0270-9139/86/0601-0142$02.00/0 HEPATOLOGY Copyright 8 1986 by the American Association for the Study of Liver Diseases

Vol. 6, No. 1, pp. 142-145,1986 Printed in U.S.A.

Hemochromatosis: How Much Iron Is Too Much?

The paper by Bassett et al. (1) in this issue of the journal is timely in addressing several remaining impor- tant controversies in the pathogenesis and diagnosis of genetic hemochromatosis (GH). As with all genetically determined metabolic diseases, the aim of early diagnosis in the asymptomatic homozygote is the prevention of tissue damage and later complications of the disease. It is evident from experience in the symptomatic patient with GH that only a few of the complications are poten- tially reversible (eg., glucose intolerance and cardiac failure); while the remainder, in particular the cirrhosis, are not correctable by phlebotomy therapy (2, 3). Al- though there are highly suggestive epidemiologic data to indicate that the toxic threshold for the development of hepatic fibrosis and cirrhosis is a function of the duration and level of tissue iron to which the liver is exposed (4, 5), there has been a lack of information on the markers of iron overload and its consequences in younger hom- ozygotes. Probands with the disease have usually pre- sented with evidence of organ failure in the fifth decade of life, when iron stores are in excess of 20 gm, and all markers of iron overload are grossly abnormal (3-5). It is, therefore, particularly valuable that genotypic detec- tion of asymptomatic relatives of patients with fully developed disease has been made possible by the evalu- ation of shared HLA haplotypes with these probands (6). With their detailed observations of the families of 179 probands over a period of almost 20 years, the Queens- land group has been able to observe the phenotypic manifestations of the disease in 114 asymptomatic rela- tives under the age of 35 years. In an earlier communi- cation, they reported the predictive accuracy of biochem- ical screening tests showing that the combination of increased transferrin saturation (>50%) and an elevated serum ferritin concentration (>200 pg per liter in men; >150 pg per liter in women) was 94% sensitive and 86% specific for the detection of GH in this group of younger homozygotes (7). It is, therefore, particularly valuable to now have available their more direct assessment of iron overload based on measurements of hepatic iron concen- tration in 30 of these asymptomatic homozygous rela- tives. This study provides unique insights into age-re- lated iron accumulation and its hepatotoxic conse- quences. Furthermore, it has yielded an essential means for the phenotypic differentiation of the homozygous state from that of the heterozygote, and perhaps more

Address reprint requests to: Anthony S. Tavill, M.D., Cleveland Metropolitan General Hospital, 3395 Scranton Road, Cleveland, Ohio 44109.

important for the differentiation of the alcoholic with biochemical and histochemical markers of iron overload from the genetic hemochromatotic who coincidentally happens to have manifestations of alcoholic liver disease. A major additional bonus is that the study clearly indi- cates that there is indeed an apparent age-related thresh- old of hepatic iron concentration above which fibrosis and cirrhosis are likely to ensue. These data add impor- tant clinical weight to current theories in which abnor- mal levels of hepatocyte iron stores are viewed as directly cytotoxic.

Circumstantial clinical evidence for the quantitative role of iron in hepatotoxicity has been provided by stud- ies of patients with GH (2, 31, Bantu hemochromatosis (8) and secondary hemochromatosis due to P-thalassemia (9-1 l), in which correlation between the hepatic iron concentration and the occurrence of liver damage has been suggested, or in which reduction of hepatic iron by either phlebotomy or chelation therapy has resulted in improved clinical outcome. The current study takes the association a step further. In GH there is an hereditary, excessive and inappropriate absorption of dietary iron resulting in progressive accumulation of storage iron predominantly in the parenchymal cells of the liver. The concept of progressive accumulation is supported by the finding of a significant correlation between age and he- patic iron concentration only in homozygotes (1). Bassett and coworkers have shown additionally that in the ab- sence of associated alcoholic injury, fibrosis or cirrhosis does not occur until a t least 22,000 pg per gm dry weight (equivalent to about 4,500 pg per gm wet weight in their study) of iron has accumulated in the liver, a figure very close to that which has been achieved in a dietary model of iron overload in the rat (12). Even in the absence of associated alcohol abuse, cirrhosis may occur as early as 35 years of age if the hepatic iron concentration is high enough. In the presence of excess alcohol intake, they observed fibrosis in three younger individuals with GH whose levels of hepatic iron were below the threshold level seen in other nonalcoholic homozygotes with fibro- sis. Although it remains to be proved experimentally, this observation suggests that there may be synergism between the hepatotoxic effects of excessive iron and alcohol (13). At this time, we have only suggestive data derived from studies in the experimental rat that iron may accentuate markers of prooxidant activity in the liver induced by acute ethanol intoxication (14, 15).

