The relation of types of dietary fat to hepatic liposis and myocardial damage in mice.код для вставкиСкачать
The Relation of Types of Dietary Fat to Hepatic Liposis and Myocardial D a m a g e in Mice' W. LANE WILLIAMS AND ROBERT I. OLIVERZ Departments of Anatomy, University of Mississippi School of Medicine, Jackson, Mississippi and University of Minnesota, Minneapolis, Minnesota.. Low protein, high fat, choline-deficient diets in which the lipid component is a natural fat, such as lard or butter, produce rapid and extensive hepatic liposis in mice (Buckley and Hartroft, '55; Meader and Williams, '57; Williams, '60). During the one month duration of a preliminary study, the substitution of three saturated triglycerides for lard as the dietary fat component greatly diminished the rate and amount of hepatic liposis (Williams, Cardle and Meader, '59). The experiments reported here included the feeding of this choline-deficient, triglyceride diet for as long as 7 months. The data thus obtained show that the responses of the livers and the hearts of these mice were very different from those in mice fed the same basic diet containing lard as the lipid component. The vitamin powder contains: Thiamine hydrochloride Riboflavin Pyridoxine hydrochloride Calcium pantothenate Nicotinic acid Powdered sugar 0.500 0.250 0.200 1.000 1.000 997.050 Fat was supplied in one diet as Iard. In the other the fat component (40% of total diet) consisted of the following saturated triglycerides: frihexanoin (50% ), trimyristin ( 2 5 % ) and tripalmitin (25%). Both diets have been used in previous studies (Meader and Williams, '57; Williams, Cardle and Mlsader, '59; Williams, '60). Within the mouse cages the food was kept in metal containers constructed with partitions and wire-mesh covers to restrict contact with the diet to the mouth and snout. A wire-mesh floor was placed above the permanent floctr of the cage to limit coprophagia. Mice were weighed daily during the first month of feeding the experimental MATERIALS AND METHODS diets and weekly thereafter. Histological technics. The methods Animals. Mice of the C (Bagg albino) stock with an initial body weight of 20- have been described in detail previously 22 gm were restricted to the choline-de- (Williams, '60). Abdominal and thoracic organs were fixed in 10% formaldehyde or ficient diets when 8 to 10 weeks of age. Diets. From weaning (30 days of age) in Lavdowsky's solution or in both. Hearts until 8 to 10 weeks of age these mice were were cut into two relatively equal frontal fed a standard laboratory ration that was sections for fixation in 10% formaldehyde adequate in choline, vitamins and min- and Lavdowsky's solution. Thigh muscle erals; and was composed of approximately from about one-half of the mice, and dia25% protein, 6% fat, and 47% carbohy- phragm from essentially all were fixed in drate. The composition of the basic cho- Lavdowsky's solution. Routine paraffin line-deficient diet used here is as follows: sections (10 v ) of all of these organs and gm tissues were si:ained with hematoxylin and Vitamin free casein 80.0 eosin. Sucrose Fat Salt mixture (no. 2, U.S.P. XIII) L-Cystine Cod liver oil (Navitol) Vitamin powder 480.5 400.0 40.0 5.0 4.5 10.0 1 Supported by grants A-2693 and H-4052 from the National Institutes of Health, U.S.P.H.S. and from the Mississippi Heart Association. 2 N.I.H. (2G-:!87) Predoctoral Trainee in the Anatomical Sciences. 97 98 W. LANE WILLIAMS Fat was demonstrated by staining frozen sections of formalin fixed material with oil red 0 in the same manner as with other Sudan dyes. It was essential to use gelatin embedding to make adequate frozen sections of the hearts. As the result of poor technic only one-third of these specimens were stained satisfactorily for fat. The PAS (periodic acid-Schiff's reagent) technic was used to show glycogen, ceroid pigment, and sites of recent myocardial injury (Kent and Diseker, '55). Ceroid pigment was demonstrated more specifically by staining dehydrated and defatted (i.e., embedded in paraffin) specimens with oil red 0 or Sudan black. The Masson (with aniline blue), the PAS and the Laidlaw reticulum