CASE REPORT

Abnormal Liver Function in Chronic Hypervitaminosis A G.C. F A R R E L L , MB, P.S. B H A T H A L , PhD, and L.W. P O W E L L , PhD, MD

Chronic hypervitaminosis A is a well-recognized but u n c o m m o n disorder (1-4). The clinical picture is varied and nonspecific, and includes skin and hair changes, h e a d a c h e , upper-gastrointestinal s y m p toms, muscle and bone pain, hypercalcemia, and psychiatric disturbances (5, 6). H e p a t o t o x i c i t y appears less c o m m o n despite the fact that the liver is the major site of vitamin A storage (7). The usual clinical manifestation of liver involvement is hepatomegaly (1, 2, 5) but cases of portal hypertension, ascites, and cirrhosis have been reported (8). We report a case of vitamin A hepatotoxicity that c a m e to our notice because of abnormalities in liver function tests detected on routine biochemical screening. The nature of the specific hepatic abnormality was further elucidated by electron microscopic studies.

CASE REPORT A 57-year-old housewife was referred to the Liver Clinic at the Royal Brisbane Hospital because of unexplained persistently abnormal liver function tests detected during the investigation of alopecia. Her main complaint was of pain in her shoulders, wrists, ankles and toes which had been present for many years. She also complained of a dry skin and progressive thinning of her hair over the previous five years. Apart from occasional attacks of lower abdominal pain she was otherwise asymptomatic. She had previously embarked upon many "health diets" for her skin and hair disorder and also had been taking many vitamin supplements including at least three vitamin A capsules per day (total, 90,000 IU or 27,000/xg) for four years. No history of alcohol consumption was elicited. On examination Heberden's nodes were apparent, her skin was dry and it had the characteristic orange-yellow appearance of xanthosis suggesting hypercar0tenemia (1). There was some thinning of the hair, particularly From the Department of Medicine, University of Queensland, Royal Brisbane Hospital, and the Department of Pathology, University of Melbourne, Australia. Address for reprint requests: Prof. L.W. Powell, Department of Medicine, Royal Brisbane Hospital, Herston 4029, Queensland, Australia.

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around the temples. Bone tenderness was not present and the optic fundi were normal. The liver was not palpable and its span was 10 cm to soft percussion. The spleen was not enlarged clinically, and there was no ascites. There were no stigmata of chronic liver disease. Investigations included serum alkaline phosphatase 108 IU/liter (normal 30--85 IU), serum aspartate transaminase 72 IU/liter (7-40), lactic dehydrogenase 218 IU/liter (125-220), 5'-nucleotidase 6 units/liter (2-15), gamma glutamyl transpeptidase 20 units/liter (5-65), total sel-um bilirubin 10 p.mole/liter (0--25), serum albumin 47 g/liter (35-45), total serum globulins 27 g/liter (21-41) and prothrombin time 13 sec (control 12 sec). Total serum carotenoids were 8.5 tzmole/liter (0.9-4.7). An oral cholecystogram showed a normally functioning gallbladder and patent intra- and extrahepatic bile ducts of normal caliber. The liver scan showed that the liver was not increased in size and had uniform isotope uptake. Radiographs of the hands showed some degenerative changes of the first metacarpophalangeal and carpometacarpal joints. Other investigations which were within normal limits included a full blood count and erythrocyte sedimentation rate, serum iron and iron-binding capacity, serum ferritin concentration, thyroid function tests, serum calcium, blood urea nitrogen, and serum creatinine. A skin biopsy showed prominent solar degeneration and mild hyperkeratosis but no keratinization of sweat glands. There was no autofluorescence. A detailed history from the patient revealed that in addition to the vitamin A capsules, her diet contained t600/xg of vitamin A, which is approximately twice the recommended daily allowance (14). She was advised to cease vitamin A supplementation and to avoid items in her diet such as vegetable oil, carrot, pumpkin, and cheese which contain large amounts of retinol and /3-carotenes. Four months after stopping her vitamin A consumption, the liver function tests returned to normal but the serum carotenoids were still elevated and the follow-up liver biopsy appearances were essentially unchanged. One year after vitamin A cessation there has been marked improvement in her skin and hair, and bone pains have also diminished. Liver function tests remain normal but the serum carotenoids are still slightly elevated (6.7/zmole/liter). Special Investigations A percutaneous liver biopsy was performed and the specimen was submitted for histological, autofluoresDigestive Diseases, Vol. 22, No. 8 (August 1977)

