Hemochromatosis Caused by Excessive Vitamin Iron Intake Gordon R. Hennigar, MD, William B. Greene, MS, Ernest M. Walker, MD, PhD, and Charlton deSaussure, MD
Rare cases of hemochromatosis have been reported in patients who underwent prolonged oral iron therapy for hemolytic anemia or prolonged self-treatment with iron pills. A proportionately large segment of the South African Bantu tribe, who ingest large quantities of an alcoholic beverage brewed in iron pots, are found to have the disease. Reports of health fadists developing hemochromatosis due to excessive dietary iron intake, however, are extremely rare. This report presents clinical considerations and pathologic findings in a compulsive health fadist who consumed large numbers of vitamins containing iron. Clinical findings included the development and progression of cirrhosis of the liver, bronzing of the skin, and diabetes mellitus, all consistent with a diagnosis of hemochromatosis. Light microscopy of liver biopsies taken late in the course of the disease revealed a massive buildup of iron in the hepatocytes, less in the Kupffer cells, and sparse deposition in the epithelial cells of the bile duct. Minimal periportal fibrosis was noted. Electron microscopy showed numerous pleomorphic siderosomes with varying degrees of crystallization and ferritin attached at uniform intervals to the membranes of residual bodies. Abundant free ferritin was observed in most cells. The aggregated and membrane-associated ferritin was verified by nondispersive x-ray analysis. An additional finding, noted only by electron microscopy, was the presence of many fat-storing cells of Ito, which are thought to be involved in the onset of fibrosis. (Am J Pathol 96:611-624, 1979)
THE TERM HEMOCHROMATOSIS was introduced by von Recklonghausen in 1889, and the disease is characterized by grossly excessive amounts of iron stores in body organs. The organs usually exhibit a diffuse fibrotic change. The underlying causes for retention of iron, resulting from multiple transfusions or excessive oral intake, may be due to inborn errors of metabolism comparable to idiopathic hemochromatosis. The evidence for genetic influence in this disorder is overwhelming. Patients presenting with hemochromatosis average a total body iron of 20-40 g, with liver iron increased by 50-100 times normal. The average liver weight is 2,400 g. Probably most of the extra iron storage is in the liver. As the concentration of unbound ferritin builds, it is transferred into membranebound hemosiderin granules, which tend to be more permanent structures. Clinical diabetes mellitus was reported in 63% (72/115) of patients at the time of diagnosis of idiopathic hemochromatosis,' and diabetes mellitus appears to occur in high incidence (47%) in close relatives of heFrom the Department of Pathology and the Department of Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina. Accepted for publication April 12, 1979. Address reprint requests to Gordon R. Hennigar, MD, Department of Pathology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29403.
0002-9440/79/0809-061 1$01.00 © American Association of Pathologists
611
612
HENNIGAR ET AL
American Journal of Pathology
mochromatotic families.2 The finding of diabetes mellitus associated with hemochromatosis may be related to concurrent liver disease and/or to excessive iron deposition in pancreatic tissue. In addition, insulin resistance has been shown to be associated with cirrhosis.3 Which disease comes first? Diabetes is present in only 20% of all the reported cases of dietary hemochromatosis. In the Bantu population, 7% of the diabetics were found to have fully developed hemochromatosis.4 Clinical Findings The patient was a 58-year-old white male locomotive operator who suffered a nervous breakdown at age 22 and shortly thereafter began a diet consisting of many raw eggs, tomatoes, prunes, raisins, and massive doses of vitamins containing iron (as many as 500 capsules during one 2week period in 1968). He was hospitalized and evaluated in 1968 for a large abdominal mass present for at least a year previously and episodes of gastroenteritis. Exploratory laparotomy and liver biopsies revealed hepatosplenomegaly with a tremendously enlarged liver presenting a roughly
granular nutmeg-appearing surface and microscopically showing extensive areas of portal fibrosis, bile-duct proliferation, and stippling of hepatic cells with brown granules, consistent with the diagnosis of hemochromatosis. Additional clinical and laboratory findings at that time (1968) were diabetes mellitus (with no previous history of the condition) and marked bronzing of the skin with dark-pigmented areolas, consistent with hemochromatosis of the skin. Medical control of the diabetes mellitus was initiated, vitamin intake was stopped, and units of blood were removed successively over a period of time to decrease the body load of iron. The patient was followed closely during the next 2-3 years (after October 1968), during which time at least four additional periods of hospitalization were required. Serum iron levels measured during this time interval did not tend to decrease, remaining within a 188-268,g/dl range (normal range 65-175 ,ug/dl), and serum iron-binding capacities remained low, in the 30-241 ,ug/dl range (normal range 250-410 ,g/dl). Serum enzymes (SGOT, LDH, alkaline phosphatase) tended to demonstrate slightly elevated levels during the same interval. The patient gradually developed cardiovascular difficulties during 1969 and presented early in 1970 with complaints and clinical findings consistent with the diagnosis of congestive heart failure, probably secondary to hepatic disease. His condition gradually worsened during the following months in spite of aggressive management with digoxin and diuretics, and he eventually died. Permission for autopsy was not granted.