Despite this clinical and experimental evidence for the hepatotoxicity of excess iron, the specific pathophysio- logic mechanisms for hepatocyte injury and hepatic fi-

142

Vol. 6, No. 1, 1986 HEMOCHROMATOSIS: HOW MlJCH IRON IS TOO MUCH? 143

brosis in chronic iron overload are not fully understood. The lack of an adequate animal model of GH has ham- pered the experimental confirmation of the role of iron in hepatotoxicity. There are two studies of parenteral iron loading which report success in reproducing exper- imentally some of the histological features of GH (16, 17). In both studies, massive doses of parenteral iron were administered to dogs or baboons over a period of several years. Other workers have produced fibrosis or cirrhosis by giving iron concomitantly with a high fat, choline-deficient diet or potent hepatotoxins such as ethionine or carbon tetrachloride (18). While the possi- bility that increased collagen deposition may occur in- dependently of cellular injury has been raised by ultra- structural and biochemical studies (19,20), it is generally agreed that a direct cytotoxic effect can result from the presence of grossly elevated levels of hepatocellular iron. In so far as transferrin-bound iron is delivered prefer- entially to the parenchymal cell (21), the excessive ab- sorption of dietary iron in GH leads to selective initial iron overloading of this cell population. In this respect, the animal models of parenteral iron overload have failed to mimic the pathophysiologic events of GH.

There are two principal theories currently being ex- plored to account for the cytotoxicity of parenchymal iron. One favors a role of hemosiderin in the destabili- zation of the lysosome, the other proposes a more gen- eralized role for a compartment of subcellular iron in peroxidative damage to the polyunsaturated fatty acids of membrane phospholipids. Initial localization of excess iron stores within lysosomes is followed by disruption of lysosomal membranes (22), and assessment of lysosomal integrity in liver biopsy specimens from patients with iron overload has demonstrated increased membrane fragility, which can be reversed by adequate phlebotomy therapy (23, 24). Hemosiderin has been shown to be the predominant form of storage iron in untreated GH with concentrations in the liver up to levels 100-fold above normal (25, 26). It has been suggested that release of iron from hemosiderin within the acid milieu of the lysosome may be responsible for the destabilization of the lysosomal membrane, releasing potentially cell-dam- aging hydrolytic enzymes into the cytosol (27). The al- ternative but mutually compatible theory that intracel- lular membrane damage is initiated by lipid peroxidation of vital membrane lipids was prompted by earlier evi- dence that experimental iron overloading by means of parenteral iron dextran was associated with evidence of hepatic lipid peroxidation (28, 29). Recently, it has been possible by dietary supplementation with elemental (car- bonyl) iron in the rat to achieve levels of hepatic iron overload, predominantly localized to the hepatocyte, which are similar to those seen in GH (12). Evidence of peroxidation in uiuo was found in microsomal and mito- chondrial lipids by the detection of conjugated dienes, products of peroxidative damage of polyunsaturated fatty acid constituents of membrane phospholipids. Further- more, it was shown that there is correlation between the threshold for iron-associated lipid peroxidation and func- tional disturbances in mitochondria and microsomes (30, 31). While it is clearly difficult to mimic in experimental animals in a few months a situation in humans in which

iron overload and its consequences are slowly cumulative over a period of many years, the dietary carbonyl iron model has afforded us useful insights into a potential pathway of cell damage. Comparison of these experimen- tal observations with the human data provided by Bas- sett and coworkers (1) justifies the feasibility of the concept of a toxic threshold coupled with the duration of exposure to cell-damaging levels of intracellular iron. We are encouraged by the observation that we have had to subject the rat liver to a t least these threshold levels to achieve hepatic fibrosis in the experimental animal (Park, C. H. et al., Hepatology 1985; 5:950, Abstract).

