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Early Fine Structural Changes in the Human Liver Induced by Alcohol
GASTROENTEROLOGY Official Publication of the American Gastroenterological Association © VOLUME
COPYRIGHT 1966 THE W,LLIAMS
&
W,LK,NS CO.
January 1967
52
NUMBER
1
EARLY FINE STRUCTURAL CHANGES IN THE HUMAN LIVER INDUCED BY ALCOHOL EM A NUEL RUBIN ,
M .D .,
AND CHARLES
S.
LIEBER ,
M .D.
Department of Pathology, The Mount Sinai School of Medicine; Liver Disease and Nutrition Unit, Cornell Medical Division, Bellevue Hospital; and Department of Medicine, Cornell University Medical College, New York, New York
Chronic alcoholism leads to fatty liver, necrosis, and inflammation in the form of alcoholic hepatitis, and finally to fibrosis and cirrhosis.l The sequential nature of these phenomena has led to the assumption that they are causally related in the order listed, and that the initiating and crucial lesion is hepatic steatosis. 2 Alcohol-induced fat accumulation in the liver, resulting from changes in fat metabolism,3 may lead to alterations of hepatocellular organelles, 4 and the escape of lipid from liver cells has been suggested as the cause of hepatic fibrosis.5 Previous ' studies of the ultrastructural hepatic alterations induced by ethanol under controlled dietary conditions in rats s. 7 and without dietary control in manS-lO have focused on the fatty liver and associated changes. It is the purpose of this study to distinguish in man hepatic ultrastructural Received May 25, 1966. Accepted August 16, 1966. Address requests for reprints to : Dr. Emanuel Rubin , D epartment of Pathology, Mount Sinai Hospital, Fifth Avenue and 100th Street, New York, NewYork 10029. This investigation was supported by Research Grants AM-03846, AM-06284, AM-10893, and AM-09536 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service. Dr. Lieber is the recipient of Research Career Development Award K3-AM22,590 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.
changes attributable directly to alcohol from those following fat accumulation or inflammation by demonstrating the early fine structural alterations in the liver which occur after isocaloric substitution of alcohol for carbohydrate, before significant hepatic steatosis or alcoholic hepatitis appears. Subjects and Procedure Two individuals, one male (subject 1), 35 years old, and one female (subject 2), 57 years old, were studied on a metabolic uni t. Both had a history of alcoholism, but they had abstained from alcohol intake during the 2- to 4-month period of hospitalization preceding the present investigation. At the time of the study, neither patient had clinical evidence of liver disease, and liver function tests were normal. During the 2 to 4 months preceding alcohol administration, both individuals were given diets with adequate content of nutrients, including amounts of vitamins, minerals, and proteins above the daily recommended allowances.ll Subject 1 received a 2500 calorie per day diet comprised of solid conventional foods, consisting of 48 % of total calories as carbohydrates, 16 % as protein, and 36% as fat, an amount of lipid less than that of the average American diet. Sixty per cent of the dietary fat consisted of corn oil, rich in unsaturated fatty acids. This solid diet consisted of an identical daily intake of the same items divided into three meals. All of the food, with the excep. tion of the lettuce, was utilized from a single lot purchased at the start of the experiment. Black coffee and tea were allowed as desired. Subject 2 received a 2100 calorie per day liquid diet in five
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RUBIN AND LIEBER
equal daily doses which had a nutrient composition similar to that of subject 1, except that, instead of corn oil, coconut oil, a highly saturated fat, was used. The diets given were similar to those administered previously, which led to the demonstration that, even with an adequate regimen, alcohol can produce a fatty liver in manP Special care was given to the maintenance of constant food intake, and the individuals had stable body weight throughout the study . After the control period, alcohol (95% ethanol), considered to have a caloric value of 7 calories per g,ll was administered in increasing amounts, isocalorically replacing carbohydrate given during the control period. During the first 4 days of alcohol administration, ethanol comprised 24 % of total calories, for the subsequent 2 days, 36%, and 46 % for the last 10 days in subj ect 1 and the last 12 days in subj ect 2. The amounts of alcohol given produced mild euphoria, without gross intoxication. When administered with the solid food to subject 1, ethanol was given in a ] 5 ,/;, aqueous solution, with various flavorings, divided in five equal daily doses. In subject 2, ethanol was incorporated in the liquid diet. The first liver biopsies were performed with a Menghini needle at the end of the control periods in both subj ects. In subj ect 1 the biopsy was repeated 1 day after the high dose of ethanol (8th day of the experiment) and 10 days after the high dose of ethanol (17th day of the experiment). In subject 2 additional biopsies were taken 3 days after the high dose of ethanol (10th day of the experiment) and 12 days after th e high dose of ethanol (19th day of the experiment).