F A R R E L L ET AL Fluorescence microscopy of an unstained frozen section showed a prominent rapidly fading green vitamin A fluorescence (11) of fat vacuoles in the perisinusoidal cells. The specimen submitted for electron microscopy was fixed by immersion in cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 3 hr, washed in buffer, postfixed in 1% osmium tetroxide in cacodylate buffer, and embedded in Spurr's low-viscosity epoxy resin medium (12). Sections 1 /xm thick stained with toluidine blue were studied with a light microscope. Ultrathin sections were stained with uranyl acetate and examined with a Siemens Elmiskop I electron microscope. These showed typical hypertrophied fat-storing cells of Ito situated in the perisinusoidal space of Disse and separated from the sinusoidal lumen by endothelial and Kupffer cells (Figure 3). The cells varied in shape

Fig 1. Portion of a liver lobule showing the characteristic vacuolated appearance produced by hyperplasia and hypertrophy of fat-storing cells (F) situated in the space of Disse; P, portal tract. (H&E, x 300)

cence, and ultrastructural studies. By light microscopy the lobular architecture was intact and there was no evidence of fibrosis. At low magnification the liver-cell plates presented a honeycombed appearance (Figure 1), which on closer inspection proved to be due to an increase in both the number and size of fat-storing cells of Ito (9). There was approximately one such cell for every six liver cells compared with the normal ratio of 1 in 20 (10). These enlarged cells possessed scalloped outlines, foamy reticulated cytoplasm, and eccentrically iocated, small, crenated nuclei. Frequently sinusoids were compressed and liver cells displaced by these cells. Scattered lymphocytes and occasional polymorphs were seen in sinusoids, and there Was a modest round-cell infiltrate in otherwise normal portal tracts. Liver cells showed moderate variation in nuclear size, slight nuclear vacuolation, a minimal amount of microvacuolar fat and a probable increase in lipofuscin. Scattered liver cells especially in central zones possessed a ground-glass cytoplasm (Figure 2). Digestive Diseases, Vol. 22, No. 8 (August 1977)

Fig 2. Epoxy-resin-embedded 1-~m-thick section showing five hypertrophied, perisinusoidal, fat-storing or vitamin A storage cells with foamy cytoplasm. Numerous lipofuscin, black-appearing, granules are seen in a pericanalicular location of hepatocytes. G, ground-glass hepatocyte; S, sinusoid containing erythrocytes. (Toluidine blue, • 8000)

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Fig 3. Electron micrograph of hypertrophied fat-storing cell of Ito containing numerous lipid vacuoles and a peripherally displaced nucleus (N). Due to the plane of sectioning the cell appears to be placed between two sinusoids and three hepatocytes (H). D, space of Disse; E, swollen sinusoidal endothelial lining; R, red blood cell. (• 6700)

from ovoid in the case of hypertrophied ones tO being more straplike and stellate in the case of the few normalsized ones (Figures 3 and 4). Hepatocytes showed normal sinusoidal, canalicular, and lateral surfaces. Scattered liver cells showed hypertrophy of the type I or vesicular (13) smooth endoplasmic reticulum. Glycogen was abundant, and peroxisomes were frequently observed. A few liver cells contained one or two lipid vacuoles. Occasional small focal areas of cytoplasmic degradation and residual bodies or lipofuscin in varying stages of formation were seen in most liver cells while some giant mitochondria containing filamentous inclusions were also seen. RESULTS AND DISCUSSION

Vitamin A hepatotoxicity was suspected in this patient because of the abnormal liver function tests, the orange-yellow discoloration of the skin, and the dietary and drug history. The bone pains, alopecia, and skin changes that were present are well-recognized features of chronic hypervitaminosis A (2-5).