Vol. 96, No. 2 August 1979
HEMOCHROMATOSIS AND IRON INTAKE
613
Materials and Methods A wedge biopsy was divided, and half was fixed in 10% phosphate-buffered formalin overnight and processed in paraffin for light microscopy. Serial sections were cut at 6 ,u and stained with hematoxylin and eosin (H&E), Masson's, and Prussian blue stains. A similar piece was minced into 1-cu mm blocks and fixed in 5% glutaraldehyde buffered with cacodylate and postfixed in 2% osmium tetroxide for 90 minutes. These blocks were dehydrated and embedded in Epon 812.' After polymerization, 0.5-1.0-,u-thick sections were cut, stained with toluidine blue, and examined with the light microscope for the determination of appropriate areas for subsequent thin sectioning. The thin sections were stained with uranyl acetate and lead citrate I and examined with a Hitachi HU-12 transmission electron microscope. A second series of epoxy sections, not stained with heavy metal solutions, were sent to the Perkin-Elmer Applications Laboratory in Mountain View, California, for energy dispersion x-ray analysis, a technique for the elemental analysis of submicroscopic structures.7
Results Light Microscopy
Light-microscopic examination of liver sections stained with H & E as well as with special stains for the demonstration of iron revealed extensive numerous granular deposits of hemosiderin within hepatocytes, at times obscuring cellular detail (Figure 4). Hemosiderin granules were present within Kupffer cells (Figure 3), bile-duct epithelium (Figures 2 and 4), and macrophages in the portal tracts, and in the areas of fibrosis. Examination of the hepatic parenchyma revealed irregular nodules of cells separated by bands of connective tissue within which the proliferation of small bile ducts was prominent (Figure 1). Degenerative changes were apparent within the majority of the liver cells (Figure 2), but an apparent relative paucity of acute and chronic inflammatory cells was seen. Nodules of regeneration and fatty changes were evident (Figure 2). Microscopically, the liver sections presented a picture of a portal type of cirrhosis. Electron Microscopy
Electron-microscopic examination of thin sections of human liver revealed large numbers of hepatocytes engorged with many pleomorphic dense granules (Figure 5) and whorl-like structures (Figures 6 and 10). Most cells contained single nuclei surrounded by small islands of disrupted rough endoplasmic reticulum (ER). There was a marked proliferation of smooth endoplasmic reticulum to the extent that some cells appeared to be completely devoid of rough ER. The dense crystalline granules were bound by a single membrane and did not contain elements of phagocytosis,8 indicating the uptake of iron by the proliferating smooth ER (Figures 7 and 8). Many of the whorled structures, when viewed at high magnification, showed a uniform spacing or beading of iron particles
614
HENNIGAR ET AL
American Journal of Pathology
on the inner aspect of the rolled-up membranes (Figure 6). The presence of iron in these structures was confirmed by nondispersive x-ray analysis. Other ultrastructural features within the affected hepatocytes appeared to be essentially normal, with the exception of a slightly decreased, though normal-appearing, mitochondrial population. Glycogen was evenly distributed throughout the cytoplasm and was intermingled with ribosomal particles, and a fine spattering of ferritin tetrads were found free in the cell cytoplasm, outside smooth ER cisternae (Figure 8). These iron particles could only be seen at high magnification and at slightly under-focus conditions. In most cells the smooth ER had completely replaced the rough ER, with only a few scattered patches remaining associated with clumps of mitochondria or closely applied to the nuclear envelope. In the hepatocytes that contained large numbers of dense bodies and whorls, the presence of smooth ER was difficult to determine. We were led to the conclusion that the vacuolar system appeared to be intimately involved in packaging the iron overload. All of the dense bodies viewed at higher magnifications were completely or partially bound by a single-unit membrane structure measuring 70 A across, which we compared with uninvolved smooth ER within the same adjacent cells. Other workers have concluded that these thin, single-membrane-bound structures are secondary lysosomes. They suggest that the thin "myelin figure" lamellas with associated ferritin molecules are a newly recognized form of intralysosomal membrane that may be specific for iron overload.9 There was much variation in the degree of organization of the dense bodies, ranging from loose, randomly spaced ferritin tetrads to highly organized crystalline structures (Figures 8, 9, and 10). There was also a large range in size in these densities, varying from 0.1 to 5 ,u, measured on the short axis, and they were dispersed randomly and not in peri-bilecanalicular locations, as is usually the case with normal hepatocytes. Cytoplasmic lipid droplets were not markedly increased, although several engorged fat-storing cells 10 were noted in the space of Disse (Figure 11). Lipid-containing lipofuscin bodies were not increased and were found among the accumulations of iron (Figure 5). The space of Disse contained increased amounts of reticulin, indicating the onset of fibrosis. This fibrosis was greatly increased in the portal areas. Lateral cytoplasmic membranes extending away from the normal-appearing bile canaliculi formed finger-like projections, indicating an early cirrhotic condition.'1 Some of the bile ducts seen with the limited sampling imposed by electron microscopy appeared normal. Others, although normal in cell structure,