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Results
Light Microscopy
The control biopsy of the liver in subject 1 (fig. 1A) taken before administration of alcohol appeared essentially normal, except for rare liver cells containing fat globules. In subject 2 the control biopsy (fig. 1D) showed occasional liver cells which contained fat globules or fine droplets of fat. The second biopsy (1 day after the full dose of ethanol) in subj ect 1 was unchanged from the control except for slightly increased fine fat droplets (fig. 1E). The second biopsy (3 days after the full dose of ethanol) in subject 2 demonstrated moderate fatty metamorphosis, with displacement of liver cell nuclei and occasional formation of fatty cysts (fig. 1E) . Ten to 12 days after the first large dose of alcohol, both patients had conspicuous fatty metamorphosis of the liver with many fatty cysts (fig. 1, C and F). In addition to the increased fat, the later biopsies revealed a moderate increase in D-PAS positive material in liver cells and a few more proliferated bile ductules in the portal tracts. N one of the specimens displayed fibrosis, necrosis, infiammation, or acidophilic bodies. With the stains employed, Mallory'S alcoholic hyalin was not observed. Electron Microscopy
The changes observed were similar in central and peripheral areas, although slightly For electron microscopy a portion of each liver more prominent in the former. The fat conbiopsy specimen was immediately fixed in ice- tent of liver cells corresponded to that seen cold 1 % osmic acid buffered with Veronal ace- with the light microscope, except for small tate at pH 7.4. The tissue was embedded in fat droplets in occasional liver cells in the Epon 812, stained with lead citrate,13 and cut on second biopsy of subject 1. a t ype 4802A LKD microtome. Semithin sections, Mitochondria. These organelles appeared 1 !J. thick, were cut from t he untrimmed blocks essentially normal in the control biopsy of and the lobular architecture identified under the 1 (fig. 2), although distortion of subj ect phase contrast microscope. Portal and central cristae in some mitochondria was noted. In areas were then cut at 0.1 !J. or thinner fo r study with an Hitachi HS-7 electron microscope. subject 2 the mitochondria were not enAnother port ion of each biopsy specimen was tirely normal. Most were of normal size, but fixed in 10 % neutral buffered formalin, after there was greater than normal variability which paraffin sections were stained with he- and a few irregular forms (fig. 3). One day matoxylin and eosin and subjected to the Gomori after a full dose of ethanol in subj ect 1, many silver impregnation for reticulum and to the mitochondria were enlarged and numerous periodic acid-Schiff reaction after digestion of' irregular forms were seen (fig. 4), some conglycogen with diasta..<;e (D-PAS). taining crystalline inclusions (fig. 5E). Three Methods
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ALCOHOL-INDUCED CHANGES IN HUMAN LIVER
days after the full dose of ethanol in subject 2, mitochondrial change was more severe with numerous large, irregularly shaped mitochondria (fig. 5A). Elongated mitochondria were frequently draped about the surface of fat droplets (fig. 6B). Many enlarged mitochondria showed loci of small crystalline inclusions, characterized by single parallel lines about 120 A wide (fig. 5C), which on cross section appeared as regularly spaced dots. The cristae of misshapen mitochondria were disoriented and were often elongated and arranged in parallel lamella resembling myelin figures, whorls, and irregular patterns. In the third biopsy specimen (after 10 to 12 days of the full dose), the mitochondrial alterations were accentuated, particularly in subject 1 in whose specimen giant mitochondria, so-called megamitochondria,6 were conspicuous (fig. 6D). These displayed increased matrix density and crystalline inclusions arranged in several loci. They were often in intimate contact with fat droplets, in some instances partially surrounding them. Some mitochondria surrounded small fat droplets (fig. 6C) and even portions of cytoplasm containing endoplasmic reticulum in both biopsy specimens after ethanol in each subject. Endoplasmic reticulum and ribosomes. N either of the control biopsy specimens was entirely normal, both showing moderately increased and vacuolated agranular endoplasmic reticulum (figs. 2 and 3). The granular endoplasmic reticulum was somewhat decreased, as were the free ribosomes. In succeeding biopsy specimens, there was a progressive vesiculation of granular and agranular endoplasmic reticulum (fig. 6A). The third biopsy (10 to 12 days after the full dose) showed a striking increase in polyribosomes (fig. 6C). Lysosomes and cytoplasmic vacuoles. Changes were present in all biopsy specimens after alcohol administration, but they were more conspicuous and progressive in subject 1 beginning 1 day after the full dose of ethanol. Lysosomes were only moderately increased. Small cytoplasmic vacuoles, limited by a single membrane, contained clumps and whorls of dense osmiophilic material. Larger,
3
polymorphous vacuoles of similar appearance were delimited by an incomplete membrane and seemed to derive from a coalescence of smaller vacuoles (figs. 4B and 7 A). Within these autophagic vacuoles or residual vacuolated bodies,14 sequestered elements of degenerated organelles, including mitochondria and endoplasmic reticulum, could be discerned (fig. 7, B, D, and E). Sequential stages in the development of residual vacuolated bodies as described by Biava14 were found in the same cell (fig. 7, B and E). Small whorls of osmiophilic material similar to those found in vacuolated bodies appeared to derive from mitochondria and occupied lacunae at the mitochondrial border. Various stages of mitochondrial degeneration were seen, with severely damaged forms isolated by single membranes (fig. 7D). Small autophagic vacuoles contained ergastoplasmic elements (fig. 7B). Finally all these structures were discernible in large autophagic vacuoles or were amorphous in residual vacuolated bodies (fig. 7E). Microbodies. These organelles were strikingly increased in number, size, and variability beginning with the early biopsies after exposure to alcohol (fig. 8). Golgi complex. After administration of alcohol, the Golgi cisternae were dilated and filled with electron-dense granules. Cell borders. Occasional cytoplasmic blebs extended into the space of Disse or impinged upon an adjacent cell (fig. 6A). Bile ductules. The bile ductules in subject 1 appeared normal. After alcohol administration in subject 2, bile ductular cells contained fat droplets (fig. 9A). Cytoplasmic blebs extended into the lumens of ductules (fig. 9C). Occasional bile ductular cells showed degenerative changes (fig. 9B). Discussion
The hepatic changes induced by iSClcaloric substitution of alcohol for carbohydrate in an otherwise complete diet include (1) fatty metamorphosis; (2) enlargement and disfiguration of mitochondria, with disorientation of cristae and crystalline inclusions; (3) vacuolization and increase in endoplasmic reticulum; (4) increase in free
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FIG. 1. Light micrographs of liver biopsies. H & E, X 240. A, Subject 1. Control biopsy. Liver cells are filled with glycogen. No fat is present. B, Subject 1, 1 day after full dose of ethanol. There is little change from the control biopsy, except for slightly increased fine droplet fat. C, Subject 1, 10 days after full dose of ethanol. Conspicuous fatty metamorpho-
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ALCOHOL-INDUCED CHANGES IN HUMAN LIVER
sis is noted. D, Subject 2, control biopsy. Essentially normal appearance with a few fat globules (arrows). E, Subject 2, 3 days after full dose of ethanol. Extensive fatty metamorphosis is present . F, Subject 2, 12 days after full dose of ethanol. Fatty metamorphosis is more severe.
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FIG. 2. Subj ect 1, co ntrol biopsy. Mitochondria are of normal s ize. Some display mild distortion of cristae. Endoplasmic reticulum is vesicular a nd slightly increased. A normal amount of glycogen (dar k dots) is present (X 10,000).
