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To investigate the role of oxygen free radicals in hepatocellular carcinoma we assayed tissue scavenger enzymes (superoxide dismutase and selenium-dependent glutathione peroxidase) in liver homogenate, plasma concentrations of vitamins A and E and the serum selenium level from 19 control patients, 23 cases of hepatocellular carcinoma and 18 cases of metastases to liver from different carcinomas. In hepatocellular carcinoma tissue the enzyme activities were all significantly lower than in control liver and in metastases-bearing liver; the enzyme activities of the latter tissues were not different from control liver. In contrast, normal liver adjacent to the hepatocellular carcinoma had decreased activity of superoxide dismutase. Serum selenium concentrations were significantly decreased in patients with hepatocellular carcinoma and those with liver metastases, while vitamin A was significantly decreased only in the former. These findings suggest that hepatocellular carcinoma develops in liver with severe impairment of cellular antioxidant systems, since, in patients with liver metastases from different cancers, despite low selenium concentrations, cellular scavenger enzymes have normal activities.

Erdheim-Chester disease is a rare disorder of unknown cause, characterized by systemic histiocytic infiltration; the long bones of the limbs are usually affected symmetrically, and a direct radiography of the involved skeletal segments is able to show a pattern of typical abnormalities. However, the patient we describe suffered of serious clinical symptoms in the lower limbs, but the direct radiography of the legs did not show any abnormality; this finding seems very remarkable and, to our knowledge, has not been reported previously in the literature. Therefore we discuss the role of the imaging procedures in the diagnosis of Erdheim-Chester disease. Differently from other authors, we did not obtain any clinical improvement in our patient by steroid treatment alone, that is generally considered the first therapeutic option for Erdheim-Chester disease with only skeletal involvement.

Intercellular adhesion molecule 1 (ICAM-1) is a marker of inflammation and tissue damage. Levels of soluble ICAM-1 (sICAM-1) were measured in 71 patients with chronic C hepatitis treated with interferon (IFN)-alpha-2a, at baseline and at every 3 months of therapy, and in 42 normal control subjects. The levels of sICAM-1 were significantly higher in the patient than in the control subject group, particularly among cirrhotics. Baseline sICAM-1 levels were similar in responders and nonresponders. By contrast, the concentration of sICAM-1 decreased significantly only in responders during the first 3 months of therapy. The probability of response to treatment, analyzed by Kaplan-Meier analysis, was much higher in the group showing a decrease of sICAM-1 than in the patients who did not show such a decrease. In conclusion, a "longitudinal" evaluation of serum levels of sICAM-1 in the first period of treatment is particularly useful in the identification of patients with high significant probability of response to treatment.

Laminin P1 (pepsin-resistant fragment of laminin) and aminoterminal peptide of type III procollagen are measurable in serum and are now considered useful serum markers of fibrogenesis and inflammation in chronic liver diseases. However, very few studies thus far have focused on assessing the diagnostic value of these markers in detecting fibrosis and necro-inflammatory activity in chronically diseased liver. The aim of the present study was therefore to investigate the correlations of laminin and type III procollagen with liver histology and to compare their diagnostic value in detecting the degree of liver fibrosis and necro-inflammatory activity in a homogeneous group of 99 patients suffering from chronic hepatitis C, and lacking other factors which can directly affect the serum levels of the two markers. Both these serum markers were measured by radioimmunoassay, employing commercially available kits. The three main aspects of liver pathology, i.e. portal-periportal activity, lobular activity and fibrosis, were histologically evaluated and semiquantitatively expressed by numerical scores. The results of this study show that laminin and type III procollagen were both positively correlated with the histological scores for portal-periportal activity and with those for fibrosis, whereas no significant correlation was observed between each of the two serum markers and the histological scores for lobular activity. The sensitivity and specificity of laminin and type III procollagen in detecting histological aspects of fibrosis and disease activity in liver, computed at various cut-off levels, showed overlapping trends for the two markers; however, the diagnostic value was in general rather low, whatever the cut-off considered. We therefore conclude that the ‘static’ measurement of both serum laminin and type III procollagen is of limited value for individual diagnosis of liver damage.

We investigated the prevalence of mixed cryoglobulinemia (MC) in 100 cases of chronic hepatitis C virus (HCV) infection and the effect of a 6-month treatment with interferon-alpha (IFN-alpha). Cryoglobulins were detected on admission in 36 of 100 patients and appeared during observation in a further 18 cases. Cryocrit ranged from 0.5% to 20%. Patients with MC were older and had a higher incidence of cirrhosis than those without MC. Immunologic characterization of the cryoprecipitate showed the presence of type II in 84% of cases and type III in 16%. The patients received IFN-alpha (6 MU three times per week) for 6 months. Fifty-seven were responders (i.e., reached normal aminotransferase levels), 26 of these relapsed within 2 months after IFN withdrawal, and 30 did not relapse. After IFN-alpha treatment, cryoglobulinemia disappeared in 11 of the 21 evaluable responders, but in none of the 15 nonresponder patients (p0.003). The clearance of MC was associated in all cases with clearance of HCV RNA. The delayed appearance of cryoglobulinemia in responders seems to be associated with a higher probability of relapse.