Vol. 96, No. 2 August 1979
HEMOCHROMATOSIS AND IRON INTAKE
615
contained numerous dense granules in the apical portion of the cytoplasm. The bile-duct lumens were open and not dilated. The Kupffer cells were relatively free of hemosiderin granules when compared to engorged hepatocytes lying adjacent to them, although some were found to contain considerable amounts of iron. Elemental X-ray Analysis
Studies to confirm the presence of iron in the dense granules, with the exception of lipofuscin bodies, were done with an energy dispersive x-ray analysis device 7 attached to a Hitachi H-500 transmission electron microscope. Figure 6 shows a whorl structure with uniformly spaced particles that were positive for iron by nondispersive x-ray analysis (Figure 12). Some of the siderosomes seemed to be more compact and denser than others, indicating the degree of crystalline organization. Discussion The membranes found surrounding the iron granules seen in these micrographs lead to speculation concerning the functional significance of these structures in iron storage and elimination.'2 Little has been reported on the excretion of large aggregates of crystalline inclusions by hepatocytes. If these structures are excretion granules, as is being proposed, the presence of closely applied membranes in these cells can be compared with membranous structures seen exiting liver cells damaged by a variety of toxic substances. One difference noted is that the toxic substance bound to the whorl is comparatively large and electron-dense. In liver cells exposed to barbiturates and carbon tetrachloride, the appearance of these whorl structures is thought to be a nonspecific reaction to injury,'13"4 but these chemicals cannot be seen with the use of the usual methods of electron microscopy. It is possible that these small molecules are present in great abundance within the folds of the whorls and are either undergoing a detoxification process by the action of smooth ER or are being rolled up into a package to be spewed out into the sinusoid (Figure 6). An alternative to this mechanistic approach is that the liver cells simply become so engorged with iron that their life-sustaining capacity is impaired and they degenerate, releasing their burden, to be taken up by surrounding liver cells, Kupffer cells, macrophages, and fibrocytes.15 It is probable that both or even more of these events take place in the injured hepatocyte. It can be further speculated that the more organized and compact the iron crystals become, the more resistant they are to dissolution and
616
HENNIGAR ET AL
American Journal of Pathology
mobilization into usable iron through the usual metabolic pathways. It also seems likely that once the crystals have formed they remain in the hepatocyte for the lifetime of the cell. It is interesting to note that investigators using intact animal models have had little or no success in duplicating hemochromatosis as seen in man 16 when using chronic and acute administrations of various iron compounds; however, if the animals are exposed to hepatotoxic substances prior to iron treatment,17 experimental iron storage can develop. Studies have also shown that iron-overloaded animals are more susceptible to toxic substances."8""9 Several workers have reported a direct relationship between iron buildup in humans and ascorbic acid deficiency.20-23 It is possible that the lack of ascorbic acid synthesis in humans, but not in most lower animals, can help to explain the resistance of the usual laboratory models to iron storage as seen in man. Kent warns that the previous animal studies of iron overloading have not been of sufficient duration, as compared with the length of time it takes for an appreciable buildup to occur in human beings.24 Many workers have shown an increase in iron stores in reticuloendothelial cells and macrophages in laboratory animals.25 Iron in doses of up to 2.16 g/kg has been administered by various routes for periods of up to 7 years. Some animals have shown a buildup of granular iron stores in the liver, spleen, and pancreas; but none have shown an associated fibrosis or cirrhosis. Eye lesions and blindness resembling retinitis pigmentosa developed in all of the dogs of a study group given acute total doses of 0.5 to 1.0 g iron/kg body weight.26 This was the only pathologic effect observed in these animals, and these findings did not vary appreciably from the majority of the others concerning the effects of dietary iron.
