Hepatic Pathology in Vitamin a Toxicity FAIRPOUR FOROUHAR, M.D.* MARTIN S

Hepatic Pathology in Vitamin A Toxicity FAIRPOUR FOROUHAR, M.D.* MARTIN S. NADEL, M.D.,t and BERNARD GONDOS, M.D.* *Department of Pathology, University of Connecticut, Farmington, CT 06032 and fDepartment of Pathology, Middlesex Memorial Hospital, Middletown, CT 06457

ABSTRACT This paper documents a case of vitamin A toxicity presenting with splenomegaly and ascites. The light microscopic, electron microscopic, and fluorescent findings are described in detail. The principal histopath ologic finding was marked perisinusoidal fibrosis. The role of Ito cells in the storage of lipid-soluble vitamins and their subsequent transformation to fibroblasts producing collagen are discussed.

tient denied use of medications but admitted long Introduction time use of a self-prescribed vitamin B1-B6 combi nation, vitamin B12, iodine, and garlic. Vitamin A Liver disease resulting from vitamin A had been taken for six years, initially 75,000 units toxicity is an uncommon but well docu daily, increased to 150,000 units daily two years prior to admission because of believed problems with night mented entity.6’1414’22’26,27 The present vision for which no medical help had been sought. case provides a good model for the study Physical examination disclosed no palpable spleno of fibrosis of the space of Disse and its megaly with liver felt one cm beneath the right costal margin. An abdominal fluid wave was present. No pathophysiologic ^consequences. Similar spider angiomata were noted, and the remainder of changes also occur in other conditions re the physical examination was unremarkable. Labo lated to chemical toxicity including al ratory exams included: white cell count 2,400; he moglobin 11.5 g; hematocrit 35.2 percent; differential coholic hepatitis,13 methotrexate tox blood count— neutrophils 56, band forms 9, lympho icity,12 carbon tetrachloride toxicity,22 cytes 27, monocytes 6, and eosinophils 2. The and veno-occlusive diseases of the liver. platelet count was 122,000; serum iron 39 meg per 100 ml; total iron binding capacity (TIBC) 183 meg per 100 ml; iron saturation 21.3 percent; total cho Case Report lesterol 136 mg per 100 ml; prothrombin time 13.1 seconds (normal 11 to 14); activated partial throm The patient is a 37-year-old white female who pre boplastin time 35.2 seconds (normal 25 to 35.1). sented with a recent 15 to 20 lb. weight gain asso Normal laboratory studies include: serum aspartate ciated with abdominal swelling. There was no known aminotransferase; serum alanine aminotransferase; history of previous illness, exposure to hepatitis, total bilirubin; gamma glutamyl transpeptidase; al jaundice, or use of birth control pills for 15 years. kaline phosphotase; ferritin; T4; T3 percent uptake; Mild alcohol intake was admitted at age 20, but al serum protein electrophoresis; ceruloplasmin; and cohol intake in the past three years was denied. thyroid stimulating hormone. A single serum vitamin There was no family history of liver disease. The pa A was 55.8 meg per dl (normal 20 to 80), but this was 304 0091-7370/84/0700-0304 $01.20 © Institute for Clinical Science, Inc. VITAMIN A TOXICITY 305 not repeated. X-ray studies disclosed a right pleural effusion, and a liver scan showed an enlarged spleen and a small liver with reversal of the colloid ratio suggestive of a cirrhotic-type pattern. A percuta neous liver biopsy was performed, and the patient was discharged pending results of pathologic exami nation which showed no significant diagnostic abnor malities. The patient was readmitted for an explor atory laparotomy and open liver biopsy. At the time of surgery, two liters of ascitic fluid were present. The liver was small without nodularity, and fluid exuded from the capsule. Wedge biopsies were ob tained from both the right and left lobes. No other intra-abdominal abnormality was noted. Laboratory Findings A portion of the liver biopsy specimen was submitted to the University of Chi cago for vitamin A analysis. Using the method described by Olson,23 a value of F ig u r e 1. Photomicrograph of a frozen section of 692 mg of vitamin A per g of liver was the liver showing intense autofluorescence, a prop erty of both hepatocytes and fat storing cells in liver obtained. An aliquot of the extract solu of patients with vitamin A toxicity. (Fluorescent mi tion was also analyzed by the method of croscopy) Neeld and Pearson21 which gave a value of 1060 mg per g. Both of these values cells averaging 5 per 20 liver cells (one were considerably above the normal per 20 is normal2) (figure 4). Many Ito level. A frozen section was studied with cells were found in the portal and peri ultraviolet light and showed strong au portal fibrotic areas (figure 5). tofluorescence characteristic of vitamin A storage in the liver (figure 1). Electron Microscopy Light Microscopy Ultrastructural examination showed good fixation. There was an accumulation Examination of the second biopsy of variable amounts of collagen and base showed that the lobular architecture of ment membrane-like material in the the liver was preserved. Patchy areas of space of Disse. Ito cells were readily perisinusoidal fibrosis with varying in identified, and some were indistinguish tensity involved the periportal and, oc able from fibroblasts. The sinusoids were casionally, pericentral zones (figures 2 partially filled by round to oval blebs of and 3). In these areas hepatocytes cytoplasm extending from the surface of showed considerable atrophy, reflected hepatocytes. The hepatocytes showed ac by their small size and accumulation of cumulation of lipofuscin pigment, nu lipofuscin pigment (figure 2). The nuclei clear glycogen and occasional fat droplets of periportal hepatocytes frequently (figure 6). showed vacuoles similar to the appear ance seen in diabetic patients (figure 2). Discussion The patchy distribution of the lesion ex plained the normal appearance of the ini The pathogenesis of liver changes in tial needle biopsy. Light microscopic ex vitamin A toxicity begins with storage of amination of plastic-embedded tissue vitamin A in Ito cells. These cells were revealed an increase in the number of Ito first described by Ito15 in 1952 and are 306 FOROUHAR, NADEL, AND GONDOS