Laminin is a major basement membrane-associated, non-collagenous glycoprotein of the extracellular matrix and is deposited in the space of Disse during sinusoidal capillarisation. Laminin P1, a pepsin-resistant fragment originating from the central portion of the cross-shaped laminin molecule, is detectable in serum and has been related to liver fibrosis and portal hypertension. In this study we investigated the behaviour of serum laminin P1, measured by radioimmunoassay, in a homogeneous group of 95 patients suffering from chronic viral hepatitis, types C or B, in order to determine the relationships between serum laminin P1 and each of the main histological aspects of the disease process (i.e. portal-periportal activity, lobular activity and fibrosis), which were assigned numerical scores. Moreover, we computed, at several cut-off levels, the sensitivity, specificity and positive and negative predictive values of laminin P1 in detecting both necroinflammatory activity and fibrosis in the liver. The results show that serum laminin P1 levels parallel the severity of liver disease, the highest laminin concentrations being observed in cirrhotic patients. They suggest also that serum laminin P1 should be considered a marker of the liver disease process as a whole, rather than a marker exclusively linked to fibrosis. Nevertheless, the usefulness of serum laminin P1 measurement, as investigated in this study, seems too limited to be recommended for routine clinical practice.

Forty patients with chronic viral hepatitis or active cirrhosis (33 anti-HCV positive) entered a recombinant human alpha 2A interferon randomized trial. Twenty-one subjects were treated with 6 million units (MU) three times per week for 6 months. Nineteen were not treated. Six months later in 12 patients of the treated group (60% of the evaluable 20) with normalized serum aminotransferases levels (responders), fibrogenesis serum markers (NPIIIP and laminin) were significantly lower than baseline. In the untreated patients and in non-responders NPIIIP and laminin were unchanged. Semi quantitative histological evaluation (allotting scores for inflammation, necrosis and fibrosis) confirmed a significant improvement of necro-inflammation in the responders. These data suggest that alpha-IFN treatment may decrease stimuli for fibrogenesis by reducing liver inflammation and necrosis, thus preventing evolution to cirrhosis.

Hepatic fibrosis is characterized by a progressive increase in extracellular matrix in the liver, formed by collagens, proteoglycans and glycoproteins, qualitatively similar but quantitatively different from that in normal liver. A great number of matrix-related substances have been investigated in serum in order to identify reliable serum markers of liver fibroplasia. Among the various cleavage products of collagen precursor, the NPIIIP collagen is at present considered the most reliable serum marker of active fibrogenesis in liver, useful in monitoring the progression of fibrosis and in assessing the therapeutic efficacy of antifibrotic drugs. Lam-P1 and type IV collagen are now regarded as putative markers of basement membrane formation and sinusoids capillarization, an important pathological process in fibrosing disease, related to the impairment of hepatic circulation. Other serum-measured matrix-related substances, e.g. enzymes involved in collagen metabolism, fibronectin and proteoglycans, have not proved to reflect liver fibroplasia reliably. In spite of the availability of useful serum markers, the assessment of hepatic fibrosis is still based on liver biopsy.

Glutathione peroxidase (GSH-Px) activity, one of the scavenger enzymes of oxygen active radicals, has been measured in hepatocellular carcinoma (HCC) of 17 patients and the values compared with the activity of adjacent tumor-free tissue and with those of 30 histologically normal livers. The results demonstrate a reduced GSH-Px activity in neoplastic tissue (21.19 vs 33.74 U/g prot.; P less than 0.001). However, the adjacent tumor-free liver also had a reduced activity when compared with normal tissue (23.15 vs 33.74 U/g prot.; P less than 0.01), but this value did not differ from that of HCC tissue. These data suggest that HCC might develop in a GSH-Px-deficient condition.