polyribosomes; (5) focal cytoplasmic degradation in the form of autophagi c vacuoles, as well as residual vacuolated bodies; (6) increased microbodies; and (7) changes in bile ductules. The mitochondrial distortions have been observed in human alcoholic hepatitis 9, 10 , 15 and alcoholic fatty livers without inflammation.8 They have also been produced in rats by isocaloric substitution of alcohol for carbohydrate6 , 7 These changes are not specific for alcohol administration, since they are seen in many dietary l6 , 17 and toxic conditions. 18 Prominent features in almost all descriptions of mitochondrial changes induced by alcohol in m an and animals are the crystalline inclusions or socalled myelin figures within mitochondria. 6 , 8 , 9, 15 Although these inclusions are regularly found in alcohol intoxication, they are also noted in other conditions19 , 20 and
rarely in normal persons. 21 The exact nature of the crystalline inclusions is not certain. They are assumed by some to be phospholipids which are unmasked as a result of disintegration of intramitochondrial components.22 The degeneration of giant mitochondria described here has been suggested as the origin of Mallory's alcoholic hyaline,6 but, in our studies of early changes, no alcoholic hyaline was seen with the light microscope, at least with the stains employed. Since in acute alcoholic hepatitis the organelle change which correlates best with the clinical condition is mitochondrial alteration,15 changes in these organelles may be the crucial effect of alcohol ingestion. In rats given long term alcohol in addition to an uncontrolled diet, isolat ed mitochondria had decreased oxidative properties. 23 Theoretically, the decreased hepatic oxidation of fatty acids3 , 24 and pyru-
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ALCOHOL-INDUCED CHANGES IN HUMAN LIVER
FIG. 3. Subject 2. Control biopsy . Mitochondria are of normal size but a few abnormal forms (.strai ght arrows) are noted . End oplasmic reticulum is slightl y in creased and vesicular. Bile canal ic ulus (c) is unremarkable. A few residual bodies (curved arrow) a re prese nt (X 9000).
FIG. 4. Subject 1, 24 hours after first full dose of ethanol. A , Mitochondria are enlarged and disfigllTed (X ()200) . B , Numerous residual vacuolated bodies are present (X 9700).
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FIG. 5. Mitochondrial changes in cells which contain no fat droplets. A, Subject 2, 3 days after first full dose of ethanol. Bizarre disfigured and enlarged mitochondria (X 14,000). B, Subject 1, 24 hours after first full dose of ethanol. Intramitochondrial crystalline inclusion (X 54,(00). C, Subject 2, 3 da ys after first full dose of ethanol. Mitochondrion shows parallel arrays of .double-membraned structures, proba bl y early crystal formation (X 17,(00) .
vate23 produced by alcohol could be related to the mitochondrial damage observed morphologically. This in turn could serve as an explanation for the hepatic accumulation of dietary lipids on prolonged ethanol intake, both in rats 25 and in man. 26 In neither of the subjects studied was the smooth endoplasmic reticulum completely normal in the control biopsy, but progressive changes occurred after alcohol administration. The vesiculation and increase in endoplasmic reticulum have also been observed in isocaloric ethanolinduced hepatic injury of rat7 and man. 27 The changes in the endoplasmic reticulum may be related to the ethanol-induced increase in lipogenesis,3 including the increased esterification by microsomes. 28 A noteworthy feature is the striking increase in polyribosomes in the third biopsy of both patients. In view of the association of polyribosomes with protein synthesis and the observation that administration of large doses of ethanol results not only in fatty liver, but also in hyperlipemia,12. 29. 30 the increase in polyribosomes may reflect increased lipoprotein
synthesis, perhaps as an adaptive mechanIsm. A striking change was the widespread focal cytoplasmic degeneration in the form of autophagic vacuoles and residual vacuolated bodies which contained remnants of degenerated organelles and ergastoplasm. These structures correspond to the D-PAS positive structures which are increased in many forms of hepatic injury.15. 31 The dense osmiophilic material within these structures may represent the liberation of the lipids of the sequestered cytoplasmic components, brought about by digestion of structural proteins. 1s Since they occur in otherwise normal cells, they probably signify increased turnover of organelles in the damaged, but surviving, cell. The function of microbodies is unknown, and consequently their increase cannot be explained. In lower vertebrates, microbodies contain uricase,a2 an enzyme absent in man. It seems probable that in man they also con· tain enzymes, perhaps catalase and d-amino oxidase. aa The changes noted in bile ductules may
FIG. 6. Mitochondrial alterations associated with fat. A, Subject 1, 10 days after full dose of alcohol. Enlarged mitochondria with bizarre shapes and abnormal cristae are arranged about fat droplets. Smooth endoplasmic reticulum is vesicular. A cytoplasmic bleb (arrow) is seen at the cell border (X 9,900). B, Subject 2, 3 days after full dose of ethanol. Elongated mitochondrion is draped around fat droplet (X 7,200). C, Subject 2, 12 days after full dose of ethanol. Mitochondrion encircles small fat droplet. Note increased polyribosomes (X 14,000). D, Subject 1, 10 days after full dose of ethanol. Megamitochondrion containing crystalline inclusions partially surrounds a fat droplet. Crystalline inclusions are also seen in other mitochondria (arrows) (X 8,700). 9
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FIG. 7. Stages in the development of autophagic vacuoles and residual vacuolated bodies in subject 1, 10 days after full dose of ethanol. A, Numero1ls residual vacuolated bodies surround a bile canaliculus, c (X 44(0). B, Autophagic vacuoles (arrows) contain ribosomes and lamellated osmiophilic material (X 27,0(0). C, Degenerating mitochondria (straight arrows) display circular, osmiophilic cristae. In more advanced degeneration, cristae are more osmiophilic and arranged concentrically (curved arrow) (X 11,000). D, Degenerated mitochondrion is enclosed within autophagic vacuole (arrow) (X 29,0(0). E, Residual vacuolated bodies are poorly delimited and contain dense , whorled osmiophilic material and degenerated mitochondrial material (straight arrow). A small autophagic vacuole (curved arrow) may merge with vacuolated body (X 14 ,(00) .
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ALCOHOL-INDUCED CHANGES I N HUMA N LIVER
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FIG. 8. Subject 1, 10 days after full dose of ethanol. Numerous clusters of microbodies are present (arrows) (X 4200) .