F ig u r e 2. Photomicrograph of the portal and periportal zone showing periportal perisinusoidal fibrosis of the space of Disse. Notice marked atrophy of entrapped hepatocytes. Arrow shows nuclear glycogen in periportal hepatocytes (Trichrom stain) also known as “fat-storing cells”2,15 be tabolism of vitamin A, protect against hy- cause their cytoplasm frequently con pervitaminosis A toxicity, and reverse tains abundant fat. The cells store a va some of the pathologic changes in rat riety of lipids and are the primary site of liver.29 This is further supported by an vitamin A storage.1,2,14,15,16,17 Toxicity re increase in smooth endoplasmic retic lated to certain chemicals, such as carbon ulum in hepatocytes,26,29 the morpho tetrachloride, Vitamin A, and metho logic equivalent of increased microsomal trexate, causes their proliferation.12,20,27 enzyme activity.9,10 Other studies have Since Ito cells are also facultative fibro emphasized that excess vitamin A results blasts,14,20,25 it is now accepted that in rapid hemolysis of erythrocytes in storage of toxic doses of vitamin A stim vitro4 and causes changes in rat liver ulates them to form collagen,16,27 which mitochondria19 and lysosomes.5 Mito leads to fibrosis of the space of Disse. chondria swell and the lysosomes release The mechanism by which vitamin A hydrolytic enzymes. This is presumably affects the Ito cells is not well under a result of changes in lipoprotein mem stood, although it is known that vitamin branes of intracellular organelles.26 A metabolism is related to the function In chronic hypervitaminosis A,5 the of microsomal enzymes.29 Chemicals that space of Disse is expanded by fibro induce these enzymes, such as pregnen- sis, deposition of basement membrane olone-16-carbonitrile, accelerate the me like substance and proliferation of Ito VITAMIN A TOXICITY 307

Figure 3. Photom i crograph shows central stellate sclerosis. Notice lack of perisinusoidal fi brosis and hepatocellular atrophy in this section. (Trichrom stain)

cells314,27 which forms a barrier between sion in the absence of cirrhosis.14 Central sinusoidal blood and hepatocytes and vein fibrosis and fibrosis of portal areas compresses the sinusoids. These changes also contribute to portal hypertension in cause hepatomegaly and portal hyperten later stages. This barrier deprives the he-

Ficure4. Photomicro graph shows proliferation of Ito cells (arrows) and ac cumulation of lipid in their cytoplasm. (Plastic em bedded, toluidine blue stained) 308 FOROUHAR, NADEL, AND GONDOS

F ig u r e 5 . Photomi crograph shows periportal fibrosis with entrapped hepatocyte (arrow head). Notice large number of Ito cells in the fibrous tis sue. (arrow) (Plastic em bedded, toluidine blue stain)

patocytes of nutrients and oxygen leading velopment of cirrhosis.14,16 The most to hepatocellular atrophy and eventual striking pathologic change in hepatocytes necrosis. Necrosis of hepatocytes usually is the formation of cytoplasmic bullae results in formation of regenerative nod which extend through the fibrous barrier ules surrounded by fibrous septa and de reaching the blood in the sinusoids like

F ig u r e 6. Electron mi crograph shows deposition of collagen in the space of Disse (arrow), an Ito cell (large arrow head) and nu merous sinusoidal cyto plasmic blebs (small arrow heads). VITAMIN A TOXICITY 309