In 55 patients with alcoholic cirrhosis and in 47 healthy individuals we assayed the concentration of selenium in serum (S-Se) by proton induced X-ray emission, the aminoterminal peptide of type III procollagen (NPIIIP) by RIA and the plasma fibronectin (FN) by immuno-nephelometry, together with routine biochemical tests. S-Se was lower in cirrhosis than in controls (0.57, SD 0.20 vs 0.92, SD 0.16 mumol/l; p less than 0.001) and was more reduced in ascitic than in compensated patients (0.50, SD 0.19 vs 0.66, SD 0.17 mumol/l; p less than 0.001). Regression analysis showed a positive correlation of S-Se with serum albumin and FN, whereas necrotic or inflammatory activity seems unrelated to S-Se; a negative correlation was found between S-Se and NPIIIP, suggesting a protective role of selenium against fibrosis.

To investigate the diagnostic and prognostic value of several biochemical tests in primary liver tumors, the authors studied 36 cases (4 cholangiocarcinomas and 32 hepatocellular carcinomas, 10 of which were associated with cirrhosis) and 47 cases of liver cirrhosis, all with morphologically proven diagnosis. Serum copper (SCu) and plasma fibrinogen (PF) appeared the most useful tests in differential diagnosis between tumors and cirrhosis. In liver tumors, mean SCu level was 200.50, standard deviation (SD) 47.17 micrograms/dl (121.40, SD 25.90 micrograms/dl in cirrhosis; P less than 0.001). PF level was 461.78, SD 151.25 mg/dl in tumors (275.30 SD, 124.40 mg/dl in cirrhosis; P less than 0.001). SCu had a good sensitivity (0.80) and a high specificity (0.92) at a cutoff value of 160 micrograms/dl; when the cutoff level was raised to 170 micrograms/dl, the specificity increased to 1, with a sensitivity of 0.77. The combination of SCu and PF improved the diagnostic value slightly. Moreover, with an estimated frequency of tumor in cirrhosis of 10%, SCu had a positive predictive value of 1 (cutoff, 170 micrograms/dl) and a negative predictive value of 0.97. In nine patients SCu levels decreased after surgical removal of tumor; five other patients, sequentially studied, showed an increase of SCu level that correlated with the progression of the disease. Finally, patients with longer survival had a lower SCu level. These findings suggest that SCu level may be used as a screening test for early detection of neoplastic degeneration, and it is correlated with the extension of tumor mass.

Liver catalase activity, one of the free-radical scavenger enzymes, has been measured in 22 normal subjects and compared with that of 13 patients suffering from hepatocellular carcinoma. The activity was estimated both in tumor tissue and in tumor-free tissue. A significant reduction of catalase activity was noted in tumor tissue (p less than 0.001) as well as in the adjacent tumor-free tissue (p less than 0.02). In patients with hepatoma, the serum iron level was lower than in normal (p less than 0.01) and was correlated with enzyme activity (r = 0.958). These findings suggest that in hepatocarcinoma the free radical scavenger system is impaired.

To evaluate the prognostic value of serum copper (S-Cu) and ceruloplasmin and their pathophysiologic significance in human hepatocellular carcinoma (HCC), we studied 49 patients with HCC (20 of which were submitted to partial hepatectomy) compared with 110 patients with liver cirrhosis. In HCC both S-Cu and ceruloplasmin were higher than in cirrhosis; moreover, S-Cu was correlated with the extension of HCC, evaluated by instrumental data and by surgical inspection. In cirrhotic patients, mean S-Cu was 122.9 micrograms/dl (SD, 29.3), in early HCC, 153.0 micrograms/dl (SD, 34.5), and in advanced HCC, 193.1 micrograms/dl (SD, 37.7). Variance analysis gave F = 59.4. In HCC patients S-Cu was positively correlated with ceruloplasmin and with fibrinogen. Survival, evaluated by Mantel's test stratified for surgical therapy, was longer in patients with S-Cu levels lower than 175 micrograms/dl and in those at an earlier stage. We therefore conclude that S-Cu has a relevant diagnostic value in detecting HCC also in early stage and allows prognostic evaluation as regards survival.

Catalase (CAT), glutathione-peroxidase (GSH-Px) activity and reduced glutathione content (GSH) were measured in patients who had hepatocellular carcinoma, and values compared with those of normal liver and liver adjacent to neoplastic tissue. The results showed a remarkable reduction of CAT in tumor and corresponding tumor-free tissue (P less than 0.001 and P less than 0.02, respectively). All neoplastic samples had a significant lower activity of CAT than the corresponding adjacent tumor-free tissue (P less than 0.05). The GSH-Px activity of tumor tissue also was lower than normal (P less than 0.001) but similar to that of adjacent tissue. No correlation was noted between the two enzyme activities. Glutathione content was extremely low in tumor (P less than 0.001) and even in tumor-free tissue (P less than 0.05) when compared with normal liver. In all cases the content of GSH in neoplastic tissue was lower than that of the corresponding tumor-free tissue (P less than 0.05). Whereas in normal liver the activity of GSH-Px was positively correlated with the content of GSH, in the neoplastic tissue such a relationship disappeared. All these findings suggest that the antioxidant system of hepatocellular carcinoma cell is severely impaired.