eventually lead to periductular inflammation and fibrosis, the so-called ductular cell reaction. 34 This reaction is assumed to be important in the eventual formation of fibrosis and cirrhosis. 35 The production of hepatic damage in normal rats after 1 to 3 weeks of alcohol administration 6, 7 suggests that chronic alcoholism is not a prerequisite for susceptibility to ethanol-induced liver damage. In human subjects in whom fatty liver was less striking than in the cases described here, and whose livers were biopsied after 18 days of alcohol administration with an adequate diet, changes in endoplasmic reticulum were particularly prominent,27 This probably reflects variation in the susceptibility to the toxin, The changes described in the subjects of this study have all been noted in chronic alcoholic injury in man, but because of the uncontrolled diets in patients with alcoholic fatty liver, with or without hepatitis, the effects of nutritional and alcoholic injury were difficult to separate. This difficulty has been overcome in this study by investigating early changes. In subject 1, ethanol produced ultrastructural hepatic damage within 7 days, and within only 1 day of the full dose (46 % of calories), at a time when fatty change was minimal. In subject 2, similar changes, which were associated with more conspicuous fatty metamorphosis, occurred within 10 days and
within only 3 days of the full dose. These observations indicate that, despite ingestion of a diet containing 16 % of calories as protein, an amount well above the recommended allowance for man,ll ethanol rapidly produces conspicuous hepatic steatosis and ultrastructural changes. It is conceivable that, in the face of a low carbohydrate-ethanol diet, protein requirements may be increased. However, in a study similar to the present one, a high protein (25 % of calories)-low fat diet failed to prevent fat accumulation in the liver. (C. S. Lieber and E. Rubin, unpublished observations). Thus alcohol appears to exert a direct toxic effect on the liver of human alcoholics which is concomitant with or may precede fatty metamorphosis. In view of the fact that alcohol produces the same effect in normal rats,6, 7 it seems likely that it is a general phenomenon. SUllllllary
The early ultrastructural changes induced by isocaloric substitution of ethanol for carbohydrate in t he livers of 2 human subjects were studied. Mitochondrial alterations and focal cytoplasmic degradation were noted within 1 d ay of the full dose of ethanol (46 % of total calories), at a time when fat accumulation was minimal. Fatty metamorphosis was prominent within 3 to 10 days of the full dose of ethanol and was associated with al-
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FIG. 9. Bile ductules. Subject 2. A, 10 days after full dose of ethanol. Fat droplet (arrow) in ductular cell (X 8200). B, 10 days after full dose of ethanol. Ductular cell (arrow) shows advanced degenerative changes (X 8500). C, 3 days after full dose of ethanol. Cytoplasmic bleb (arrow) extends into lumen of ductule (X 16,000).
terations of mitochondria, endoplasmic reticulum, autophagic vacuoles, microbodies, and ductular cells. It is concluded that alcohol exerts a direct toxic action on the liver which precedes or is concomitant with fatty metamorphosis. REFERENCES 1. Popper, H., P. B. Szanto, and H. Elias. 1955.
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Transition of fatty liver into cirrhosis. Gastroenterology 28: 183-192. 2. Himsworth, H. P. 1960. The liver and its diseases. Harvard University Press, Cambridge. 3. Lieber, C. S. 1966. Hepatic and metabolic effects of alcohol. Gastroenterology 50: 119133. 4. Popper, H. 1961. Morphological and biochemical aspects of fatty liver. Acta Hepatosplen. 8: 29-292. 5. Hartroft, W. S., and J. H. Ridout. 1951. Pathogenesis of the cirrhosis produced by choline deficiency; escape of lipid from fatty hepatic cysts into the biliary and vascular systems. Amer. J. Path. 27: 951-989. 6. Porta, E. A., W. S. Hartroft, and F. A. de la Iglesia. 1965. Hepatic changes associated with chronic alcoholism in rats. Lab. Invest. 14: 1437-1455. 7. Iseri, O. A., C. S. Lieber, and L. S. Gottlieb. 1966. The ultrastructure of fatty liver induced by prolonged ethanol ingestion. Amer. J. Path. 48: 535--555. 8. Svoboda, D. J., and R. T. Manning. 1964. Chronic alcoholism with fatty metamorphosis of the liver. Mitochondrial alterations in hepatic cells. Amer. J. Path. 44: 645--662. 9. Porta, E. A., B. J. Bergman, and A. A. Stein. 1965. Acute alcoholic hepatitis. Amer. J. Path. 46: 657-689. 10. Kiessling, K. H., L. Lindgren, B. Strandberg, and U. Tobe. 1964. Electron microscopic study of liver mitochondria from human alcoholics. Acta Med. Scand. 176: 595--598. 1l. Food and Nutrition Board. 1964. Recommended Dietary Allowances, publication 1146, Ed. 6, National Academy of Sciences, National Research Council. Washington, D. C. 12. Lieber, C. S., D. P. Jones, and L. M. DeCarli. 1965. Effects of prolonged ethanol intake: Production of fatty liver despite adequate diets. J. Clin. Invest. #: 1009-1O2l. 13. Reynolds, E. S. 1963. The use of lead citrate as an electron opaque stain in electron microscopy. J. Cell BioI. 17: 208-212. 14. Biava, C. 1965. Electron microscopic studies on periodic acid-Schiff positive nonglycogenic structures in human liver cells. Amer. J. Path. 46: 435--465. 15. Schaffner, F., A. Lobel, H. A. Weiner, and T. Barka. 1963. Hepatocellular cytoplasmic changes in acute alcoholic hepatitis. J. A. M. A. 183: 343-346. 16. David, H. 1961. Die Leber bei Nahrungsmangel und Mangelernahrung, p. 56. Akademie Verlag, Berlin.
