British Journal of Haematology, 1976, 34, I.

Anno tation

METABOLIC CONSEQUENCES OF IRON OVERLOAD

In normal individuals the blue-staining material detected with Pcrls’ reagent in reticulo- endothelial cells and in liver parenchymal cells is believed to represent storage iron. In iron overload large amounts of this blue staining material are found in many tissucs and thcre is a good correlation between the amount of staining and tlic iron coiitciit of tlic tissue. Although the nature of the iron is only poorly understood it is assumed to be eithcr ‘fcrritin’ or ‘haemosiderin’. Ferritin is a complex molccule and its bioclicmistry has bcen dcalt with elsewhere (Jacobs & Worwood, 1975). Hacniosiderin has never been clearly defined although Richter & Bessis (1965) demonstrated that it is more of a visual impression than a biochcmical entity. It is sometimes present as dcnse iiisolublc iron rich masses, sometinics as membrane- bound aggregations of closely packcd ferritin niolcculcs and somctimcs as a mixture of iron compounds, cell debris and other abnormal deposits. Thcy considcrcd tlic isolated substaiicc to be something of an artefact. It is known that iiicrcased cell iron uptakc is associated with an increase in ferritin synthesis (Drysdale & Munroc, 1966; Harrison ct nl, 1974) but the nature of the iiitracellular transit form of iron that cxists bcforc its incorporation into ferritin (White cf al , 1976) is unknown. Increased iron loading lcads to a progressive conver- sion of ferritin to an iiisolublc iron rich substance and therc is now a little data suggesting how this might occur.

Closely packed ferritin iiiolecules seen on electron microscopy liavc a 50 A gap between each iron core because of the 25 A thickness of each protein shcll whilc in haemosiderin thc iron cores sit more closely together because of protciii loss (Fischbach ct a/, 1971). Ferritiii degradation may be related simply to close packing, possibly to polymer formation and perhaps to enzymatic attack following incorporation into lysosomcs. Starch gel elcctro- phorcsis of purified ferritin gives rise to a number of bands reprcsciiting monomers, diniers and trimers. Oligomers containing up to five molecules can bccii sccii on electron microscopy (Williams & Harrison, 1968) and cannot bc dissociated except uiidcr conditions leading to disaggregation of subunits. The intercore distances may be slightly less than $0 A in some cases, consistent with some degree of condensation of the protein shells. The appearance of ferritiii polymers in extracts prepared by different techniques from different tissue suggests that they probably exist within cells in vim. Niitsu & Listowsky (1973) noted the high iron content of polymeric ferritin and suggested that iron loading may play a part in their forma- tion but Lee & Richter (1976), on the other hand, suggest that the concentration of ferritiii may be a major factor, association or dissociation being reversible according to the conditions in solution, and this may be relevant to the molecular packing that occurs in cytoplasmic vesicles. When the iron cores of ferritin and haemosiderin are isolated in a test tube and examined by X-ray diffraction, Mossbauer spectroscopy and electron microscopy they show a similar atomic structure with only a slightly smaller particle size in haemosiderin (Fischbach et al, 1971). These results also suggest that haemosiderin is formed by the denaturation ofthe

Correspondence: Professor Allan Jacobs, Department of Haeinatology, Welsh National School of Medicine, Health Park, Cardiff CF4 4XW, Wales.

2 Annotation

ferritin protein shell leaving a slightly smaller iron core. While there are indications that different isoferritins may have functional differences (Jacobs & Worwood, 1975) there is no clearly defined change in tissue isoferritin patterns in the iron overload state.

When there is a continuous process of ferritin synthesis it appears to be balanced by its conversion into haemosiderin. It has been suggested that iron overload is easily produced in the liver because its considerable capacity to synthesize ferritin cannot be matched by its ability to process the product in secondary lysosomes prior to excretion. Instead the lysosomal accumulation of ferritin behaves as a sump which gradually converts the protein to hacmo- siderin which then remains in sittr (Trump et a/, 1973). Although in the early experiments of Golbcrg et a! (1957) high doses of parenteral iron resulted in increased activity of hepatic lysosomal activity, recent studies have shown that it is possible to iron load rats in this way with no subsequent liver abnormality except a massive lysosomal accumulation of iron granules (Arborgh e t a ] , 1974). Similar lysosomal accumulations can be found in the pancreatic acini (Pechet, 1969). When cell death occurs in cxperirnental iron overload the microscopic picture may show no specific changes. In addition to thc inevitable secondary lysosomes containing amorphous electron-dcnse debris, remains of partly digestcd organelles and thc contents of the cell sap, there may be degcnerate organelles in the cytoplasm with widespread evidcnce of membrane damagc, myelin whorls, lipid droplets and rcsidual bodies.