There is no direct evidence that excessive iron stores in the liver of human beings causes fibrosis or cirrhosis. In the cases where these conditions do exist, it could be argued that iron was deposited as a result of tissues damaged by fibrosis, a theory that animal experimentation tends to support.27
References 1. Dymock IW, Cassar J, Pyke DA, Oakley WG, Williams R: Observations on the pathogenesis, complications, and treatment of diabetes in 115 cases of haemochromatosis. Am J Med 52:203-210, 1972 2. Balcerzak SP, Mintz DH, Westerman MP: Diabetes mellitus and idiopathic hemochromatosis. Am J Med Sci 255:53-62, 1968 3. Megyesi C, Samols E, Marks V: Glucose tolerance and diabetes in chronic liver disease. Lancet 2:1051-1055, 1967
Vol. 96, No. 2 August 1979
HEMOCHROMATOSIS AND IRON INTAKE
617
4. Seftel HC, Keeley KJ, Isaacson C, Bothwell TH: Siderosis in the Bantu: The clinical incidence of hemochromatosis in diabetic subjects. J Lab Clin Med 58:837844, 1961 5. Luft JH: Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409-414, 1961 6. Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208-212, 1963 7. Bender SL, Duff RH: Energy dispersion x-ray analysis with the transmission electron microscope, Publication of the Symposium on Energy Dispersion X-ray Analysis. American Society for Testing and Materials, 1970, Publication Number 485. 8. Arborg BAM, Glaumann H, and Ericsson JLE: Studies on Iron loading of Rat Liver Lysosomes: Effects on the liver and distribution and fate of iron. Lab Invest 30:664673, 1974 9. Iancu TC, Neustein HB, Landing BH: The liver in thalassemia major: Ultrastructural observations, Iron Metabolism, Ciba Foundation, Amsterdam, Elsevier Excerpta Medica, North-Holland, 1977 10. Ito T, Shibasaki S: Electron microscopic study on the hepatic sinusoidal wall and the fat storing cells in the normal human liver. Arch Histol Jpn 29:137-192, 1968 11. Steiner PE: Precision in the classification of cirrhosis of the liver. Am J Pathol 37:21-47, 1960 12. Puro DG, Richter GW: Ferritin synthesis by free and membrane bound (poly) ribosomes of rat liver: Proc Soc Exp Biol Med 138:399-403, 1971 13. Ortega P: Light and electron microscopy of dichlorodiphenyltrichloroethane (DDT) poisoning in the rat liver. Lab Invest 15:657-679, 1966 14. Greene WB, Walker EM, Gadsden RH, Klobukowski CJ, Gale GR, Hennigar GR: PCB-DDT toxicity in mouse liver cells: An electron microscopic study, Pesticides and the Environment: A Continuing Controversy. Edited by WB Deichmann. New York, International Medical Book Corp., 1973, pp 137-150 15. Berry CL, Marshall WC: Iron distribution in the liver of patients with thalassemia major. Lancet 1:1031-1033, 1967 16. Polson CJ: The failure of prolonged administration of iron to cause hemochromatosis. Br J Exp Pathol 14:73-76, 1933 17. Kent G, Volini FT, Minick OT, Orfei E, deLa Huerga J: Effect of hepatic injuries upon iron storage in the liver. Lab Invest 12:1094-1101, 1963 18. Witzleben CL, Chaffey NJ: A study of iron-induced liver damage. J Exp Med 115:723-729, 1962 19. MacDonald RA, Pechet GS: Experimental hemochromatosis in rats. Am J Pathol 46:85-109, 1965 20. Lipschitz DA, Bothwell TH, Seftel HC, Wapnick AA, Charlton RW: The role of ascorbic acid in the metabolism of storage iron. Br J Hematol 20:155-163, 1971 21. Lynch SR, Seftel HC, Torrance JD, Charlton RW, Bothwell TH: Accelerated oxidative catabolism of ascorbic acid in sideratic Bantu. Am J Clin Nutr 20:641-647, 1967 22. Wapnick AA, Lynch SR, Charlton RW, Seftel HC, Bothwell TH: The effect of ascorbic acid deficiency on desferrioxamine-induced urinary iron excretion. Br J Haematol 17:563-568, 1969 23. Wapnick AA, Lynch SR, Seftel HC, Charlton RW, Bothwell TH: The effect of siderosis and ascorbic acid depletion on bone metabolism, with special reference to osteoporosis in the Bantu. Br J Nutr 25:367-376, 1971 24. Kent G, Volini FT, Minick OT, Orfeti E, deLa Huerga J: Effect of iron loading upon the formation of collagen in the hepatic injury induced by carbon tetrachloride. Am J Pathol 45:129-155, 1964 25. Nissin JA: Experimental siderosis: A study of the distribution, delayed effects and
618
HENNIGAR ET AL
American Journal of Pathology
metabolism of massive amounts of various iron preparations. J Pathol 66:185-204, 1953 26. Brown EB, Jr, Dubach R, Smith DE, Reynafarje CJ, Moore CV: Studies in iron transportation and metabolism: X. Long-term iron overload in dogs. J Lab Clin Med 50:862-893, 1957 27. Goldberg L, Smith JP: Iron overloading and hepatic vulnerability. Am J Pathol 36:125-149, 1960
r jl EIy : t
Figure 1-Hepatocytes containing numerous hemosiderin granules, in some cases large enough to distort cellular architecture. Fibrous tissue (band) containing proliferating bile ducts. (H&E, X80) Figure 2-Fatty changes with distortion of cellular architecture and degenerative changes. Marked distortion of hepatic parenchyma by fibrous bands. Chronic inflammatory cells are present as are areas of regeneration. Small bile duct with fine granules of hemosiderin within bile-duct epithelium. (Masson's stain, X200)
Figure 3-Hepatic cells containing small nuclei with prominent nucleoli and clear areas of cytoplasm, often globular in appearance. Prominent sinusoid (lower left) with Kupffer cells containing hemosiderin granules, in some cases almost crystalline in appearance. Sinusoid containing inflammatory cells (lower right). (H&E, X400) Figure 4-Hepatocytes with ballooned cytoplasm and granular material (hemosiderin), which obscures nuclei in many instances, and with small nuclei containing prominent nucleoli. Several small bile ducts (lower left) with hemosiderin granules in cytoplasm of bile-duct epithelium. (Prussian blue, X200)
lbaw %
Figure 5-Low-power micrograph of hepatocyte showing numerous pleomorphic dense granules, Figure 6-Whorl structure whorled structures (arrows), and scant smooth and rough ER. (X6000) at higher power showing uniform-sized iron particles, evenly spaced and aligned on membranes.