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17. Theron, J. J., A. O. Hawtrey, N. Liebenberg, and _V. Schirren. 1963. The pathogenesis of experimental dietary siderosis of the liver. Amer. J. Path . 43: 73-91. 18. David, H., and L. -H. Kettler. 1960-61. Degenerat ion von Lebermitochondrien nach Ammonium-Intoxikation . Z. Zellforsch . 53: 857-866. 19. Jezequel, A. M. 1959. Degenerescence myelinique des mitochondries de foie humaine dans un epithelioma du choledoque et un ict{~re viral. Etude au microscope electronique. J. Ultrastruct. Res. 3: 210-215. 20. Laguens, R., and N _Bianchi. 1963 . Fine structure of the liver in human idiopathic diabetes. I. P arenchymal cell mitochondria. Exp. Molec. Path. 2: 203-214. 21. Wills, E. J. 1965. Crystalline structures in the mitochondria of normal human liver parenchymal cells. J. Cell. BioI. 24: 511- 514. 22. Fawcett, D. W., and S. Ito . 1958. Observations on the cytoplasmic membranes of testicular celis, examined by phase contrast and electron microscopy . J. Biophys . Biochem. Cytol. 4: 135-142. 23. Kiessling , K. H ., and K. Tilander. 1963 . The effect of prolonged a lcohol treatment on the respiration of the liver and brain mitochondria from male and female rats. Exp. Cell Res . 30: 476-480. 24. Lieber, C. S., and R. Schmid . 1961. The effect of ethanol on fatty ac id metabolism . Stimulation of hep atic fatty acid synthesis in vitro . J. Clin. Invest. 40: 393-399. 25. Lieber, C. S., N . Spritz , and L. M. DeCarli. 1966. Role of dietary, adipose and endogenousl y synthesized fatty acids in the pathogenesis of the alcoholic fatt y liver. J. Clin. Invest . 45: 51-62 _ 26 . Lieber, C. S., and N. Spritz. 1966. Effects of prolonged ethanol intake in man : Role of dietary, adipose and endogenously synthe-
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sized fatty acids in the pathogenesis of the alcoholic fatty liver. J. Clin . Invest. 45: 1400-1411 . Lane, B. P., and C. S. Lieber . 1966 . Ultrastructural alterations in human hepatocytes following ingestion of ethanol with adequate diets. Amer. J. Path. 49: 593-603. Scheig, R., and K. J . Isselbacher. 1965. Pathogenesis of ethanol-induced fatty liver. III. In vivo and in vitro effects of ethanol on hepatic fatty acid metabolism in rat. J . Lipid Res. 6: 269-277. Lieber, C. S., D. P. Jones, J . Mendelson, and L. M. DeCarli. 1963. Fatty liver, hyperlipemia and hyperuricemia produced by prolonged alcohol consumption despite ade quate dietary intake. Trans. Assn. Amer. Physicians 76: 289-300. Shapiro, R. H ., R. L. Scheig, G. D. Drummey, J . H. Mendelson, and K . J . Isselbacher. 1965. Effects of prolonged ethanol ingestion on the transport and metabolism of lipids in man. New Eng. J. Med. 272: 610-615. Popper, H ., F . Paronetto, and T. Barka . 1960. PAS-positive structures of non-glycogenic character in normal and abnormal liver. Arch. Path. 70: 300-313. Hruban, Z., and H. Swift. 1964. Uricase: Localization in hepatic microbodies. Science 146: 1316- 1317. Fawcett, D. W. 1966. The cell . Its organelles and inclusions , p. 213. W. B. Saunders Company, Philadelphia. Popper, H., G. Kent, and R. Stein . 1957. Ductular cell reaction in the liver in hepatic injury. J . Mount Sinai Hosp . N .Y. 24: 551-556. Rubin, E., F. Hutterer, and H. Popper. 1963. Cell proliferation and fiber formati on in chronic carbon tetrac hloride intoxication. A morphologic and chemical study. Amer. J. Path. 42: 715-728 .
COPYRIGHT 1966 THE W,LLIAMS
&
W,LK,NS CO.
January 1967
52
NUMBER
1
EARLY FINE STRUCTURAL CHANGES IN THE HUMAN LIVER INDUCED BY ALCOHOL EM A NUEL RUBIN ,
M .D .,
AND CHARLES
S.
LIEBER ,
M .D.
Department of Pathology, The Mount Sinai School of Medicine; Liver Disease and Nutrition Unit, Cornell Medical Division, Bellevue Hospital; and Department of Medicine, Cornell University Medical College, New York, New York
Chronic alcoholism leads to fatty liver, necrosis, and inflammation in the form of alcoholic hepatitis, and finally to fibrosis and cirrhosis.l The sequential nature of these phenomena has led to the assumption that they are causally related in the order listed, and that the initiating and crucial lesion is hepatic steatosis. 2 Alcohol-induced fat accumulation in the liver, resulting from changes in fat metabolism,3 may lead to alterations of hepatocellular organelles, 4 and the escape of lipid from liver cells has been suggested as the cause of hepatic fibrosis.5 Previous ' studies of the ultrastructural hepatic alterations induced by ethanol under controlled dietary conditions in rats s. 7 and without dietary control in manS-lO have focused on the fatty liver and associated changes. It is the purpose of this study to distinguish in man hepatic ultrastructural Received May 25, 1966. Accepted August 16, 1966. Address requests for reprints to : Dr. Emanuel Rubin , D epartment of Pathology, Mount Sinai Hospital, Fifth Avenue and 100th Street, New York, NewYork 10029. This investigation was supported by Research Grants AM-03846, AM-06284, AM-10893, and AM-09536 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service. Dr. Lieber is the recipient of Research Career Development Award K3-AM22,590 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.
changes attributable directly to alcohol from those following fat accumulation or inflammation by demonstrating the early fine structural alterations in the liver which occur after isocaloric substitution of alcohol for carbohydrate, before significant hepatic steatosis or alcoholic hepatitis appears. Subjects and Procedure Two individuals, one male (subject 1), 35 years old, and one female (subject 2), 57 years old, were studied on a metabolic uni t. Both had a history of alcoholism, but they had abstained from alcohol intake during the 2- to 4-month period of hospitalization preceding the present investigation. At the time of the study, neither patient had clinical evidence of liver disease, and liver function tests were normal. During the 2 to 4 months preceding alcohol administration, both individuals were given diets with adequate content of nutrients, including amounts of vitamins, minerals, and proteins above the daily recommended allowances.ll Subject 1 received a 2500 calorie per day diet comprised of solid conventional foods, consisting of 48 % of total calories as carbohydrates, 16 % as protein, and 36% as fat, an amount of lipid less than that of the average American diet. Sixty per cent of the dietary fat consisted of corn oil, rich in unsaturated fatty acids. This solid diet consisted of an identical daily intake of the same items divided into three meals. All of the food, with the excep. tion of the lettuce, was utilized from a single lot purchased at the start of the experiment. Black coffee and tea were allowed as desired. Subject 2 received a 2100 calorie per day liquid diet in five
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RUBIN AND LIEBER
equal daily doses which had a nutrient composition similar to that of subject 1, except that, instead of corn oil, coconut oil, a highly saturated fat, was used. The diets given were similar to those administered previously, which led to the demonstration that, even with an adequate regimen, alcohol can produce a fatty liver in manP Special care was given to the maintenance of constant food intake, and the individuals had stable body weight throughout the study . After the control period, alcohol (95% ethanol), considered to have a caloric value of 7 calories per g,ll was administered in increasing amounts, isocalorically replacing carbohydrate given during the control period. During the first 4 days of alcohol administration, ethanol comprised 24 % of total calories, for the subsequent 2 days, 36%, and 46 % for the last 10 days in subj ect 1 and the last 12 days in subj ect 2. The amounts of alcohol given produced mild euphoria, without gross intoxication. When administered with the solid food to subject 1, ethanol was given in a ] 5 ,/;, aqueous solution, with various flavorings, divided in five equal daily doses. In subject 2, ethanol was incorporated in the liquid diet. The first liver biopsies were performed with a Menghini needle at the end of the control periods in both subj ects. In subj ect 1 the biopsy was repeated 1 day after the high dose of ethanol (8th day of the experiment) and 10 days after the high dose of ethanol (17th day of the experiment). In subject 2 additional biopsies were taken 3 days after the high dose of ethanol (10th day of the experiment) and 12 days after th e high dose of ethanol (19th day of the experiment).
