Is serum hepcidin causative in hemochromatosis ? Novel analysis from a liver transplant with hemochromatosis

1Department of Medicine; 2Department of Surgery; 3Department of Pathology, University of Western Ontario, London, Ontario Correspondence: Dr Paul C Adams, Department of Medicine, University Hospital, 339 Windermere Road, London, Ontario N6A 5A5. Telephone 519-685-8500 ext 35375, fax 519-663-3549, e-mail padams@uwo.ca Received for publication January 15, 2008. Accepted April 11, 2008 PC Adams, V McAlister, S Chakrabarti, M Levstik, P Marotta. Is serum hepcidin causative in hemochromatosis? Novel analysis from a liver transplant with hemochromatosis. Can J Gastroenterol 2008;22(10):851-853.


Is serum hepcidin causative in hemochromatosis?
Novel analysis from a liver transplant with hemochromatosis T he pathogenesis of genetic hemochromatosis remains elu- sive despite the discovery of the HFE gene in 1996 (1).Several investigators have suggested that serum hepcidin -a hepatic hormone that controls iron balance (2) -may be the primary abnormality in patients with genetic hemochromatosis.An inappropriately low hepcidin level has been demonstrated in serum from hemochromatosis patients.A low serum hepcidin leads to an increase in intestinal iron absorption and subsequent hepatic deposition of dietary iron (3-6).There have been previous reports of liver transplantation occurring from a C282Y homozygous donor into a recipient (7) and transplantation of a normal liver into a C282Y homozygous recipient.
Iron reaccumulation has been an inconsistent observation, although the follow-up time may not have been adequate (8,9).The simultaneous transplantation of the liver and small intestine from a C282Y homozygous donor has led to iron overload in the recipient within two years (10).The case described in the present report provides a clinical opportunity to observe the effects of liver transplantation of a C282Y homozygous liver into an unaffected recipient without phlebotomy treatment, and without anemia or clinical bleeding.The development of a new assay for serum hepcidin provides new information on the role of hepcidin in the pathogenesis of hemochromatosis.

METHODS
Liver iron absorption was measured by atomic absorption spectrophotometry.Genetic testing for the C282Y and H63D mutation of the HFE gene was done from a buffy coat prep on the liver donor and recipient, as previously described (11).Serum hepcidin was measured using an enzyme-linked assay, utilizing a polyclonal antibody developed by Dr Tomas Ganz (Intrinsic LifeSciences, USA) (12).The assay is available for research purposes only, and it has been estimated that a typical serum hepcidin level is 50 ng/mL in women and 100 ng/mL in men.

CASE PRESENTATION
A 62-year-old woman underwent an elective liver transplantation for chronic hepatitis C cirrhosis in May 2005.She had been successfully treated for hepatitis C before the liver transplantation and was HCV RNA-negative.The explanted liver demonstrated no evidence of iron overload.The donor was an 89-year-old woman who had a cerebral aneurysm and no clinical evidence of liver disease.At the time of donor liver retrieval, an analysis of a frozen section demonstrated iron overload (Figure 1).Post-transplantation review of the donor biopsy confirmed iron overload with a liver iron concentration of 326 μmol/g (normal range 0 μmol/g to 35 μmol/g).The donor was confirmed to carry two copies of the C282Y mutation of the HFE gene (C282Y homozygote) and the recipient carried neither the C282Y nor the H63D mutation (wild-type).Transplant surgery was complicated by hepatic artery stenosis and dissection.Therefore, on day 2, intraoperative repair was performed.A liver biopsy was also performed, which demonstrated a liver iron concentration of 333 μmol/g.The subsequent course of the patient as an outpatient was dominated by multiple endoscopic retrograde cholangiopancreatography examinations to dilate a persistent biliary anastomotic stricture, which was likely secondary to early hepatic ischemia.At day 652, a liver biopsy was performed because of a new pattern of elevated liver enzymes.The liver iron concentration was measured at 253 μmol/g with no fibrosis.At day 935 post-transplant, the patient was noted to have a serum ferritin level of 4999 μg/L (reference range 15 μg/L to 200 μg/L) and a transferrin saturation of 100% (reference range, 20% to 55%).Other blood test results at day 935 included the following: hemoglobin 149 g/L, aspartate aminotransferase 30 U/L, alanine aminotransferase 34 U/L, alkaline phosphatase 53 U/L, erythrocyte sedimentation rate 1 mm/h and C-reactive protein less than 3 mg/L.Serum hepcidin in the transplant recipient at day 940 was 111 ng/mL.Serum hepcidin was measured in three other ambulatory liver transplant patients without iron overload and was 66 ng/mL, 76 ng/mL and 81 ng/mL.The patient has begun a program of 500 mL phlebotomy every two weeks to decrease liver iron.

DISCUSSION
In the present case, it is intriguing to speculate on whether we have transplanted the genetic defect of hemochromatosis into the recipient.An HFE knockout mouse specific for hepatocytes has been demonstrated to develop iron overload (13).In the present patient, the absence of progression of iron overload over 1.8 years goes against this hypothesis.A novel aspect of the present case is the ability to measure serum hepcidin.It has been proposed that hepcidin and iron are analogous to insulin and glucose as regulatory hormones and substrates (4).In this hypothesis, patients with typical hemochromatosis and some other forms of iron overload have a low and inappropriate serum hepcidin level, which leads to increased intestinal iron absorption.In the present case, our patient had the highest serum hepcidin of the four liver transplant recipients that were tested.An elevated serum hepcidin would be expected to decrease iron absorption, which is consistent with the absence of iron accumulation in our patient.It is possible that liver transplantation, rejection and inflammation could elevate hepcidin, but presumably this would apply to all of the transplant patients, and our patient had a low erythrocyte sedimentation rate and a low C-reactive protein level, without significant inflammation on liver biopsies.If the recipient had received the primary defect of hemochromatosis as a result of the transplanted liver, she would be expected to have a low serum hepcidin and progressive iron overload over time.The present report raises the possibility that other factors control the regulation of hepcidin in hemochromatosis, and extrahepatic production of hepcidin may be another explanation.We have previously reported progressive iron overload after the simultaneous transplantation of the liver and intestine from a C282Y homozygous donor (7), suggesting that there may be a signal from the hemochromatosis intestine that affects hepcidin and iron absorption.

CONCLUSIONS
Based on the present case, it seems unlikely that hepcidin insufficiency is the primary genetic defect of C282Y-linked hemochromatosis.

Figure 1 )
Figure 1) Liver biopsy at the time of liver transplantation demonstrated a liver iron concentration of 326 μmol/g (reference range 0 μmol/g to 35 μmol/g; Perl's stain for iron)