Section 4 -

Iron Overload Linda D. Ferrell University of California San Francisco, CA

Introduction Iron metabolism is a complex process that is currently becoming more understood, but many aspects of these processes still remain unknown. The details of iron metabolism is beyond what can be discussed, but D Trinder, et al have written a good review of the current status of these investigations (see Ref 2). Iron balance is essentially controlled by the regulation of iron absorption by the duodenum and proximal jejunum, but no good mechanism is present for the excretion of excess iron stores. Iron is a potentially highly toxic chemical, so it must be sequestered in non-toxic forms while still available for synthesis of such iron-containing proteins as hemoglobin, myoglobin, and catalase. Iron stores in the liver are present primarily as ferritin; hemosiderin is also present but to a much lesser degree. The ferritin is present both as free ferritin within the cytosol and within siderosomes. The free cytosolic ferritin is seen by light microscopy as the blue blush of liver cells by Perls' stain, while the granules are the siderosomes, a form of secondary lysosome filled with predominantly ferritin. With increased iron stores, the granules become more prominent, forming first near the bile canalicular membrane in hepatocytes nearest to the portal zone (zone 1). As iron stores become more pronounced within hepatocytes, this storage extends to zone 2 and then zone 3 (central zone). The free ferritin within the cytosol reaches a plateau beyond which the ferritin is than transferred to siderosome. The amount of iron/ferritin in the siderosomes, however, can increase indefinitely. Mechanisms of Iron-Induced Fibrosis and Injury The iron-loaded hepatocytes may release profibrogenic substances, which activate hepatic stellate cells (which in turn, are thought to be the principal sources of collagen and other matrix proteins in chronic liver disease), or they may release substances which stimulate Kupffer cells to produce profibrogenic substances which in turn activate the stellate cells. Iron in toxic amounts can also induce lipid peroxidation of organic membranes, leading to cell injury and death. These lipid peroxidation products can also stimulate stellate cells to produce collagen and can stimulate Kupffer cells to stimulate the stellate cells as well. In addition, the increased iron stores in hepatocytes leads to catalyzed oxidative destabilization of lysosomes, which then leak digestive enzymes and cause cell death. Assessment of Iron Light microscopic evaluation of the iron stores in the liver can provide rapid and valuable information about the levels of iron stores. Three main elements need to be assessed: the grade (or amount) of stainable iron, its distribution in the various cell types of the parenchyma or portal zones, and the degree of fibrosis. We commonly use a methodology of 0-4+ for grading of iron stores, by assessing the overall amount of iron in all cell types together. One practical method of how to determine the various levels in a relatively reproducible manner is given in the Table 1 below. Table 1. Histologic Grading of Iron Stores Grade Magnification (eyepiece and objective) required for observation 0 Granules absent or barely seen x 400 1+ Barely seen x250, easily seen x400 2+ Discrete granules seen x100 3+ Discrete granules seen x25 4+ Masses visible x10, or naked eye Adapted from MacSween: Pathology of the Liver, Chap 5., Ref 1 Hepatic iron concentration can be obtained by measuring the dry weight of iron in the liver tissue. Tissue that has been previously embedded in paraffin will still work well for this determination. Using the iron concentration, the hepatic iron index (HII) can be determined by dividing the concentration (in umol/g dry weight) by the age of the patient (in years). Values more than or equal to about 1.9 to 2 can be used to separate patients who are homozygous for the hemochromatosis gene from the heterozygotes, and can be helpful to identify the homozygous patients from other causes of iron overload in many instances as well. However, genetic testing is now the preferred methodology to determine hereditary hemochromatosis. Classification of Iron Overload Iron overload can be divided into familial or hereditary forms versus acquired forms. The following table lists many of the currently known iron overload states (adapted from ref 2 and 5). Table 2. Classification of Iron Overload Familial or hereditary forms of hemochromatosis Hereditary hemochromatosis (HHC, HFE1, HFE gene and Hfe protein defect) C282Y homozygosity, C282/H63D heterozygosity, other HFE gene mutations Juvenile hemochromatosis (HFE2, Type 2A: HJV gene and hemojuvelin protein;Type 2B: HAMP gene and hepcidin protein defects) Transferrin receptor 2 mutation (HFE3, TFR2 gene and transferring receptor 2 protein defect) Ferroportin mutation (HFE4, SCL40A1 gene and ferroportin 1 protein defect) Aceruloplasminemia Atransferrinemia Autosomal dominant hemochromatosis (from Soloman Islands) Acquired iron overload Iron loading anemias, such as Thalassemia major, sideroblastic anemias, chronic hemolytic anemias Dietary iron overload (African iron overload) Iron via parenteral route, with blood transfusions, parenteral iron medications Miscellaneous Chronic liver disease such as end-stage cirrhosis, hepatitis C, alcoholic liver disease, non-alcoholic steatohepatitis Neonatal hemochromatosis Porphyria cutanea tarda Hereditary Hemochromatosis (HHC, HFE1) Hereditary Hemochromatosis, commonly known in the past as primary hemochromatosis, is the most common form of iron overload, and is an autosomal recessive disorder seen in Anglo-Celtic/Northern European