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This diagnosis was confirmed by the elevated level of serum carotenoids, the unusual but characteristic appearances of the liver biopsy, and particularly strong vitamin A fluorescence of the unstained frozen section. Electron microscopy showed the presence of numerous hypertrophied fat-storing cells of Ito which are readily distinguished from Kupffer and endothelial cells by their perisinusoidal location in the space of Disse and the absence of phagocytic activity (9, 10, 15). Ultrastructural change s in hepatocytes were similar to those described by others (7, 17, 18) and included focal cytoplasmic degradation, lipid droplets, excessive accumulation of lipofuscin, giant mitrochondria with paracrystalline inclUsions, and hypertrophy of smooth endoplasmic reticulum. The latter is an adaptive response to increased metabolism of vitamin A since both synthesis (19) and splitting (20) of the ester are microsomal functions. The presence of filamentous inDigestive Diseases, Vol. 22, No. 8 (August 1977)

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Fig 4. Straplike Ito cell and the cytoplasmic process (p) of such a cell, containing a few small vacuoles and bundles of microfibrils attached to dense zones beneath the plasma membrane (large arrows). In addition there is a small amount of collagen (C) and basement-membrane-like material (small arrows) in the space of Disse (D). E, Pale cytoplasmic processes of endothelial or Kupffer cells; H, hepatocyte, K, Kupffer cell body; L, lipolysosome; R, red blood cell in sinusoid. (x 12,600)

clusions in o c c a s i o n a l giant m i t o c h o n d r i a is considered to be within normal limits (21, 22). There was only minor evidence of hepatotoxicity which took the form of occasional small foci of cytoplasmic degradation and the accumulation of residual bodies. Vitamin A is primarily stored in the liver as the biologically inactive palmitate ester (23). Hepatic esterases convert it to the biologically active retinol which is released into the blood. In vitro excess free vitamin A induces changes in isolated rat liver mitochondria (24) and lysosomes (25), causing swelling of mitochondria and release of hydrolytic enzymes from lysosomes, respectively. Previous case reports have emphasized hepatomegaly as a common feature (2). Our patient is unusual in this regard and probably represents an early case. The total amount of vitamin A ingested on an average daily basis was about 30,000 ~g, which is more than double the amount thought by some to cause significant human toxicity (2, 4). Chronic hypervitaminosis A may exist for many Digestive Diseases, Vol. 22, No. 8 (August 1977)

years without recognition (2), eventually resulting in hepatocellular damage, hepatic fibrosis, portal hypertension, and ascites (8). The last two have been reported in the absence of cirrhosis and have been attributed to obstruction of sinusoids, obliteration of the space of Disse by collagen, and the greatly increased number and size of fat-storing cells of Ito (8). These cells appear to be able to transform into fibroblasts and may be important in the pathogenesis of hepatic fibrosis (26, 27). The presence in fat-storing cells of microfibrils at the cytoplasmic periphery and of convoluted nuclei indicate some resemblance between these cells and myofibroblasts (28, 29). The possibility of transformation of Ito cells to myofibroblasts was raised by Hruban et al (18). In the present case the absence of portal and perisinusoidal fibrosis suggests the patient was discovered at an early stage. This is also consistent with the length of the history, the average daily intake of vitamin A, and the clinical findings. As mobilization of large hepatic stores of vitamin A takes years (30), it is possible that further hepatic damage

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may occur in our patient despite stopping vitamin A ingestion. Vitamin A supplements are widely advertised as general health tonics, for prophylaxis against the common cold, and for skin disorders. Such advertising occurs without adequate warning to the public regarding possible toxicity. The case described here illustrates the importance of restricting the sale of vitamin A preparations without prescription. Vitamin A must be included in the growing list of drugs and other hepatotoxins that have to be considered in assessing any patient with acute or chronic liver disease. SUMMARY A case of chronic hypervitaminosis A is reported in a 57-year-old woman who took vitamin preparations for alopecia. Liver biopsy of the patient showed both an increase in the number and size of fat-storing cells on light microscopy and rapidly fading green autofluorescence of vitamin A. Electron microscopy confirmed the presence of engorged fatstoring cells in the space of Disse and minor toxic changes in hepatocytes. This case illustrates the early hepatic changes of chronic hypervitaminosis A and indicates a need for restriction of sales of vitamin preparations. REFERENCES 1. Josephs HW: Hypervitaminosis A and carotenemia. Am J Dis Child 67:33--43, 1944 2. Stimson WH: Vitamin A intoxication in adults. Report of a case with a summary of the literature. N Engl J Med 265:369-373, 1961 3. Soler-Bechara J, Soscia JL: Chronic hypervitaminosis A. Arch Intern Med 112:462-466, 1963 4. Bergen SS, Roels OA: Hypervitaminosis A: Report of a case. Am J Clin Nutr 16:265-269, 1965 5. Muenter MD, Perry HO, Ludwig J: Chronic vitamin A intoxication in adults: Hepatic, neurologic and dermatologic complications. Am J Med 50:129-136, 1971 6. Katz CM, Tzagournis M." Chronic adult hypervitaminosis A with hypercalcemia. Metabolism 21:1171-1176, 1972 7. Lane BP: Hepatic microanatomy in hypervitaminosis A in man and rat. Am J Pathol 53:591-598, 1968 8. Russell RM, Boyer JL, Bag,heft SA, Hruban Z: Hepatic injury from chronic hypervitaminosis A resulting in portal hypertension and ascites. N Engl J Med 291:435-440, 1974 9. Ito T, Nemoto M: r die kupfferschen Steruzellen und die "FettspeicherungszeUen" ("fat-storing cells") in der Blutkapillarenwand der menschlichen Leber. Okajimas Folia Anat Jpn 24:243-258, 1952