(X 100, O)
@'sWe
-8iy
Rare cases of hemochromatosis have been reported in patients who underwent prolonged oral iron therapy for hemolytic anemia or prolonged self-treatment with iron pills. A proportionately large segment of the South African Bantu tribe, who ingest large quantities of an alcoholic beverage brewed in iron pots, are found to have the disease. Reports of health fadists developing hemochromatosis due to excessive dietary iron intake, however, are extremely rare. This report presents clinical considerations and pathologic findings in a compulsive health fadist who consumed large numbers of vitamins containing iron. Clinical findings included the development and progression of cirrhosis of the liver, bronzing of the skin, and diabetes mellitus, all consistent with a diagnosis of hemochromatosis. Light microscopy of liver biopsies taken late in the course of the disease revealed a massive buildup of iron in the hepatocytes, less in the Kupffer cells, and sparse deposition in the epithelial cells of the bile duct. Minimal periportal fibrosis was noted. Electron microscopy showed numerous pleomorphic siderosomes with varying degrees of crystallization and ferritin attached at uniform intervals to the membranes of residual bodies. Abundant free ferritin was observed in most cells. The aggregated and membrane-associated ferritin was verified by nondispersive x-ray analysis. An additional finding, noted only by electron microscopy, was the presence of many fat-storing cells of Ito, which are thought to be involved in the onset of fibrosis. (Am J Pathol 96:611-624, 1979)
THE TERM HEMOCHROMATOSIS was introduced by von Recklonghausen in 1889, and the disease is characterized by grossly excessive amounts of iron stores in body organs. The organs usually exhibit a diffuse fibrotic change. The underlying causes for retention of iron, resulting from multiple transfusions or excessive oral intake, may be due to inborn errors of metabolism comparable to idiopathic hemochromatosis. The evidence for genetic influence in this disorder is overwhelming. Patients presenting with hemochromatosis average a total body iron of 20-40 g, with liver iron increased by 50-100 times normal. The average liver weight is 2,400 g. Probably most of the extra iron storage is in the liver. As the concentration of unbound ferritin builds, it is transferred into membranebound hemosiderin granules, which tend to be more permanent structures. Clinical diabetes mellitus was reported in 63% (72/115) of patients at the time of diagnosis of idiopathic hemochromatosis,' and diabetes mellitus appears to occur in high incidence (47%) in close relatives of heFrom the Department of Pathology and the Department of Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina. Accepted for publication April 12, 1979. Address reprint requests to Gordon R. Hennigar, MD, Department of Pathology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29403.
0002-9440/79/0809-061 1$01.00 © American Association of Pathologists
611
612
HENNIGAR ET AL
American Journal of Pathology
mochromatotic families.2 The finding of diabetes mellitus associated with hemochromatosis may be related to concurrent liver disease and/or to excessive iron deposition in pancreatic tissue. In addition, insulin resistance has been shown to be associated with cirrhosis.3 Which disease comes first? Diabetes is present in only 20% of all the reported cases of dietary hemochromatosis. In the Bantu population, 7% of the diabetics were found to have fully developed hemochromatosis.4 Clinical Findings The patient was a 58-year-old white male locomotive operator who suffered a nervous breakdown at age 22 and shortly thereafter began a diet consisting of many raw eggs, tomatoes, prunes, raisins, and massive doses of vitamins containing iron (as many as 500 capsules during one 2week period in 1968). He was hospitalized and evaluated in 1968 for a large abdominal mass present for at least a year previously and episodes of gastroenteritis. Exploratory laparotomy and liver biopsies revealed hepatosplenomegaly with a tremendously enlarged liver presenting a roughly
granular nutmeg-appearing surface and microscopically showing extensive areas of portal fibrosis, bile-duct proliferation, and stippling of hepatic cells with brown granules, consistent with the diagnosis of hemochromatosis. Additional clinical and laboratory findings at that time (1968) were diabetes mellitus (with no previous history of the condition) and marked bronzing of the skin with dark-pigmented areolas, consistent with hemochromatosis of the skin. Medical control of the diabetes mellitus was initiated, vitamin intake was stopped, and units of blood were removed successively over a period of time to decrease the body load of iron. The patient was followed closely during the next 2-3 years (after October 1968), during which time at least four additional periods of hospitalization were required. Serum iron levels measured during this time interval did not tend to decrease, remaining within a 188-268,g/dl range (normal range 65-175 ,ug/dl), and serum iron-binding capacities remained low, in the 30-241 ,ug/dl range (normal range 250-410 ,g/dl). Serum enzymes (SGOT, LDH, alkaline phosphatase) tended to demonstrate slightly elevated levels during the same interval. The patient gradually developed cardiovascular difficulties during 1969 and presented early in 1970 with complaints and clinical findings consistent with the diagnosis of congestive heart failure, probably secondary to hepatic disease. His condition gradually worsened during the following months in spite of aggressive management with digoxin and diuretics, and he eventually died. Permission for autopsy was not granted.