populations due to mutations in the HFE gene found on chomosome 6p. The HFE gene product is a transmembrane glycoprotein homologous to Class I major histocompatibility complex. Mutation of the gene prevents the formation of a disulfide bond that disrupts an important association with a B2microglobulin, which in turn alters its normal cell surface expression. HFE also associates with transferrin receptors, and so mutations may decrease the affinity for iron-bound transferrin, altering intestinal absorption. Two major gene defects have been specifically identified. The most common defect is a single G to A mutation at position 282, resulting in a cysteine to tyrosine substitution, designated as the C282Y mutation. The second most common defect is the H63D mutation, which results in a histadine to aspartic acid substitution, and causes a reduced function of the HFE gene product rather than the more severe complete loss of function seen with the C282Y mutation. Pathology - Homozygotes for C282Y The patients with both gene copies of the C282Y mutation may have the full-blown syndrome of hereditary hemochromatosis, but the actual penetrance of disease is very low (ref 6), and additional factors such as alcohol exposure may be factors that enhance the development of disease. In patients with liver disease, iron deposits can be seen in teenage years in periportal hepatocytes, but no Kupffer cell iron or fibrosis is typically present. Young males typically have more iron stores than young females of the same age. With increasing age, iron deposits become heavier in parenchymal cells, with extension from the periportal zone to the central zone (Zone 1-3 gradient), and Kupffer cells as well as other cell types such as portal tract macrophages show increased iron stores as well. Fibrosis then is likely to begin, which probably corresponds to the development of mesenchymal iron deposits. The fibrosis begins in the periportal areas, causing portal-to-portal septa and a portal-based pattern of fibrosis. The cirrhosis is typically micronodular in type, with only mild to moderate inflammatory changes mostly associated with foci of hepatocyte damage and necrosis, and no significant bile ductular proliferation. The hepatocyte damage (sideronecrosis) is a late stage finding seen in the highest levels of iron overload. Iron-free foci In later stages of iron overload, small areas of liver parenchyma with very little to no iron can be found. The Kupffer cells in these foci will contain iron. This change can also be seen in severe non-hereditary forms of iron overload (secondary hemochromatosis) as well. Some consider these to be "dysplastic" foci. Effects of Iron removal Therapeutic phlebotomy is the treatment of choice for these patients. The removal of iron occurs in the reverse order of the accumulation pattern, beginning in the central zone, with the last cells to lose iron in the periportal area (zone 1). However, the largest clusters of iron deposits may still remain after the smaller granules are removed, so this should probably be taken into account in the grading scheme. The reversal of fibrosis has been reported. There is well-accepted evidence that there are changes in the pattern of fibrosis and in the parenchymal architecture. The nodules transform to macronodules, which contain increased numbers of central veins and tiny portal tract-like structures. These macronodules are partially separated by thin septa, as seen in the incomplete septal type of cirrhosis. It is suggested that the iron removal allows the liver to undergo gradual repair and regrowth of normal structures that expand the size of the original nodules and lead to thinning of the fibrous septa. However, only a very rare case of restoration of a cirrhotic liver to near normal structure has been documented. Predisposition for HCC Homozygotes may have a 2-4x increased risk for hepatocellular carcinoma (HCC) over those patients with other forms of cirrhosis, and there is definitely a >200x increase incidence over patients without cirrhosis. The HCC essentially always occurs in patients with cirrhosis (but rare cases with fibrosis short of cirrhosis have been described). The removal of iron apparently does not reverse this tendency. Pathology – Heterozygotes for C282Y 5-10% of C282Y heterozygotes show increased iron stores sufficient to meet the criteria for hereditary hemochromatosis. These patients will typically have the H63D mutation as the other copy of the gene. True C282Y heterozygotes with a normal second copy of the gene may show some mild increase in iron stores, with varying amounts of iron seen from 0-2+, but have no evidence of clinical disease. Other Familial Forms of Hemochromatosis The juvenile pattern (HFE2) is an autosomal recessive disorder with the same pattern of iron overload as seen in the homozygous C282Y form, but instead, the iron overload occurs much sooner, in the second and third decades. Two gene defects have been identified: HJV on chromosome 1 that codes for hemojuvelin and HAMP on chromosome 19 that codes for hepcidin. The patients have hypogonadism, other endocrine manifestations, and cardiac failure as well as cirrhosis. A defect in the gene coding for transferrin receptor 2 (TfR2) has been labelled as HFE3, a defect in ferroportin gene as HFE4. The mechanisms underlying the iron overload in these three entities are unknown. However, it is thought that ferroportin may be key in transporting iron out of the cell, so lack of this protein may be the cause of the overload in these patients. A severe defect in ceruloplasmin function (aceruloplasminemia) reduces the rate of iron oxidation (Iron is oxidased by the ceruloplasmin so that it can be bound to plasma transferrin) and