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10. Bronfenmajer S, Schaffner F, Popper H: Fat-storing cells (lipocytes) in human liver. Arch Pathol 82:447-453, 1966 11. Popper H: Distribution of vitamin A in tissue as visualised by fluorescence microscopy. Physiol Rev 24:205-224, 1944 12. Spurr AR: A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31-43, 1969 13. J6z6quel AM, Koch M, Orlandi F: A morphometric study of the endoplasmic reticulum in human hepatocytes. Correlation between morphological and biochemical data in subjects under treatment within certain drugs. Gut 15:737-747, 1974 14. Scrimshaw NS: Hypovitaminosis A. Textbook of Medicine, 13th ed. PB Beeson, W McDermott (eds). Philadelphia, W. B. Saunders, 1971, pp 1438-1440 15. Widmann J-J, Cotran RS, Fahimi HD: Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat liver sinusoids by endogenous peroxidase activity. J Cell Biol 52:159-170, 1972 16. Kobayashi K, Takahashi Y, Shibasaki S: Cytological studies of fat-storing cells in the liver of rats given large doses of vitamin A. Nature (London), New Biol 243:186-188, 1973 17. Rubin E, Florman AL, Degnan T, Diaz J: Hepatic injury in chronic hypervitaminosis A. Am J Dis Child 119:132-138, 1970 18. Hruban A, Russell RM, Boyer JL, Glagov S, Bagheri SA: Ultrastructural changes in livers of two patients with hypervitaminosis A. Am J Pathol 76:451-468, 1974 19. Futterman S, Andrews JS: The composition of liver vitamin A ester and the synthesis of vitamin A ester by liver microsomes. J Biol Chem 239:4077-4080, 1964 20. Ganguly J, Deuel HJ Jr: Intracellular distribution of vitamin A esterase activity in rat liver. Nature 172:120--121, 1953 21. Wills EJ: Crystalline structures in the mitochondria of normal human liver parenchymal cells. J Cell Biol 24:511-514, 1965 22. Ma MH, Biempica L: The normal human liver cell. Cytochemical and ultrastructural studies. Am J Pathol 62:353390, 1971 23. Mandel HG: Fat-soluble vitamins, I. Vitamin A. The Pharmacological Basis of Therapeutics. LS Goodman, A Gilman (eds). New York, Macmillan, 1970, pp 1672-1679 24. Lucy JA, Luscombe Mollie, Dingle JT: Studies on the mode of action of excess of vitamin A. 8. Mitochondrial swelling. Biochem J 89:419--425, 1963 25. Dingle JT: Studies on the mode of action of excess vitamin A.3. Release of bound protease by the action of vitamin A. Biochem J 79:509-512, 1961 26. Popper H, Udenfriend S: Hepatic fibrosis: Correlation of biochemical and morphological investigation. Am J Med 49:707-721, 1970 27. McGee JO'D, Patrick RS: The role of perisinusoidal cells in hepatic fibrogenesis. An electron microscopic study of acute carbon tetrachloride liver injury. Lab Invest 26:429-440, 1972 28. Gabbiani G, Ryan GB, Majno G: Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 27:549-550, 1971 29. Bhathal PS: Presence of modified fibroblasts in cirrhotic liver in man. Pathology 4:139-144, 1972 30. Nieman C, Klein Obbink HJ: The biochemistry and pathology of hypervitaminosis A. Vitam Horm 12:69-99, 1954

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