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Results
Light Microscopy
The control biopsy of the liver in subject 1 (fig. 1A) taken before administration of alcohol appeared essentially normal, except for rare liver cells containing fat globules. In subject 2 the control biopsy (fig. 1D) showed occasional liver cells which contained fat globules or fine droplets of fat. The second biopsy (1 day after the full dose of ethanol) in subj ect 1 was unchanged from the control except for slightly increased fine fat droplets (fig. 1E). The second biopsy (3 days after the full dose of ethanol) in subject 2 demonstrated moderate fatty metamorphosis, with displacement of liver cell nuclei and occasional formation of fatty cysts (fig. 1E) . Ten to 12 days after the first large dose of alcohol, both patients had conspicuous fatty metamorphosis of the liver with many fatty cysts (fig. 1, C and F). In addition to the increased fat, the later biopsies revealed a moderate increase in D-PAS positive material in liver cells and a few more proliferated bile ductules in the portal tracts. N one of the specimens displayed fibrosis, necrosis, infiammation, or acidophilic bodies. With the stains employed, Mallory'S alcoholic hyalin was not observed. Electron Microscopy
The changes observed were similar in central and peripheral areas, although slightly For electron microscopy a portion of each liver more prominent in the former. The fat conbiopsy specimen was immediately fixed in ice- tent of liver cells corresponded to that seen cold 1 % osmic acid buffered with Veronal ace- with the light microscope, except for small tate at pH 7.4. The tissue was embedded in fat droplets in occasional liver cells in the Epon 812, stained with lead citrate,13 and cut on second biopsy of subject 1. a t ype 4802A LKD microtome. Semithin sections, Mitochondria. These organelles appeared 1 !J. thick, were cut from t he untrimmed blocks essentially normal in the control biopsy of and the lobular architecture identified under the 1 (fig. 2), although distortion of subj ect phase contrast microscope. Portal and central cristae in some mitochondria was noted. In areas were then cut at 0.1 !J. or thinner fo r study with an Hitachi HS-7 electron microscope. subject 2 the mitochondria were not enAnother port ion of each biopsy specimen was tirely normal. Most were of normal size, but fixed in 10 % neutral buffered formalin, after there was greater than normal variability which paraffin sections were stained with he- and a few irregular forms (fig. 3). One day matoxylin and eosin and subjected to the Gomori after a full dose of ethanol in subj ect 1, many silver impregnation for reticulum and to the mitochondria were enlarged and numerous periodic acid-Schiff reaction after digestion of' irregular forms were seen (fig. 4), some conglycogen with diasta..<;e (D-PAS). taining crystalline inclusions (fig. 5E). Three Methods
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days after the full dose of ethanol in subject 2, mitochondrial change was more severe with numerous large, irregularly shaped mitochondria (fig. 5A). Elongated mitochondria were frequently draped about the surface of fat droplets (fig. 6B). Many enlarged mitochondria showed loci of small crystalline inclusions, characterized by single parallel lines about 120 A wide (fig. 5C), which on cross section appeared as regularly spaced dots. The cristae of misshapen mitochondria were disoriented and were often elongated and arranged in parallel lamella resembling myelin figures, whorls, and irregular patterns. In the third biopsy specimen (after 10 to 12 days of the full dose), the mitochondrial alterations were accentuated, particularly in subject 1 in whose specimen giant mitochondria, so-called megamitochondria,6 were conspicuous (fig. 6D). These displayed increased matrix density and crystalline inclusions arranged in several loci. They were often in intimate contact with fat droplets, in some instances partially surrounding them. Some mitochondria surrounded small fat droplets (fig. 6C) and even portions of cytoplasm containing endoplasmic reticulum in both biopsy specimens after ethanol in each subject. Endoplasmic reticulum and ribosomes. N either of the control biopsy specimens was entirely normal, both showing moderately increased and vacuolated agranular endoplasmic reticulum (figs. 2 and 3). The granular endoplasmic reticulum was somewhat decreased, as were the free ribosomes. In succeeding biopsy specimens, there was a progressive vesiculation of granular and agranular endoplasmic reticulum (fig. 6A). The third biopsy (10 to 12 days after the full dose) showed a striking increase in polyribosomes (fig. 6C). Lysosomes and cytoplasmic vacuoles. Changes were present in all biopsy specimens after alcohol administration, but they were more conspicuous and progressive in subject 1 beginning 1 day after the full dose of ethanol. Lysosomes were only moderately increased. Small cytoplasmic vacuoles, limited by a single membrane, contained clumps and whorls of dense osmiophilic material. Larger,
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polymorphous vacuoles of similar appearance were delimited by an incomplete membrane and seemed to derive from a coalescence of smaller vacuoles (figs. 4B and 7 A). Within these autophagic vacuoles or residual vacuolated bodies,14 sequestered elements of degenerated organelles, including mitochondria and endoplasmic reticulum, could be discerned (fig. 7, B, D, and E). Sequential stages in the development of residual vacuolated bodies as described by Biava14 were found in the same cell (fig. 7, B and E). Small whorls of osmiophilic material similar to those found in vacuolated bodies appeared to derive from mitochondria and occupied lacunae at the mitochondrial border. Various stages of mitochondrial degeneration were seen, with severely damaged forms isolated by single membranes (fig. 7D). Small autophagic vacuoles contained ergastoplasmic elements (fig. 7B). Finally all these structures were discernible in large autophagic vacuoles or were amorphous in residual vacuolated bodies (fig. 7E). Microbodies. These organelles were strikingly increased in number, size, and variability beginning with the early biopsies after exposure to alcohol (fig. 8). Golgi complex. After administration of alcohol, the Golgi cisternae were dilated and filled with electron-dense granules. Cell borders. Occasional cytoplasmic blebs extended into the space of Disse or impinged upon an adjacent cell (fig. 6A). Bile ductules. The bile ductules in subject 1 appeared normal. After alcohol administration in subject 2, bile ductular cells contained fat droplets (fig. 9A). Cytoplasmic blebs extended into the lumens of ductules (fig. 9C). Occasional bile ductular cells showed degenerative changes (fig. 9B). Discussion
The hepatic changes induced by iSClcaloric substitution of alcohol for carbohydrate in an otherwise complete diet include (1) fatty metamorphosis; (2) enlargement and disfiguration of mitochondria, with disorientation of cristae and crystalline inclusions; (3) vacuolization and increase in endoplasmic reticulum; (4) increase in free
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FIG. 1. Light micrographs of liver biopsies. H & E, X 240. A, Subject 1. Control biopsy. Liver cells are filled with glycogen. No fat is present. B, Subject 1, 1 day after full dose of ethanol. There is little change from the control biopsy, except for slightly increased fine droplet fat. C, Subject 1, 10 days after full dose of ethanol. Conspicuous fatty metamorpho-
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ALCOHOL-INDUCED CHANGES IN HUMAN LIVER
sis is noted. D, Subject 2, control biopsy. Essentially normal appearance with a few fat globules (arrows). E, Subject 2, 3 days after full dose of ethanol. Extensive fatty metamorphosis is present . F, Subject 2, 12 days after full dose of ethanol. Fatty metamorphosis is more severe.