The biochemical mechanisms responsible for lipid per- oxidation in iron overload are unknown. It may be that, in situations of genetic or acquired hemochromatosis, the ability of the hepatocyte to maintain iron in a nontoxic protein-bound ferr’ic ( Fe3+) form is exceeded, resulting either in small amounts of ferrous (Fe2+) iron or leading to an expansion of the low molecular weight iron chelate pool (32-35). It has been postulated also that nonregu- lated release of iron from ferritin or hemosiderin could provide a “delocalized,” toxigenic form of nonprotein- bound iron. Iron in these forms could play a role in the generation of free hydroxyl radicals (OH. ) by reacting with H202 via the Fenton reaction or by reacting with the superoxide radical (02-) via the modified (iron- catalyzed) Haber-Weiss reaction (36, 37). Alternatively, ferric iron itself, released from ferritin or hemosiderin, could serve to promote superoxide-dependent lipid per- oxidation (38) or could generate ferry1 or perferryl radi- cals capable of attacking polyunsaturated fatty acids directly (36). Since experimental work in uitro suggests that Fez+ and not Fe’+ is probably the catalytically active form of iron in free-radical generation (39-41), the fact that the liver has to be heavily overloaded with iron in uiuo before lipid peroxidation is initiated is a tribute to those mechanisms operating to keep the concentration of Fez+ ions at zero to submicromolar levels (42).

A final dilemma which is addressed by Bassett et al. (I) is the differentiation of GH in the alcoholic from alcoholic liver disease with apparent iron overload. This confusion has arisen from two main factors. First is the high prevalence of excessive alcohol intake in GH (25 to 40%) (43), and second is the misinterpretation of the significance of stainable hepatic iron coupled with the relatively low sensitivity and specificity of indirect tests for assessment of iron overload in alcoholic liver disease (44). While it has been shown previously in older indi- viduals that quantitative liver iron determination can differentiate alcoholics with significant stainable hepatic iron from GH (45), it was not clear that such measure- ments would be discriminant in presymptomatic, younger patients with GH. In that study, levels of hepatic iron exceeded 10,000 pg per gm dry weight in all 15 subjects with GH, and those with cirrhosis tended to have higher levels than those with fibrosis alone. In contrast, although alcoholics with stainable iron had elevated hepatic iron concentrations, they never ex- ceeded 10,000 pg per gm, and there was no correlation between iron levels and the presence of cirrhosis. Those alcoholics with total iron stores in excess of 10 to 15 gm and hepatic iron concentrations >10,000 pg per gm

144 TAVILL AND BACON HEPATOLOGY

dry weight are probably examples of GH, as evidenced by family studies and the high prevalence of associated HLA-A3 histocompatibility antigens (13, 46). In con- trast, alcoholics with minor degrees of iron overload do not have GH, even as the result of heterozygous pheno- typic expression (47). The current study by Bassett and coworkers (1) shows that patients with alcoholic liver disease even with significant stainable iron never ex- ceeded an hepatic iron concentration of about 5,000 pg per gm dry weight. However, there were eight homozy- gotes with GH (of a total of 30) whose liver iron levels were also less than this. These were all younger individ- uals identified by elevated serum transferrin saturation or ferritin concentration and confirmed by HLA studies as genetic homozygotes, who over the course of time would have progressively accumulated enough iron to reach hepatotoxic levels. However, the application of the hepatic iron index [hepatic iron (pmoles per gram)/age (years)] clearly separated these early GH patients from alcoholic liver disease by showing that relative to dura- tion of life, the GH subject had a much higher hepatic iron concentration. Every patient with GH had an he- patic iron index above 2.0, while every patient with alcoholic liver disease had an index below 2.0. The sig- nificant positive correlation between hepatic iron con- centration and age in this group, in contrast to the genetic heterozygotes and the alcoholics, clearly maps out the fate of the untreated homozygote. What this study does, that no previous study has been able to achieve, is to offer the strongest possible clinical evidence for the presence of a toxic threshold for iron in the liver, to support the value of appropriate biochemical and genetic screening in first degree family members of the proband (from the age of about 10 years onward) and to promote what was implied but not explicitly stated, namely that these individuals should be prophylactically treated by attempting to normalize all the markers of iron overload. Although it is axiomatic in the context of this inherited metabolic disease that any amount of excess storage iron is too much, it is fortunate that the process of iron accumulation is relatively gradual and that we have available both direct and indirect means for quantifying liver iron deposition (48,49) for genotyp- ing presymptomatic homozygotes and for predicting the risk of tissue damage before iron overload has produced irreversible consequences.

ANTHONY S. TAVILL BRUCE R. BACON Case Western Reserve

University Cleveland Metropolitan

General Hospital Cleveland, OH 44109

1.

2.

REFERENCES Bassett ML, Halliday JW, Powell LW. Value of hepatic iron measurements in early hemochromatosis and determination of the critical iron level associated with fibrosis. Hepatology 1986; 624- 29. Bomford A, Williams R. Long-term results of venesection therapy in idiopathic hemochromatosis. Q J Med NS 1976; 45:611-623.

