Iron accumulation in the liver of male patients with Wilson's disease.

OBJECTIVES: There is accumulating evidence that ceruloplasmin, a copper protein with ferroxidase activity, plays an important role in iron metabolism. The genetic disorder, aceruloplasminemia, can lead to tissue storage of iron as in hemochromatosis. Because most patients with Wilson's disease, a genetic copper toxicosis, have hypoceruloplasminemia, some could be affected by iron overload. METHODS: Four male patients with Wilson's disease were enrolled in this study of pre- and post-treatment iron metabolism. RESULTS: Pretreatment copper contents of the liver were high in all four male patients studied as diagnostic of Wilson's disease. Genetic analysis supported their clinical diagnosis of Wilson's disease without a background of hemochromatosis. Pretreatment serum ceruloplasmin levels were <20 mg/dl in all four patients. A standard penicillamine treatment for 3-8.5 yr further decreased their serum ceruloplasmin levels. Post-treatment serum ferroxidase activity was low as was the serum ceruloplasmin protein. Copper contents in the liver decreased after treatment in all subjects. In contrast, nonheme iron in the liver increased during treatment. Pretreatment liver specimens were positive for histochemical iron in two patients, and post-treatment specimens were positive in all four patients. In two patients, serum aminotransferase levels rebounded with elevation of serum ferritin concentration during the treatment period. Subsequent iron reduction by phlebotomy ameliorated their biochemical liver damage. CONCLUSION: Iron overload related to hypoceruloplasminemia may be clinically important, particularly in male patients with Wilson's disease.

C282Y and H63D mutations in the HFE gene have no effect on iron overload disorders in Japan.

OBJECTIVE: The gene responsible for hereditary hemochromatosis close to the human leukocyte antigen A locus was previously identified and designated as HFE. This study was performed to evaluate the clinical significance of two mutations, C282Y and H63D of HFE, in Japanese patients with hepatic iron overload. PATIENTS AND METHODS: We examined C282Y and H63D in 11 patients with primary hemochromatosis, 94 patients with chronic hepatitis C, 54 patients with miscellaneous liver diseases, and 151 healthy volunteers. The HFE gene region of DNA samples extracted from peripheral leukocytes was amplified by polymerase chain reaction. Restriction enzyme analysis was performed using SnaBI for C282Y and BclI for H63D. Direct sequence analysis was then performed when products suggested the presence of a mutation. RESULTS: All the subjects studied were free from C282Y. None of the patients with hemochromatosis had H63D. One patient with chronic hepatitis C was homozygous, and 4 patients were heterozygous for H63D. Two patients with alcoholic liver disease were heterozygous for H63D. The prevalence of chromosomes with H63D was 6/188 (3.2%) in patients with chronic hepatitis C, 2/108 (1.9%) in patients with miscellaneous liver diseases, and 8/302 (2.6%) in healthy volunteers. These differences were not significant. CONCLUSION: Our results suggested that neither C282Y nor H63D in HFE affect Japanese patients with hemochromatosis or chronic hepatitis C.

Body iron stores and iron restoration rate in Japanese patients with chronic hepatitis C as measured during therapeutic iron removal revealed neither increased body iron stores nor effects of C282Y and H63D mutations on iron indices.

Information on the level of iron stores in chronic hepatitis C is clinically important because its reduction is technically simple and therapeutically effective. This study was performed to measure the levels of iron stores from the total amounts of hemoglobin removed during iron reduction therapy. The C282Y and H63D mutations of HFE gene were analyzed in 94 patients. All of the patients were negative for C282Y mutation. One patient was homozygous, and 4 patients were heterozygous for H63D mutation. The body iron stores and iron restoration rate were measured in 59 patients in serial courses of iron reduction therapy. Mean values of body iron stores in the two groups with and without H63D mutation were 890 and 606 mg, while those of iron restoration rate were 1.85 and 1.52 mg/day, respectively. None of the indices of iron metabolism were different from the reference values measured similarly in healthy subjects, suggesting that the iron deposition in chronic hepatitis C is limited to the liver, probably due to changes in the iron distribution in tissues.

Combined phlebotomy and ursodeoxycholic acid treatment in the patients with chronic hepatitis C.

As an option to interferon, either iron removal by phlebotomy or ursodeoxycholic acid administration has been recommended for patients with chronic hepatitis C. Some patients, however, show only partial responses to such monotherapy. In the present study, we investigated the effects of a combination of phlebotomy and ursodeoxycholic acid in the patients who did not show normalization of serum alanine aminotransferase levels by either phlebotomy or ursodeoxycholic acid monotherapy. The combination of these therapies in any order additively improved the biochemical parameter to the upper normal range. There were no statistically significant differences between the results of the two combination treatments. The combination treatment was also effective in decreasing serum alpha fetoprotein levels.