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FIG. 2. Subj ect 1, co ntrol biopsy. Mitochondria are of normal s ize. Some display mild distortion of cristae. Endoplasmic reticulum is vesicular a nd slightly increased. A normal amount of glycogen (dar k dots) is present (X 10,000).
polyribosomes; (5) focal cytoplasmic degradation in the form of autophagi c vacuoles, as well as residual vacuolated bodies; (6) increased microbodies; and (7) changes in bile ductules. The mitochondrial distortions have been observed in human alcoholic hepatitis 9, 10 , 15 and alcoholic fatty livers without inflammation.8 They have also been produced in rats by isocaloric substitution of alcohol for carbohydrate6 , 7 These changes are not specific for alcohol administration, since they are seen in many dietary l6 , 17 and toxic conditions. 18 Prominent features in almost all descriptions of mitochondrial changes induced by alcohol in m an and animals are the crystalline inclusions or socalled myelin figures within mitochondria. 6 , 8 , 9, 15 Although these inclusions are regularly found in alcohol intoxication, they are also noted in other conditions19 , 20 and
rarely in normal persons. 21 The exact nature of the crystalline inclusions is not certain. They are assumed by some to be phospholipids which are unmasked as a result of disintegration of intramitochondrial components.22 The degeneration of giant mitochondria described here has been suggested as the origin of Mallory's alcoholic hyaline,6 but, in our studies of early changes, no alcoholic hyaline was seen with the light microscope, at least with the stains employed. Since in acute alcoholic hepatitis the organelle change which correlates best with the clinical condition is mitochondrial alteration,15 changes in these organelles may be the crucial effect of alcohol ingestion. In rats given long term alcohol in addition to an uncontrolled diet, isolat ed mitochondria had decreased oxidative properties. 23 Theoretically, the decreased hepatic oxidation of fatty acids3 , 24 and pyru-
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ALCOHOL-INDUCED CHANGES IN HUMAN LIVER
FIG. 3. Subject 2. Control biopsy . Mitochondria are of normal size but a few abnormal forms (.strai ght arrows) are noted . End oplasmic reticulum is slightl y in creased and vesicular. Bile canal ic ulus (c) is unremarkable. A few residual bodies (curved arrow) a re prese nt (X 9000).
FIG. 4. Subject 1, 24 hours after first full dose of ethanol. A , Mitochondria are enlarged and disfigllTed (X ()200) . B , Numerous residual vacuolated bodies are present (X 9700).
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FIG. 5. Mitochondrial changes in cells which contain no fat droplets. A, Subject 2, 3 days after first full dose of ethanol. Bizarre disfigured and enlarged mitochondria (X 14,000). B, Subject 1, 24 hours after first full dose of ethanol. Intramitochondrial crystalline inclusion (X 54,(00). C, Subject 2, 3 da ys after first full dose of ethanol. Mitochondrion shows parallel arrays of .double-membraned structures, proba bl y early crystal formation (X 17,(00) .
vate23 produced by alcohol could be related to the mitochondrial damage observed morphologically. This in turn could serve as an explanation for the hepatic accumulation of dietary lipids on prolonged ethanol intake, both in rats 25 and in man. 26 In neither of the subjects studied was the smooth endoplasmic reticulum completely normal in the control biopsy, but progressive changes occurred after alcohol administration. The vesiculation and increase in endoplasmic reticulum have also been observed in isocaloric ethanolinduced hepatic injury of rat7 and man. 27 The changes in the endoplasmic reticulum may be related to the ethanol-induced increase in lipogenesis,3 including the increased esterification by microsomes. 28 A noteworthy feature is the striking increase in polyribosomes in the third biopsy of both patients. In view of the association of polyribosomes with protein synthesis and the observation that administration of large doses of ethanol results not only in fatty liver, but also in hyperlipemia,12. 29. 30 the increase in polyribosomes may reflect increased lipoprotein
synthesis, perhaps as an adaptive mechanIsm. A striking change was the widespread focal cytoplasmic degeneration in the form of autophagic vacuoles and residual vacuolated bodies which contained remnants of degenerated organelles and ergastoplasm. These structures correspond to the D-PAS positive structures which are increased in many forms of hepatic injury.15. 31 The dense osmiophilic material within these structures may represent the liberation of the lipids of the sequestered cytoplasmic components, brought about by digestion of structural proteins. 1s Since they occur in otherwise normal cells, they probably signify increased turnover of organelles in the damaged, but surviving, cell. The function of microbodies is unknown, and consequently their increase cannot be explained. In lower vertebrates, microbodies contain uricase,a2 an enzyme absent in man. It seems probable that in man they also con· tain enzymes, perhaps catalase and d-amino oxidase. aa The changes noted in bile ductules may
FIG. 6. Mitochondrial alterations associated with fat. A, Subject 1, 10 days after full dose of alcohol. Enlarged mitochondria with bizarre shapes and abnormal cristae are arranged about fat droplets. Smooth endoplasmic reticulum is vesicular. A cytoplasmic bleb (arrow) is seen at the cell border (X 9,900). B, Subject 2, 3 days after full dose of ethanol. Elongated mitochondrion is draped around fat droplet (X 7,200). C, Subject 2, 12 days after full dose of ethanol. Mitochondrion encircles small fat droplet. Note increased polyribosomes (X 14,000). D, Subject 1, 10 days after full dose of ethanol. Megamitochondrion containing crystalline inclusions partially surrounds a fat droplet. Crystalline inclusions are also seen in other mitochondria (arrows) (X 8,700). 9
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FIG. 7. Stages in the development of autophagic vacuoles and residual vacuolated bodies in subject 1, 10 days after full dose of ethanol. A, Numero1ls residual vacuolated bodies surround a bile canaliculus, c (X 44(0). B, Autophagic vacuoles (arrows) contain ribosomes and lamellated osmiophilic material (X 27,0(0). C, Degenerating mitochondria (straight arrows) display circular, osmiophilic cristae. In more advanced degeneration, cristae are more osmiophilic and arranged concentrically (curved arrow) (X 11,000). D, Degenerated mitochondrion is enclosed within autophagic vacuole (arrow) (X 29,0(0). E, Residual vacuolated bodies are poorly delimited and contain dense , whorled osmiophilic material and degenerated mitochondrial material (straight arrow). A small autophagic vacuole (curved arrow) may merge with vacuolated body (X 14 ,(00) .