3. Milder MS, Cook JD, Stray S, et al. Idiopathic hemochromatosis, an interim report. Medicine 1980; 59:39-49.

4. Cartwright GE, Edwards CQ, Kravitz K, et al. Hereditary hemo- chromatosis. Phenotypic expression of the disease. N Engl J Med 1979; 301: 175-179.

5. Edwards CQ, Cartwright GE, Skolnick MH, et al. Homozygosity for hemochromatosis: clinical manifestations. Ann Intern Med 1980; 93:519-525.

6. Simon M, Bourel M, Genetet B, et al. Idiopathic hemochromatosis: demonstration of recessive transmission and early detection by family HLA typing. N Engl J Med 1977; 297:1017-1021.

7. Bassett ML, Halliday JW, Ferris RA, et al. Diagnosis of hemo- chromatosis in young subjects: predictive accuracy of biochemical screening tests. Gastroenterology 1984; 87:628-633.

8. Isaacson C, Seftel HC, Keeley KJ, et al. Siderosis in the Bantu: the relationship between hepatic iron overload and cirrhosis. J Lab Clin Med 1961; 58:845-953.

9. Risdon RA, Barry M, Flynn DM. Transfusion iron overload: the relationship between tissue iron concentration and hepatic fibrosis in thalassemia. J Pathol 1975; 11683-95.

10. Barry M, Flynn DM, Letsky EA, et al. Long-term chelation therapy in thalassemia major: effect on liver iron concentration, liver histology and clinical progress. Br Med J 1975; 2:16-20.

11. Cohen A, Martin M, Schwartz E. Depletion of excessive liver iron stores with desferrioxamine. Br d Haematol 1984; 58369-373.

12. Bacon BR, Tavill AS, Brittenham GM, et al. Hepatic lipid perox- idation in uioo in rats with chronic iron overload. J Clin Invest

13. Powell LW. The role of alcoholism in hepatic iron storage disease. Ann NY Acad Sci 1975; 252124-134.

14. Valenzuela A, Fernandez V, Videla LA. Hepatic and biliary levels of glutathione and lipid peroxides following iron overload in the rat: effect of simultaneous ethanol administration. Toxicol Appl Pharmacol 1983; 70:87-95.

1.5. Videla LA. Hepatic antioxidant-sensitive respiration. Effect of ethanol, iron and mitochondria1 uncoupling. Biochem J 1984; 223:885-891.

16. Lisboa PE. Experimental hepatic cirrhosis in dogs caused by chronic iron overload. Gut 1971; 12:363-368.

17. Brissot P, Campion JP, Guillozo A, et al. Experimental hepatic iron overload in the baboon: results of a two-year study. Evolution of biological and morphologic hepatic parameters of iron overload. Dig Dis Sci 1983; 28:616-624.

18. Richter GW. The iron-loaded cell-The cytopathology of iron storage. A review. Am J Pathol 1978; 91:363-396.

19. Iancu T, Neustein HB, Landing BH. The liver in thalassemia major: ultrastructural observations in iron metabolism. CIBA Foundation Symposium 1977; 51:293-307.

20. Weintraub LR, Goral A, Grasso J, et al. Pathogenesis of hepatic fibrosis in experimental iron overload. Br J Haematol 1985; 59:321- 33 1.

’21. Hershko C, Cook JD, Finch CA. Storage iron kinetics. I11 Study of desferrioxamine action by selective radioiron labels of reticuloen- dothelial and parenchymal cells. J Lab Clin Med 1973; 81:876-886.

22. Trump BF, Valigorsky JM, Arstila AA, et al. The relationship of intracellular pathways of iron metabolism to cellular iron overload and iron storage diseases. Am J Pathol 1973; 72:295-324.

23. Peters Td, Seymour CA. Acid hydrolase activities and lysosomal integritv in liver biopsies from patients with iron overload. Clin Sci Molec Med 1976; 5075-78.

24. Seymour CA, Peters TJ. Organelle pathology in primary and sec- ondary haemochromatosis with special reference to lysosomal changes. Br J Haematol 1978: 40:239-253.

25. Beaumont C , Simon M, Smith PM, et al. Hepatic and serum ferrit in concentrations in patients with idiopathic hemochroma- tosis. Gastroenterology 1980; 79:877-883.