The advantage of gastrectomized patients in management of their chronic hepatitis C.

Because the majority of patients with chronic hepatitis C do not respond to interferon, alternative treatments need to be established. Several lines of evidence suggest that iron depletion is beneficial for such patients. Thus, gastrectomized patients with a reduced capacity for iron absorption might have an advantage in treatment of their liver damage over patients with intact gastrointestinal tracts. Four male gastrectomized patients had post-transfusion chronic hepatitis C. The iron load in three patients was adjusted below 10 ng/ml of serum ferritin level by phlebotomy. Subsequent interferon treatment for the four patients without iron load cleared circulating hepatitis C virus RNA in one patient only. However, serum ferritin concentrations were stabilized at low levels without maintenance phlebotomy, and sustained normalization of serum liver enzyme activities was obtained in all four patients. Similar treatments were done for 10 male patients with intact gastrointestinal tracts. The amount of removed iron from these patients was more than that from gastrectomized patients. Interferon also failed to clear circulating hepatitis C virus RNA except in one case. Low ferritin levels and sustained normalization of liver enzymes were seen in three patients. A transient elevation of ferritin levels with low enzyme activities was seen in two patients. Relapsing hepatitis was seen in five of the seven patients who needed maintenance phlebotomy due to a rebound in serum ferritin levels, probably because of active iron absorption from the intestine. Our data suggest that depletion of cytotoxic iron is a key to managing patients with chronic hepatitis C.

Serum aminotransferase levels as an indicator of the effectiveness of venesection for chronic hepatitis C.

BACKGROUND/AIMS: Iron cytotoxicity may play an important role in chronic hepatitis C. The effects of venesection suggest that a slight iron overload contributes to hepatic injury in subjects infected with hepatitis C virus. A better indication of the efficacy of venesection was studied in patients with and without overt iron overloading. METHODS: All 40 patients had chronic hepatitis C but none had hemochromatosis of a known etiology. A serum ferritin level of 10 ng/ml or less was chosen as the treatment goal. A mean blood volume of 2400 +/- 1100 ml was removed during treatments lasting 5 +/- 3 months. RESULTS: Treatment significantly reduced the mean serum levels of alanine aminotransferase activity from 128 +/- 74 to 63 +/- 28 IU/l (p < 0.01). The baseline enzyme activity was highly correlated with reduction in activity after treatment (r = 0.94, p < 0.01), but the baseline levels of ferritin and histochemistry for iron showed poor correlations with the reduction in enzyme activity (r = 0.63 with p < 0.01 and r = 0.38 with p < 0.05, respectively). CONCLUSIONS: Thus, serum levels of aminotransferases were a more important indicator for venesection than conventional indices of iron overload, probably because cytotoxic iron includes some reactive iron species rather than stored iron alone.

Biochemical improvement of chronic hepatitis C after gastrointestinal bleeding.

Although chronic hepatitis C is frequently complicated by iron overload, it remains unclear whether iron cytotoxicity is involved in the disease process. Five patients with chronic hepatitis C showed rapid reduction of serum aminotransferase activity after gastrointestinal bleeding. Posthemorrhagic reduction of liver enzyme levels lasted for more than one week. Anemia was associated with a reduction of serum ferritin concentration. Considering the short half-lives of circulating liver enzymes, reduced release of enzymes, that is inactivation of cell lysis, is the likely cause of the improved biochemical indices. Reactive iron, which is cytotoxic for patients infected by HCV, may be rapidly incorporated into hemoglobin when erythropoiesis is stimulated. Our observation also suggests that intensive iron removal by phlebotomy is a safe, economic treatment for patients with chronic hepatitis C.

Correlation between serum levels of alanine aminotransferase and ferritin in male blood donors with antibody to hepatitis C virus.

Chronic hepatitis C has been demonstrated to be associated with hepatic iron overload, and the hypothesis that the disease activity of hepatitis C is associated with iron cytotoxicity was tested in male volunteer blood donors. Sera with either antibody to hepatitis C virus or hepatitis B surface antigen were selected for determination of ferritin concentration and alanine aminotransferase activity. A correlation between serum ferritin concentration (Y; microgram/l) and alanine aminotransferase activity (X; IU/l) was found in donors with antibody to hepatitis C (log Y = 0.65 x log X + 0.98, r = 0.53, and P < 0.01). The correlation was lower in donors with hepatitis B surface antigen (r = 0.37; P < 0.01). Hepatitis C virus infection probably induces time-dependent iron accumulation associated with the progression of disease activity, while hepatitis B virus infection results in a variety of iron loads with different clinical features. The high disease activity related to hyperferritinemia suggests the presence of iron-induced liver damage in donors with hepatitis C.