Most accounts of chronic iron toxicity point, directly or indirectly, to evidence of in- creased lipid peroxidation and consequent membrane damage. Golberg & Smith (1958) found cytological changes closcly resembling thosc seen in vitamin E deficicncy and were able to prevent these changes by administration of vitamin E. In iron loaded patients with thalas- sacmia major excess lipid peroxidation in crythrocytes is associated with reduced lcvcls of vitaniin E (Rachmikwitz ct al, 1976). Peroxidation results in the destruction of sulphhydryl groups (Lewis & Wills, 1962) and the mitochondria1 nicmbraiie damage that occurs is associated with loss of components of the electron transport pathway and the inactivation of a number of other cnzyme systems including parts of the Krcbs cycle (Hunter et ul, 1963 ; McKnight & Hunter, 1966). Biochemical damage to suspensions of mitochondria is demon- strable at I ,UM iron conccntrations (McKnight el a / , 1965).

Microsomal lipid peroxidation in the rat liver appears to be dcpendcnt on a non-fcrritin, non-haem iron component and the process can be inhibited by iron chelators such as desfcrri- oxamine (Wills, 1969). Increasing the inorganic iron content of the suspending medium in- creases the rate of lipid peroxidation up to maximum levels at 30 pmol/l Fe. Ascorbic acid stimulates a marked increase in lipid peroxidation in liver, heart and kidney and although ferritin is inactive in promoting thc oxidation of fatty acids it has a marked cffcct wlieii ascorbic acid is present (Wills, 1966). The cffcct of ascorbate is attributed to its ability to mobilize ferritin iron into a low molecular weight catalytic form. Iron overload in rats is associated not only with increased lipid pcroxide formation in hepatic endoplasniic reticulum but with impaired amino-pyrine metabolism and presumably otlier detoxicating rcactioiis (Wills, 1972). The role of ascorbic acid in lipid peroxidation descrvcs to bc rcnicmbered in discussions of its therapeutic use in increasing the availability of iron for chclation or countcr- ing the effects of iron-induced scurvy.

Lysosomal abnormalities in experimental iron overload arc wcll recognized and Pctcrs & Seymour (1976) have shown that lysosomal enzyincs are increased in livcr biopsy specimens

Annotation 3

from patients with both primary and secondary overload. The lysosomes appear to be abnormally fragile, having both a low latency and a low sedimentable &$xosaminidase activity. It is tempting to consider that the degraded ferritin and amorphous iron deposits physically damage the organelle with intracellular release of its enzymes. An alternative ex- planation for lysosome-mediated cell damage is the gradual solubilization of its iron deposits at a relatively low pH with the release of chemically reactive iron stimulating lipid peroxidation both of the lysosomal membrane and beyond.

Lynch et a1 (1967a) reported accelerated oxidative catabolism of ascorbic acid in siderotic subjects who had a higher plasma clearance and lower excretion of ascorbic acid, together with higher oxalic acid excretion than normal subjects. The ascorbic acid deficiency that appears to be universal in heavily iron loaded patients leads to osteoporosis (Lynch et a l , 1967b; Wapnick et al , 1971), probably through defective collagen and osteoid formation and failure of osteoblast maturation. A similar syndrome can be induced in guinea-pigs by experimental iron loading with iron-dextran (Wapnick et a2, 1971).

The clinical importancc of iron loading and toxicity is in the increasing number of patients with refractory anaemia being treated by repeated blood transfusion. Transfusional iron overload has a poor prognosis (Barry et 01, 1974; Modell, 1975) and treatment has been on an empirical basis. It is to be hoped that identification of intracellular iron compounds and their role in tissue toxicity may result in more effective therapy.

Departnierit of Haenzatology, Welsh National School of Medicine, Cardiff

ALLAN JACOBS

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