Vol. 96, No. 2 August 1979
HEMOCHROMATOSIS AND IRON INTAKE
613
Materials and Methods A wedge biopsy was divided, and half was fixed in 10% phosphate-buffered formalin overnight and processed in paraffin for light microscopy. Serial sections were cut at 6 ,u and stained with hematoxylin and eosin (H&E), Masson's, and Prussian blue stains. A similar piece was minced into 1-cu mm blocks and fixed in 5% glutaraldehyde buffered with cacodylate and postfixed in 2% osmium tetroxide for 90 minutes. These blocks were dehydrated and embedded in Epon 812.' After polymerization, 0.5-1.0-,u-thick sections were cut, stained with toluidine blue, and examined with the light microscope for the determination of appropriate areas for subsequent thin sectioning. The thin sections were stained with uranyl acetate and lead citrate I and examined with a Hitachi HU-12 transmission electron microscope. A second series of epoxy sections, not stained with heavy metal solutions, were sent to the Perkin-Elmer Applications Laboratory in Mountain View, California, for energy dispersion x-ray analysis, a technique for the elemental analysis of submicroscopic structures.7
Results Light Microscopy
Light-microscopic examination of liver sections stained with H & E as well as with special stains for the demonstration of iron revealed extensive numerous granular deposits of hemosiderin within hepatocytes, at times obscuring cellular detail (Figure 4). Hemosiderin granules were present within Kupffer cells (Figure 3), bile-duct epithelium (Figures 2 and 4), and macrophages in the portal tracts, and in the areas of fibrosis. Examination of the hepatic parenchyma revealed irregular nodules of cells separated by bands of connective tissue within which the proliferation of small bile ducts was prominent (Figure 1). Degenerative changes were apparent within the majority of the liver cells (Figure 2), but an apparent relative paucity of acute and chronic inflammatory cells was seen. Nodules of regeneration and fatty changes were evident (Figure 2). Microscopically, the liver sections presented a picture of a portal type of cirrhosis. Electron Microscopy
Electron-microscopic examination of thin sections of human liver revealed large numbers of hepatocytes engorged with many pleomorphic dense granules (Figure 5) and whorl-like structures (Figures 6 and 10). Most cells contained single nuclei surrounded by small islands of disrupted rough endoplasmic reticulum (ER). There was a marked proliferation of smooth endoplasmic reticulum to the extent that some cells appeared to be completely devoid of rough ER. The dense crystalline granules were bound by a single membrane and did not contain elements of phagocytosis,8 indicating the uptake of iron by the proliferating smooth ER (Figures 7 and 8). Many of the whorled structures, when viewed at high magnification, showed a uniform spacing or beading of iron particles
614
HENNIGAR ET AL
American Journal of Pathology
on the inner aspect of the rolled-up membranes (Figure 6). The presence of iron in these structures was confirmed by nondispersive x-ray analysis. Other ultrastructural features within the affected hepatocytes appeared to be essentially normal, with the exception of a slightly decreased, though normal-appearing, mitochondrial population. Glycogen was evenly distributed throughout the cytoplasm and was intermingled with ribosomal particles, and a fine spattering of ferritin tetrads were found free in the cell cytoplasm, outside smooth ER cisternae (Figure 8). These iron particles could only be seen at high magnification and at slightly under-focus conditions. In most cells the smooth ER had completely replaced the rough ER, with only a few scattered patches remaining associated with clumps of mitochondria or closely applied to the nuclear envelope. In the hepatocytes that contained large numbers of dense bodies and whorls, the presence of smooth ER was difficult to determine. We were led to the conclusion that the vacuolar system appeared to be intimately involved in packaging the iron overload. All of the dense bodies viewed at higher magnifications were completely or partially bound by a single-unit membrane structure measuring 70 A across, which we compared with uninvolved smooth ER within the same adjacent cells. Other workers have concluded that these thin, single-membrane-bound structures are secondary lysosomes. They suggest that the thin "myelin figure" lamellas with associated ferritin molecules are a newly recognized form of intralysosomal membrane that may be specific for iron overload.9 There was much variation in the degree of organization of the dense bodies, ranging from loose, randomly spaced ferritin tetrads to highly organized crystalline structures (Figures 8, 9, and 10). There was also a large range in size in these densities, varying from 0.1 to 5 ,u, measured on the short axis, and they were dispersed randomly and not in peri-bilecanalicular locations, as is usually the case with normal hepatocytes. Cytoplasmic lipid droplets were not markedly increased, although several engorged fat-storing cells 10 were noted in the space of Disse (Figure 11). Lipid-containing lipofuscin bodies were not increased and were found among the accumulations of iron (Figure 5). The space of Disse contained increased amounts of reticulin, indicating the onset of fibrosis. This fibrosis was greatly increased in the portal areas. Lateral cytoplasmic membranes extending away from the normal-appearing bile canaliculi formed finger-like projections, indicating an early cirrhotic condition.'1 Some of the bile ducts seen with the limited sampling imposed by electron microscopy appeared normal. Others, although normal in cell structure,