6. F a r r e l l , C. C., B h a t h a l , P. S., and P o w e l l , pseudopodia. This change represents an L. W.: Abnormal liver function in chronic hy- attempt by the hepatocytes to reach the pervitaminosis A. Dig. Dis. 22:724-8, 1977. nutrients and oxygen in the sinuses. 7. F e l d m a n , G., D u d e a , P., M o l a s , G., D o m a r t - D u d e a , M. C., and F a u v e r t , R.: L’ultrastruc- Other hepatocellular changes such as in ture des hepatocyte on cours der cirrhoses al- crease in fat, smooth endoplasmic retic coolique. Presse Med. 78:409-444, 1970. ulum, lipofuscin, and lysosomes, and 8. F u t t e r m a n , S. and A n d r e w s , S.: The composi focal degradation of cytoplasmic organ tion of liver vitamin A ester and the synthesis of vitamin A ester by liver. 239:4077-4080, 1964. elles are nonspecific and probably re 9. G a r y , B. D., B l a s c h e c k , J. A., and K o v a c s , K.: lated to chronic ischemia. However, they A comparative ultrastructural study on drug in may also reflect toxic effects of intracel duced proliferation of smooth surface endo plasmic reticulum in hepatocytes. Tohoku J. lular release of the biologically active al Exp. Med. 104:205- 214, 1971. cohols of vitamin A.26 10. G a r y , B. D., K o v a c s , K., and B l a s c h e c k , J. A.: Ultrastructural changes induced by pregneno The delayed metabolism of excess vi lone nitrate in the rat liver. J. Pharm. Pharmacol. tamin A in the liver raises an important 22:872-873, 1970. question, that is, whether or not the pro 11. H e r d s o n , P. B., G a r v i n , P. J., and Je n n i n g s , R. B.: Fine structural changes in rat liver in cess of fibrogenesis would continue after duced by phenobarbital. Lab Invest. 73:1032— ingestion of vitamin A is discontinued. 1037, 1964. There is a well documented case re 12. H o p w o o d , D. and N y f o r s , A. Effect of metho ported by Russell et al27 describing pro trexate therapy in psoriatics on the Ito cells in liver biopsies, assessed by point counting. J. gression of fibrogenesis after discontin Clin. Path. 29:698- 703, 1976. uation of vitamin A. In our case, the first 13. H o r v a t h , E., K o v a c s , K., and Ross, R. L.: Al coholic liver lesion frequency and diagnostic needle biopsy showed no significant ab value of fine structural alterations in hepato normality, but the second open biopsy cytes, Beitr. Pathol. Anat. Allgem. Pathol. showed considerable fibrosis. This is 148:67-85. probably more a result of sampling than 14. H r u b a n , Z., R u s s e l , R., B o y e r , J., G l a g o r , S., and B a g h e r i , S.: Ultrastructural changes in liver progression of fibrosis, although, it is of two patients with hypervitaminosis A. Am. J. possible that fibrosis could progress Path. 76:451-468, 1974. 15. It o , T. and N e m o t o , M.: Über die kupfFerschen within this period. Regardless of the ex Sternzeller and die “Fett speicherungszeller” in planation, this unique situation empha der Blutkapillare nwand der menschlichen leber. sizes the significance of repeated biopsies Okajimas Folia Anat. Jap. 24:243-258, 1952. when a high index of suspicion exits clin 16. Ja q u e s , E. A., B u s c h m a n n , R. J., and L a y d e n , T. J.: Histopathologic progression of vitamin A ically. The autofluorescence of vitamin A induced hepatic injury. Gastroenterology is very helpful when light microscopy is 76:599-602, 1979. non-diagnostic. 17. K e n t , G., In o u y e , T., B a h u , R., M in ic k, T., P o p p e r , H.: Vitamin A containing lipocytes and formation of type III collagen in liver injury. Proc. Natl. Acad. Sei. USA 73:3719-3722, 1976. References 18. K l i o n , F. M. and S c h a f f n e r , F.: Ultrastructural studies in alcoholic liver disease. Digestion 1:2- 1. B h a t h a l , P. S., B ronfenmajor , S.,: Presence of 14, 1968. modified fibroblases in cirrhotic livers in man. 19. L u c y , J. A., L u s c o m b e , M., and D i n g l e , J. T.: Pathology 4:139-144, 1972. Studies on the mode of action of excess vitamin 2. B ronfenmajors , S., Sc h a f f n e r , F., and P o p p e r A. Biochem J. 89:419-425, 1963. H.: Fat storing cells (lipocytes) in human liver. 20. M cGree, J. O. and Patrick, R.: The role of peri- Arch. Path. 83:447-453, 1966. sinusoidal cells in hepatic fibrinogenesis: an elec 3. C o s s e l , L.: Über akutes Auftreten von Basal tron microscopic study of acute carbon tetrachlo membranen an den Lebersinusoiden. Beitr. ride liver injury. Lab. Invest. 26:429-440, 1972. Pathol. Anat. Allgems Pathol. 734:103-122, 21. N e a l d , J. B. and P e a r s o n , W. N.: Macro- and 1966. micromethods for determination of serum vi 4. D i n g l e , J. T. and L u c y , J. A.: Studies on the tamin A using trifluoracetic acid. J. 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