not the same. The cut-off point will depend on the aetiology of liver disease and on the level of sensitivity and specificity that an investigator is looking for [14, 15, 23]. Our study showed that the cut-off point of 126.4 ngm/ml could differential extensive fibrosis from milder ones with sensitivity of 90.9% and specificity of 98.1% and at the same level could differentiate extensive inflammation from milder ones with sensitivity of 63.6% and specificity of 92.6% in patients with chronic hepatitis B. Also, it should be noted that the cut-off value for a given variable depends upon the sample in which it has been identified. In order to be reproducible, a cut-off value should be obtained in a sample representative of the population with the disease.
Interestingly enough our results showed that, serum hyaluronate had best predictive value for the fibrosis and inflammation comparing to the other variables. This is in agreement with report of Ding H et al in which serum hyaluronate reflect both inflammation and fibrosis in HBV infection . Currently it is believed that serum hyaluronate is a marker of liver fibrosis rather than inflammation. To what extent increased level of hyaluronate is due to inflammation alone without fibrosis, is difficult question to answer at the moment. It is because of complex interrelation of fibrogenesis and inflammatory process in vivo, which makes separation of pure fibrosis and inflammation impossible in a clinical setting. Elucidation of this complex issue requires further work. As stated above, regression analysis showed that only serum hyaluronate was associated with significant liver fibrosis. It should not be misinterpreted that other important factors (e.g. age, serum albumin, etc) are not associated with liver fibrosis in CHB. Since, the association of such a powerful factor (e.g. serum hyaluronate) was so close to the outcome (liver fibrosis) that the effects of other factors were excluded from the regression model.
Our study has some limitations. First and the most important limitation is that our study is considered a training study, and our data should be validated in another set of patients with chronic hepatitis B. Secondly, number of our patients with extensive fibrosis was relatively small. Further works are underway to validate this test, and also to find out other markers of liver fibrosis and inflammation in patients infected with hepatitis B virus.