In order to assess the prognostic value of clinical and laboratory variables in liver cirrhosis, 36 of these variables were statistically analyzed in 151 patients followed up for 8 years. The ‘survival time’ was taken as the reference variable. In a first step we analyzed by log-rank test and by Cox’s proportional hazard regression model the data of 98 patients (study group), obtaining 7 prognostically significant variables (age, leukocytes, calcium, potassium, globulins, cholesterol and previous diagnosis). From the regression coefficients of these variables, a risk score was obtained for each patient. To validate the prognostic value of this score, we computed it, using the same coefficients obtained in the study group, in 53 subsequently examined patients (control group) showing that the prognostic score allows the classification of these patients in 3 risk classes with different observed survival times.

42 different samples of human erythrocytes were tested for glutathione-peroxidase activity (GSH-Px) in an attempt to study the inhibitory effect of Bromsulphalein (BSP). The mean activity of the enzyme was 11.90 +/- 3.61 U/g Hb, with no significant difference between males and females. BSP was used at different concentrations from 1 to 45 mM and inhibited GSH-Px activity; the inhibition curve showed a sinusoidal pattern. The major effect was obtained at 30 mM BSP when almost 65% of the initial activity was inhibited. The inhibition of GSH-Px by BSP has also been confirmed using partially purified GSH-Px obtained from human erythrocytes, as well as purified bovine GSH-Px. Some difference was noted between males and females: females may be divided into two subgroups, one with a lower and a second with a higher level of GSH-Px. 1 mM BSP increased the activity in the first group, whereas it reduced the activity in the second group. The inhibition by BSP was positively correlated with the basal value of GSH-Px and this effect was particularly evident in females (r = 0.865; p less than 0.001). The possibility that GSH-Px may be inhibited by BSP would be of some importance considering the strategic role of GSH-Px in protecting the cell from oxidative attack.

In order to clarify the significance of procollagen III peptide (PIIIP) and fibronectin (FN) blood concentration in alcohol related chronic liver disease (ALD), we have investigated their relationships with histological liver features and biochemical liver tests in 44 ALD patients. PIIIP was measured in serum by radioimmunoassay whereas FN was determined in plasma using an immunonephelometric method. In each liver biopsy, steatosis, portal infiltrate, lobular necro-inflammation, portal fibrosis and lobular fibrosis were semiquantitatively assessed by scoring from 0 to 3. A close correlation of PIIIP was found with morphological features of fibrosis (both of lobular and portal type), but not with necro-inflammation or steatosis. PIIIP was also positively correlated with ALP and GGT and exhibited a good diagnostic value in liver fibrosis. On the contrary, FN did not distinguish between normals and patients and was not correlated with any morphological liver feature or biochemical liver test. We also conclude that serum NP3P effectively reflects liver fibrosis, whereas plasma FN seems not related to any of the main histological aspects of liver damage in ALD.

In a group of 23 patients with advanced liver cirrhosis we have found a statistically significant positive correlation (r = 0.746; p less than 0.0001) between fibronectin and prealbumin levels measured in plasma by immunonephelometric methods and found significantly lower than in healthy controls (p less than 0.001). On the contrary, no correlation of fibronectin neither to albumin nor to the presence of an enlarged spleen was observed. Since the sensitivity of prealbumin as an index of liver function is believed to be higher than that of albumin, our results support the view that the decreased fibronectin in advanced cirrhotics is mainly due to their liver failure, an enlarged spleen playing only a minor role.

Plasma fibronectin (FN) has been measured by immunonephelometric method in 100 cirrhotic patients and compared with that of 77 normal subjects and with that of 57 patients suffering from liver disorders different from cirrhosis. Both, compensated and decompensated cirrhotics had lower plasma FN than controls (31.14 +/- 11.42 and 20.88 +/- 10.43 respectively vs 40.13 +/- 8.58 mg/dl; rho less than 0.02 and rho less than 0.001). FN in ascitic patients was lower than in non-ascitic (rho less than 0.001). These differences were not due to different weight or age of patients. It appears, therefore, that FN parallels in cirrhosis the grade of liver function impairment. No significant difference has been noted between plasma FN of patients with liver diseases different from cirrhosis and control subjects. In cirrhosis, a positive relation has been observed among FN and other parameters of liver function such as serum albumin, cholinesterase activity, fibrinogen and prothrombin time. Plasma FN has a low sensitivity but a high specificity and a good positive predictive value in distinguishing normals and patients with liver disorders different from cirrhosis. This diagnostic value is similar to that of serum albumin.