Improvement of serum aminotransferase levels after phlebotomy in patients with chronic active hepatitis C and excess hepatic iron.

OBJECTIVES: Iron metabolism may be altered in patients with chronic active hepatitis C. In an attempt to evaluate whether excess iron contributes to liver injury, we used phlebotomy for removal of iron from patients with chronic hepatitis C. METHODS: All 10 patients had histochemically detectable iron in the liver and underwent an initial period of weekly or monthly phlebotomy of 200 or 400 ml. A serum ferritin level of 10 ng/ml or less was chosen as the endpoint, and maintenance phlebotomy was performed if the level rebounded. RESULTS: The treatment reduced mean serum alanine aminotransferase activity from 152 +/- 49 to 55 +/- 32 IU/L; this level became normal in five of the 10 patients. Anti-HCV antibodies could be detected in all patients throughout the study. Histologic abnormalities of the liver were unchanged except for disappearance of iron deposits from seven of the patients studied. CONCLUSIONS: Our findings suggest that iron removal may be beneficial for patients with chronic active hepatitis C and histochemical iron in the liver.

Hepatic copper accumulation in patients with primary biliary cirrhosis.

Liver biopsy specimens from 18 patients with primary biliary cirrhosis were examined histochemically and by energy-dispersive x-ray microanalysis. Using two indices, we classified hepatic copper accumulation into three stages based on the Cu x-ray intensity of cuproproteins that had accumulated in hepatocyte lysosomes and on the binding ratio of postulated copper transfer proteins between the cytosol and lysosomes. Eight patients were in stage 1 with an initial accumulation of lysosomal cuproproteins, mediated by transfer proteins not saturated with copper. Two patients were in stage 2, in which transfer proteins were saturated with copper. The first two stages gave negative results for histochemical copper. The remaining eight patients were in stage 3, in which copper accumulation detected by histochemical included transfer proteins saturated with copper and large amounts of lysosomal cuproproteins. Five patients (one each in stages 1 and 2, and three in stage 3) underwent a second liver biopsy after treatment with 600 mg of ursodeoxycholic acid daily for 14 to 39 months. Results of blood chemistry tests improved, but liver histologic findings and copper accumulation were unchanged in all five patients. It seems likely that ursodeoxycholic acid does not affect the copper accumulation in hepatocyte lysosomes that reflects the state of cholestasis in patients with primary biliary cirrhosis.

Intrahepatocellular localization of copper in Wilson's disease.

It is well known that excess copper plays a role in the pathogenesis of Wilson's disease; however, the hepatic copper contents, determined either histochemically or biochemically, are not always correlated with the activity of Wilson's disease. To better understand copper-induced cytotoxicity, intrahepatocellular localization of copper was studied in five patients with Wilson's disease. The liver specimens were obtained by biopsy to confirm the clinical diagnosis of Wilson's disease before treatment. Neither hepatic copper content nor histochemical copper deposits were correlated with any of the biochemical indices studied. Energy-dispersion X-ray microanalysis was done on the nuclei, lysosomes and lysosome-free cytoplasm of hepatocytes. Lysosomes had the highest Cu X-ray intensity but no correlation was shown between the lysosomal copper content and biochemical indices. The nucleus/lysosome-free cytoplasm ratio of the copper content was correlated with the serum levels of aminotransferases. These results suggest that the copper increasing gradient from the lysosome-free cytoplasm to the nucleus is associated with hepatocyte necrosis, and probably causes irreversible nuclear damage.

Suppressive effect of superoxide dismutase on adriamycin nephropathy.

A single intravenous injection of adriamycin (ADR) results in marked proteinuria and glomerular morphological changes that are similar to minimal-change disease in humans. We examined the effect of superoxide dismutase (SOD) on ADR-induced proteinuria. ADR in a dose of 7.5 mg/kg body weight significantly increased urinary protein by day 14; proteinuria rapidly increased thereafter. Concurrent administration of SOD (50 mg/kg) over 30 min prior to and 30 min following ADR injection markedly reduced proteinuria. Twenty-one days after the treatment with SOD, the amount of urinary protein was 108.6 +/- 43.1 mg/24 h in the experimental animals, while it was 221.6 +/- 102.9 mg/24 h in the ADR control group (p less than 0.05). There were also less severe glomerular morphologic changes in the SOD group versus ADR controls. The protective effects of SOD provide indirect evidence that oxygen free radicals are important mediators of ADR-induced proteinuria.