Vol. 96, No. 2 August 1979
HEMOCHROMATOSIS AND IRON INTAKE
615
contained numerous dense granules in the apical portion of the cytoplasm. The bile-duct lumens were open and not dilated. The Kupffer cells were relatively free of hemosiderin granules when compared to engorged hepatocytes lying adjacent to them, although some were found to contain considerable amounts of iron. Elemental X-ray Analysis
Studies to confirm the presence of iron in the dense granules, with the exception of lipofuscin bodies, were done with an energy dispersive x-ray analysis device 7 attached to a Hitachi H-500 transmission electron microscope. Figure 6 shows a whorl structure with uniformly spaced particles that were positive for iron by nondispersive x-ray analysis (Figure 12). Some of the siderosomes seemed to be more compact and denser than others, indicating the degree of crystalline organization. Discussion The membranes found surrounding the iron granules seen in these micrographs lead to speculation concerning the functional significance of these structures in iron storage and elimination.'2 Little has been reported on the excretion of large aggregates of crystalline inclusions by hepatocytes. If these structures are excretion granules, as is being proposed, the presence of closely applied membranes in these cells can be compared with membranous structures seen exiting liver cells damaged by a variety of toxic substances. One difference noted is that the toxic substance bound to the whorl is comparatively large and electron-dense. In liver cells exposed to barbiturates and carbon tetrachloride, the appearance of these whorl structures is thought to be a nonspecific reaction to injury,'13"4 but these chemicals cannot be seen with the use of the usual methods of electron microscopy. It is possible that these small molecules are present in great abundance within the folds of the whorls and are either undergoing a detoxification process by the action of smooth ER or are being rolled up into a package to be spewed out into the sinusoid (Figure 6). An alternative to this mechanistic approach is that the liver cells simply become so engorged with iron that their life-sustaining capacity is impaired and they degenerate, releasing their burden, to be taken up by surrounding liver cells, Kupffer cells, macrophages, and fibrocytes.15 It is probable that both or even more of these events take place in the injured hepatocyte. It can be further speculated that the more organized and compact the iron crystals become, the more resistant they are to dissolution and
616
HENNIGAR ET AL
American Journal of Pathology
mobilization into usable iron through the usual metabolic pathways. It also seems likely that once the crystals have formed they remain in the hepatocyte for the lifetime of the cell. It is interesting to note that investigators using intact animal models have had little or no success in duplicating hemochromatosis as seen in man 16 when using chronic and acute administrations of various iron compounds; however, if the animals are exposed to hepatotoxic substances prior to iron treatment,17 experimental iron storage can develop. Studies have also shown that iron-overloaded animals are more susceptible to toxic substances."8""9 Several workers have reported a direct relationship between iron buildup in humans and ascorbic acid deficiency.20-23 It is possible that the lack of ascorbic acid synthesis in humans, but not in most lower animals, can help to explain the resistance of the usual laboratory models to iron storage as seen in man. Kent warns that the previous animal studies of iron overloading have not been of sufficient duration, as compared with the length of time it takes for an appreciable buildup to occur in human beings.24 Many workers have shown an increase in iron stores in reticuloendothelial cells and macrophages in laboratory animals.25 Iron in doses of up to 2.16 g/kg has been administered by various routes for periods of up to 7 years. Some animals have shown a buildup of granular iron stores in the liver, spleen, and pancreas; but none have shown an associated fibrosis or cirrhosis. Eye lesions and blindness resembling retinitis pigmentosa developed in all of the dogs of a study group given acute total doses of 0.5 to 1.0 g iron/kg body weight.26 This was the only pathologic effect observed in these animals, and these findings did not vary appreciably from the majority of the others concerning the effects of dietary iron.
There is no direct evidence that excessive iron stores in the liver of human beings causes fibrosis or cirrhosis. In the cases where these conditions do exist, it could be argued that iron was deposited as a result of tissues damaged by fibrosis, a theory that animal experimentation tends to support.27
References 1. Dymock IW, Cassar J, Pyke DA, Oakley WG, Williams R: Observations on the pathogenesis, complications, and treatment of diabetes in 115 cases of haemochromatosis. Am J Med 52:203-210, 1972 2. Balcerzak SP, Mintz DH, Westerman MP: Diabetes mellitus and idiopathic hemochromatosis. Am J Med Sci 255:53-62, 1968 3. Megyesi C, Samols E, Marks V: Glucose tolerance and diabetes in chronic liver disease. Lancet 2:1051-1055, 1967