the subsequent cellular release of iron causes progressive accumulation in the liver, pancreas and brain, resulting in diabetes and neurodegenerative disease. A defect resulting in lack of plasma transferrin (atransferrinemia) causes increased iron absorption, and subsequent iron deposits in liver, pancreas, and heart as well. An autosomal dominant pattern of inheritance with disease effect similar to HHC has been noted in Melanesians from the Soloman Islands. The genetic defect remains unknown. Acquired Iron Overload The acquired iron overload syndromes are typically those associated with chronic anemias, dietary overload, and transfusional or parenteral overload. For the hematologic diseases (such as thalassemia major and most sideroblastic anemias) that are characterized by severely ineffective erythropoiesis, with markedly hyperplastic marrow and high plasma iron turnover, the increased iron stores result from both increased absorption and increased transfusional iron in combination. In the chronic anemias that do not require transfusion therapy, such as beta thalassemia minor or hereditary spherocytosis, occasional patients develop severe iron overload that is not apparently routinely associated with HFE gene defect; thus, it is though iron absorption may be increased in these cases. In the aplastic anemias or hypoplastic anemias, the intestinal absorption does not appear to be increased to any significant degree, so iron overload in these patients is transfusional. Transfusional overload as well as parenteral overload will show heavy preferential early loading of the Kupffer and other mesenchymal cells such as portal tract macrophages and endothelial sinusoidal cells as well as iron in hepatocytes. Fibrosis/cirrhosis is extremely rare in these patients. Excess dietary overload Iron overload in patients without chronic anemia but who are self-medicating with iron compounds has been described. Whether these patients are heterozygotes or not for the HFE gene defects is unknown. The most studied group is the African population whose source of excess iron was found within the containers used for brewing their traditional beers. The excess iron storage is predominantly in bone marrow, spleen, and liver. In the liver, the storage is mainly within macrophages in portal areas and Kupffer cells, with less storage in hepatocytes. Fibrosis occurs in these patients related to the dense deposits in portal tracts. Miscellaneous Iron Storage Conditions Chronic Liver Disease It is known that end-stage cirrhosis due to a variety of causes (including alcohol, nonalcoholic steatohepatitis, HBV, and HCV) can show increased iron stores even without genetic evidence of HFE gene defects. In these patients, the hepatic iron index (HII) can be >1.9. These cases typically show prominent hepatocyte iron, lack vascular or biliary epithelial iron deposits, and only have minimal mesenchymal iron within portal zones, macrophages, or Kupffer cells (unlike hereditary hemochromatosis which involves all these sites by the cirrhotic stage). The deposits of iron are also more scattered, with considerable variation in the amount of iron seen from nodule to nodule. It is also thought that the presence of one copy of the HFE gene mutation (C282Y) can cause mild iron overload in patients with other forms of liver disease (HBV, HCV, steatohepatitis), but most note that this degree of iron overload does not enhance the progression of the primary disease. Neonatal hemochromatosis A current debate still exists as to whether this entity is hereditary or acquired as a secondary effect to severe antenatal liver disease resulting in feto-placental iron processing, with subsequent iron deposition in a variety of tissues, including liver, heart, gastric and bronchial mucosa, and endocrine tissues such as pancreas and thyroid. These infants present with hypoglycemia, hyperbilirubinemia, and other signs of liver failure. The condition may recur in siblings, suggesting a hereditary factor. Histologically, the liver shows extensive fibrosis and loss of hepatocytes, with siderosis. Some giant hepatocytes as well as nodular formation can be seen. Porphyria Cutanea Tarda (PCT) A common feature of PCT is the deposition of iron in periportal hepatocytes and Kupffer cells. The degree of deposition is usually only mild, but more severe storage has been noted in patients with alcoholic liver disease. Some have suggested that heterozygotes for the HFE mutation at C282Y leads to the iron overload. References: Reviews Iron Storage Diseases, Chap 5, In MacSween (Eds), et al. Pathology of the Liver, 2002. Trinder D, Fox C, Vautier G, Olynyk JK. Molecular pathogenesis of iron overload. Gut 2002;51:290-295. Olynyk JK. Hereditary Haemochromatosis: diagnosis and management in the gene era. Liver 1999;19:73-80. Britton RS, et al. Pathogenesis of Hereditary Hemochromatosis: genetics and beyond. Sem Gastrointeint Dis 2002;13:68-79. Roetto A. New insights into iron homeostasis through the study of non-HFE hereditary haemochromatosis. Best Practice & Research Clinical Haematology 2005;18(2):235-50. Waalen J, Nordestgaard BG, Beutler E. The penetrance of hereditary hemochromatosis. Best Practice & Research Clinical Haematology 2005;18(2):203-20. Pathology focus Deugnier YM, et al. Liver pathology in genetic hemochromatosis: a review of 135 homozygous cases and their bioclinical correlations. Gastroenterol 1992;102:2050-2059. Nash S, et al. Role of liver biopsy in the diagnosis of hepatic iron overload in the era of genetic testing. Amer J Clin Pathol 2002;118:73-81. Hohler T, et al. Heterozygosity of the hemochromatosis gene in liver disease- prevalence and effects on liver histology. Loiver 2000;20:482-486.