26. Selden C, Owen M, Hopkins JMP, et al. Studies on the concentra- tion and intracellular localization of iron proteins in liver biopsy specimens from patients with iron overload with special reference to their role in lysosomal disruption. Br J Haematol 1980; 44:593- 603.

27. O’Connell M, Ward RJ, Baum H, et al. Role of iron in ferritin and haemosiderin-mediated lipid peroxidation in liposomes. Biochem

28. Golberg L, Marti LE, Batchelor A. Biochemical changes in the

1983; 71:429-439.

J 1985; 229:135-139.

Vol. 6, No. 1, 1986 HEMOCHROMATOSIS: HOW MI'CH IRON IS TOO MUCH? 145

tissues of animals injected with iron: 3. Lipid peroxidation. Bio- chem J 1962; &3:291-298.

29. Wills ED. Effects of iron overload on lipid peroxide formation and oxidative demethylation of the liver endoplasmic reticulum. Bio- chem Pharmacol 1972; 21:239-247.

30. Bacon BR, Park CH, Brittenham GM, et al. Hepatic mitochondria1 oxidative metabolism in rats with chronic dietary iron overload. Hepatology 1985 5:789-797.

31. Bacon BR, Healey JF, Brittenham GM, et al. Hepatic microsomal function in rats with chronic dietarv iron overload. Gastroenter- ology 1986 (in press).

32. Jacobs A. Low molecular weight intracellular iron transport rom- pounds. Blood 1977; 50433-439.

33. Grohlich D, Morley CGD, Bezkorovainy A. Some aspects of' iron uptake by rat hepatocytes in suspension. Int .J Biochem 1959;

34. Morley CGD, Rewers KA, Bezkorovainy A. Iron metabolism path- ways in the rat hepatocyte. Clin Physiol Biochem 1983; 1:3-11.

35. Bacon BR, Tavill AS. The role of the liver in normal iron metah- olism. Semin Liver Dis 1984; 4:181-192.

36. Halliwell B, Gutteridge JMC. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem d 1984; 219:l-14.

37. Starke PE, Farber JL. Ferric iron and superoxide ions are required for the killing of cultured hepat.ocytes by hydrogen peroxide. Evi- dence for the participation of hydroxyl radicals formed by an iron- catalyzed Haber-Weiss reaction. J Biol Chem 1985; 260:10099- 10104.

38. Thomas CE, Morehouse LA, Aust SD. Ferritin and superoxide- dependent lipid peroxidation. J Biol Chem 1985; 2603275-3280.

39. Pederson TC, Aust SD. The mechanism of liver microsomal lipid

10797-802.

peroxidation. Biochim Biophys Acta 1975; 386:232-241. -10. Kornhrust DJ. Mavis RD. Microsomal lipid peroxidation. I. Char-

acterization of the role of iron and NADPH. Molec Pharmacol 1980: 17:400-407.

-1 1. Mak I T . Weglicki WB. Characterization of iron mediated peroxi- dative injurv in isolated hepatir Ivsosomes. J Clin Invest 1 9 S ; 75:58-6:3.

42. Bacon HR, Tavill AS, Recknagel RO. Lipid peroxidation and experimental iron overload. In: Zirnmerman H, ed. Proceedings of International Symposium on Hepatotoxicity, Rouen, France, 1986 (in press).

43. Powell LW, Hassett MI,, Halliday JW. Hemochromatosis: 1980 update. Gastroenterology 1980; 78374-381.

44. Brissot 1'. Bourel M, Henry D, et al. Assessment of' liver iron content in 271 patients; a re-evaluation of direct and indirect methods. Gastroenterology 1981; 80557-565.

4.5. Chapman RW, Morgan MY, Laulicht M, et al. Hepatic iron stores and markers of iron overload in alcoholics and patients with idiopathic hemochromatosis. Dig Dis Sci 1982; 27:909-916.

4ci. LeSage GD, Baldus WP, Fairbanks VF, et al. Hemochromatosis: genetic or alcohol-induced? Gastroenterology 1983; 84:1471-1477.

47. Simon M, Bourel M, Genetet B, et al. Idiopathic hemochromatosis and iron overload in alcoholic liver disease: differentiation by HLA phenotype. Gastroenterology 1977; 73655-658.

48. (;allan JL. Diagnosis of hemochromatosis (editorial). Gastroenter- ology 1983; 84:418-431.

49. Hrittenham GM, Danish EH, Harris dW. Assessment of bone marrow and body iron smres: old techniques and new technologies. Semin Heniatol 1981: 18:194-2'21.