Vol. 96, No. 2 August 1979
HEMOCHROMATOSIS AND IRON INTAKE
617
4. Seftel HC, Keeley KJ, Isaacson C, Bothwell TH: Siderosis in the Bantu: The clinical incidence of hemochromatosis in diabetic subjects. J Lab Clin Med 58:837844, 1961 5. Luft JH: Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409-414, 1961 6. Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208-212, 1963 7. Bender SL, Duff RH: Energy dispersion x-ray analysis with the transmission electron microscope, Publication of the Symposium on Energy Dispersion X-ray Analysis. American Society for Testing and Materials, 1970, Publication Number 485. 8. Arborg BAM, Glaumann H, and Ericsson JLE: Studies on Iron loading of Rat Liver Lysosomes: Effects on the liver and distribution and fate of iron. Lab Invest 30:664673, 1974 9. Iancu TC, Neustein HB, Landing BH: The liver in thalassemia major: Ultrastructural observations, Iron Metabolism, Ciba Foundation, Amsterdam, Elsevier Excerpta Medica, North-Holland, 1977 10. Ito T, Shibasaki S: Electron microscopic study on the hepatic sinusoidal wall and the fat storing cells in the normal human liver. Arch Histol Jpn 29:137-192, 1968 11. Steiner PE: Precision in the classification of cirrhosis of the liver. Am J Pathol 37:21-47, 1960 12. Puro DG, Richter GW: Ferritin synthesis by free and membrane bound (poly) ribosomes of rat liver: Proc Soc Exp Biol Med 138:399-403, 1971 13. Ortega P: Light and electron microscopy of dichlorodiphenyltrichloroethane (DDT) poisoning in the rat liver. Lab Invest 15:657-679, 1966 14. Greene WB, Walker EM, Gadsden RH, Klobukowski CJ, Gale GR, Hennigar GR: PCB-DDT toxicity in mouse liver cells: An electron microscopic study, Pesticides and the Environment: A Continuing Controversy. Edited by WB Deichmann. New York, International Medical Book Corp., 1973, pp 137-150 15. Berry CL, Marshall WC: Iron distribution in the liver of patients with thalassemia major. Lancet 1:1031-1033, 1967 16. Polson CJ: The failure of prolonged administration of iron to cause hemochromatosis. Br J Exp Pathol 14:73-76, 1933 17. Kent G, Volini FT, Minick OT, Orfei E, deLa Huerga J: Effect of hepatic injuries upon iron storage in the liver. Lab Invest 12:1094-1101, 1963 18. Witzleben CL, Chaffey NJ: A study of iron-induced liver damage. J Exp Med 115:723-729, 1962 19. MacDonald RA, Pechet GS: Experimental hemochromatosis in rats. Am J Pathol 46:85-109, 1965 20. Lipschitz DA, Bothwell TH, Seftel HC, Wapnick AA, Charlton RW: The role of ascorbic acid in the metabolism of storage iron. Br J Hematol 20:155-163, 1971 21. Lynch SR, Seftel HC, Torrance JD, Charlton RW, Bothwell TH: Accelerated oxidative catabolism of ascorbic acid in sideratic Bantu. Am J Clin Nutr 20:641-647, 1967 22. Wapnick AA, Lynch SR, Charlton RW, Seftel HC, Bothwell TH: The effect of ascorbic acid deficiency on desferrioxamine-induced urinary iron excretion. Br J Haematol 17:563-568, 1969 23. Wapnick AA, Lynch SR, Seftel HC, Charlton RW, Bothwell TH: The effect of siderosis and ascorbic acid depletion on bone metabolism, with special reference to osteoporosis in the Bantu. Br J Nutr 25:367-376, 1971 24. Kent G, Volini FT, Minick OT, Orfeti E, deLa Huerga J: Effect of iron loading upon the formation of collagen in the hepatic injury induced by carbon tetrachloride. Am J Pathol 45:129-155, 1964 25. Nissin JA: Experimental siderosis: A study of the distribution, delayed effects and
618
HENNIGAR ET AL
American Journal of Pathology
metabolism of massive amounts of various iron preparations. J Pathol 66:185-204, 1953 26. Brown EB, Jr, Dubach R, Smith DE, Reynafarje CJ, Moore CV: Studies in iron transportation and metabolism: X. Long-term iron overload in dogs. J Lab Clin Med 50:862-893, 1957 27. Goldberg L, Smith JP: Iron overloading and hepatic vulnerability. Am J Pathol 36:125-149, 1960
r jl EIy : t
Figure 1-Hepatocytes containing numerous hemosiderin granules, in some cases large enough to distort cellular architecture. Fibrous tissue (band) containing proliferating bile ducts. (H&E, X80) Figure 2-Fatty changes with distortion of cellular architecture and degenerative changes. Marked distortion of hepatic parenchyma by fibrous bands. Chronic inflammatory cells are present as are areas of regeneration. Small bile duct with fine granules of hemosiderin within bile-duct epithelium. (Masson's stain, X200)
Figure 3-Hepatic cells containing small nuclei with prominent nucleoli and clear areas of cytoplasm, often globular in appearance. Prominent sinusoid (lower left) with Kupffer cells containing hemosiderin granules, in some cases almost crystalline in appearance. Sinusoid containing inflammatory cells (lower right). (H&E, X400) Figure 4-Hepatocytes with ballooned cytoplasm and granular material (hemosiderin), which obscures nuclei in many instances, and with small nuclei containing prominent nucleoli. Several small bile ducts (lower left) with hemosiderin granules in cytoplasm of bile-duct epithelium. (Prussian blue, X200)
lbaw %
Figure 5-Low-power micrograph of hepatocyte showing numerous pleomorphic dense granules, Figure 6-Whorl structure whorled structures (arrows), and scant smooth and rough ER. (X6000) at higher power showing uniform-sized iron particles, evenly spaced and aligned on membranes.
(X 100, O)
@'sWe
-8iy