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ALCOHOL-INDUCED CHANGES I N HUMA N LIVER
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FIG. 8. Subject 1, 10 days after full dose of ethanol. Numerous clusters of microbodies are present (arrows) (X 4200) .
eventually lead to periductular inflammation and fibrosis, the so-called ductular cell reaction. 34 This reaction is assumed to be important in the eventual formation of fibrosis and cirrhosis. 35 The production of hepatic damage in normal rats after 1 to 3 weeks of alcohol administration 6, 7 suggests that chronic alcoholism is not a prerequisite for susceptibility to ethanol-induced liver damage. In human subjects in whom fatty liver was less striking than in the cases described here, and whose livers were biopsied after 18 days of alcohol administration with an adequate diet, changes in endoplasmic reticulum were particularly prominent,27 This probably reflects variation in the susceptibility to the toxin, The changes described in the subjects of this study have all been noted in chronic alcoholic injury in man, but because of the uncontrolled diets in patients with alcoholic fatty liver, with or without hepatitis, the effects of nutritional and alcoholic injury were difficult to separate. This difficulty has been overcome in this study by investigating early changes. In subject 1, ethanol produced ultrastructural hepatic damage within 7 days, and within only 1 day of the full dose (46 % of calories), at a time when fatty change was minimal. In subject 2, similar changes, which were associated with more conspicuous fatty metamorphosis, occurred within 10 days and
within only 3 days of the full dose. These observations indicate that, despite ingestion of a diet containing 16 % of calories as protein, an amount well above the recommended allowance for man,ll ethanol rapidly produces conspicuous hepatic steatosis and ultrastructural changes. It is conceivable that, in the face of a low carbohydrate-ethanol diet, protein requirements may be increased. However, in a study similar to the present one, a high protein (25 % of calories)-low fat diet failed to prevent fat accumulation in the liver. (C. S. Lieber and E. Rubin, unpublished observations). Thus alcohol appears to exert a direct toxic effect on the liver of human alcoholics which is concomitant with or may precede fatty metamorphosis. In view of the fact that alcohol produces the same effect in normal rats,6, 7 it seems likely that it is a general phenomenon. SUllllllary
The early ultrastructural changes induced by isocaloric substitution of ethanol for carbohydrate in t he livers of 2 human subjects were studied. Mitochondrial alterations and focal cytoplasmic degradation were noted within 1 d ay of the full dose of ethanol (46 % of total calories), at a time when fat accumulation was minimal. Fatty metamorphosis was prominent within 3 to 10 days of the full dose of ethanol and was associated with al-
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FIG. 9. Bile ductules. Subject 2. A, 10 days after full dose of ethanol. Fat droplet (arrow) in ductular cell (X 8200). B, 10 days after full dose of ethanol. Ductular cell (arrow) shows advanced degenerative changes (X 8500). C, 3 days after full dose of ethanol. Cytoplasmic bleb (arrow) extends into lumen of ductule (X 16,000).
terations of mitochondria, endoplasmic reticulum, autophagic vacuoles, microbodies, and ductular cells. It is concluded that alcohol exerts a direct toxic action on the liver which precedes or is concomitant with fatty metamorphosis. REFERENCES 1. Popper, H., P. B. Szanto, and H. Elias. 1955.
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Transition of fatty liver into cirrhosis. Gastroenterology 28: 183-192. 2. Himsworth, H. P. 1960. The liver and its diseases. Harvard University Press, Cambridge. 3. Lieber, C. S. 1966. Hepatic and metabolic effects of alcohol. Gastroenterology 50: 119133. 4. Popper, H. 1961. Morphological and biochemical aspects of fatty liver. Acta Hepatosplen. 8: 29-292. 5. Hartroft, W. S., and J. H. Ridout. 1951. Pathogenesis of the cirrhosis produced by choline deficiency; escape of lipid from fatty hepatic cysts into the biliary and vascular systems. Amer. J. Path. 27: 951-989. 6. Porta, E. A., W. S. Hartroft, and F. A. de la Iglesia. 1965. Hepatic changes associated with chronic alcoholism in rats. Lab. Invest. 14: 1437-1455. 7. Iseri, O. A., C. S. Lieber, and L. S. Gottlieb. 1966. The ultrastructure of fatty liver induced by prolonged ethanol ingestion. Amer. J. Path. 48: 535--555. 8. Svoboda, D. J., and R. T. Manning. 1964. Chronic alcoholism with fatty metamorphosis of the liver. Mitochondrial alterations in hepatic cells. Amer. J. Path. 44: 645--662. 9. Porta, E. A., B. J. Bergman, and A. A. Stein. 1965. Acute alcoholic hepatitis. Amer. J. Path. 46: 657-689. 10. Kiessling, K. H., L. Lindgren, B. Strandberg, and U. Tobe. 1964. Electron microscopic study of liver mitochondria from human alcoholics. Acta Med. Scand. 176: 595--598. 1l. Food and Nutrition Board. 1964. Recommended Dietary Allowances, publication 1146, Ed. 6, National Academy of Sciences, National Research Council. Washington, D. C. 12. Lieber, C. S., D. P. Jones, and L. M. DeCarli. 1965. Effects of prolonged ethanol intake: Production of fatty liver despite adequate diets. J. Clin. Invest. #: 1009-1O2l. 13. Reynolds, E. S. 1963. The use of lead citrate as an electron opaque stain in electron microscopy. J. Cell BioI. 17: 208-212. 14. Biava, C. 1965. Electron microscopic studies on periodic acid-Schiff positive nonglycogenic structures in human liver cells. Amer. J. Path. 46: 435--465. 15. Schaffner, F., A. Lobel, H. A. Weiner, and T. Barka. 1963. Hepatocellular cytoplasmic changes in acute alcoholic hepatitis. J. A. M. A. 183: 343-346. 16. David, H. 1961. Die Leber bei Nahrungsmangel und Mangelernahrung, p. 56. Akademie Verlag, Berlin.
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