The patient was a 55-year old female with a 10-year history of excessive alcohol consumption who, following hospitalization due to decompensation and alcoholic hepatitis, became abstinent in May 2005. She remained stable until March 2006 when she developed worsening jaundice. Hematological findings indicated that hemolysis was a significant contributory factor in the patient’s jaundice. Subsequent investigations confirmed the diagnosis of SCA in the context of cirrhosis. She was referred for transplantion assessment in November 2006. She had an unconjugated hyperbilirubinaemia (230 U/L) and raised reticulocyte count (125 × 109/L). Hepatic synthetic function was relatively preserved with an INR of 1.5 and serum albumin concentration of 38 g/L. A blood film showed many acanthocytes with polychromasia and spherocytes. A transjugular liver biopsy showed micronodular cirrhosis and grade 1 siderosis. Her serum ferritin level at this time was greatly elevated at 600 ng/mL.
The patient decompensated further and was listed for transplantation. The blood film immediately before transplantation showed a predominance of acanthocytes (70%-80% of total red blood cells (RBC), and there was a gradual reduction in acanthocytes over the 48 h following transplantation so that examination of a blood film at 48 h after surgery indicated that acanthocytes made up 50%-60% of total circulating RBCs.
The patient gave consent for blood and urine samples collection for the study (LREC 08/MRE09/2). Samples were taken immediately before and at 12, 36, 48 h and 2 mo after transplantation. Blood was analyzed for ferritin, hemoglobin, erythropoietin, IL-6 and CRP to assess the extent of hemolysis and inflammation. Hepcidin was measured by SELDI-TOF using Cu2+
loaded IMAC ProteinChip Arrays and stable isotope labeled hepcidin as an internal standard as previously described[10
] and available via http://www.hepcidin.bham.ac.uk/
. Urine samples were normalized with respect to protein concentration (20 μg/mL, Bradford assay).
A sample of the explant liver was analyzed for hepcidin mRNA by q-RTPCR and this was compared with samples taken from 8 normal livers.
The hepcidin level in relation to hematological parameters is shown in Table . Hepcidin levels before transplantation were very low compared with the elevated ferritin. Following transplantation, hepcidin production rose with falling ferritin. At 2 mo after transplantation, the hepcidin production had fallen in the context of the normal serum ferritin. The urinary hepcidin/ferritin ratio rose progressively following transplantation.
Urinary hepcidin levels in relation to haemoglobin and ferritin
Figure shows that the rise and subsequent fall in urinary hepcidin following transplantation does not seem to be related to erythropoietin production which remains quite stable (except for one dip at 12 h coincident with a two unit peri-operative blood transfusion). Similarly, IL6 levels did not vary greatly. CRP rose as expected in relation to the operation. This rise was not immediately accompanied with the dramatic rise in hepcidin production which was delayed for 12 h. Figure illustrates a close correlation between urinary and serum hepcidin levels in this patient (Pearson correlation coefficient r = 0.958).
Variation in iron parameters with time.
Correlation between urinary and serum ferritin.
We compared hepcidin mRNA expression in the explant liver with that in 8 normal livers from transplant donor tissues. Hepcidin mRNA expression in the explant liver from our patient was significantly lower than that in normal liver (data not shown).