methods were used to show fibrosis, and the von Kossa and the alizarin technics to demonstrate minerals. Selection of mice for study. At the intervals shown in table 2, mice were killed by digital compression of the cervical spinal column. The usual procedure was to select those showing weakness, decreased activity, and respiratory difficulties; and also extreme weight loss, provided the other signs of ill health just mentioned were evident. During the first 8 weeks of the experiment, such evidence of ill health was relatively infrequent and mice were killed on a schedule that produced a chronologic survey of changes in the livers and hearts of the members of the two dietary groups. Approximately 80% of the mice killed after 8 weeks of feeding were females. Of those killed earlier, one-half were males. The males were difficult to use because of their activity in upsetting and burrowing into the food containers and in becoming entrapped during such activities. Study of the material Livers. One large lobe was fixed in Lavdowsky's solution (for subsequent paraffin-embedding) and another in 10% formaldehyde. Six to 8 sections of the latter (across the entire lobe) were stained to show fat. Complete sections of the paraffin embedded specimens were processed as follows: 6 to 8 sections, H. and E.; 4 to 6 , PAS; and when so indica.ted by the results of staining with H. and E. or PAS, 4 to 6 AND ROBERT I. OLIVER additional sections were stained by the Laidlaw reticulum method. Ceroid pigment is recognizable in H. and E. and in PAS preparations and also in frozen sections stained with Sudan dyes. When the pigment was so observed more sections (paraffin) were cut and then stained with Sudan dyes (Meader and Williams, '57; Williams, '60). Hearts. One-half of each heart (complete frontal or sagittal section) was embedded in paraffin and the other was kept in formalin (10% ) for frozen sections and also as reserve material. At least 80% of every heart and all of a majority were completely consumed in making the following preparations, all of which had some value in identifying myocardial lesions: H. and E., Masson, PAS, Laidlaw reticulum (in a variety of combinations with PAS and H. and E.), von Kossa or alizarin, and frozen sections stained with oil red 0. Subsequent to frozen sectioning the remaining portion of the formalin fixed half of the heart was dehydrated and embedded in para& and prepared for study by the staining procedures mentioned above. These sections permitted study of structures located at or near the cardiac base, including aorta, vena cava, pulmonary vessels, thymus, lymph nodes, nerves, autonomic ganglia, uniloculax and multilocular fat, esophagus, bronchi and a large number and variety of small blood vessels within or near these several structures. Other organs and tissues. Adrenals and kidneys as a single 'block" were cut into two equal portions in the usual fashion for fixation in Lavdowsky and in 10% formaldehyde. Two to 4 sections were stained with H. and E. and a similar number with PAS. Skeletal muscle (diaphragm, thigh and esophagus) and gonads and spleen were studied in two to 4 sections stained with H. and E. In most instances two to 4 sections of adrenals (embedded in paraffin and included in same section as kidney) and gonads were stained with Sudan black to demonstrate ceroid pigment. The simple statistical analysis (means and standard deviations) of the data on body weights (tables 1, 2 and 4 ) was based on the methods and tables presented by Croxton ( ' 5 3 ) . 99 HEPATIC FAT AND CARDIAC DAMAGE TABLE 1 Weight changes in mice fed choline-deficient, low protein-high fat diets Lard as dietary fat Months fed Total notof mce 1 84 Mean weight change Saturated triglycerides as dietary fat Per cent of mice : $%$ Total no. of mice weight Lost Mean weight change % 2 41 (6.1)2 2 2.9 29 (10) zt 7.3 45 (9.5) 26 52 (9.8) rt 7.2 44 (8.3) r+ 5.2 33.3 (7.7) rfr 2.4 8.5 (3.2) -4.2 f 9.8 3 - 1.0 56 f11.1 4 -3.9 48 2 14.8 5 -4.3 40 2 11.9 6 - 5.0 24 2 9.4 7 - 10.4 21 -t 7.9 8 15 9 8 10 6 z;kt % - 1.2' 2 6.53 69 Per cent of mice: $2: -11.7 -C 13.1 -20.0 8.4 - 24.2 e4.1 * - 1.3 0 47 (7.7) f4.6 60 (12) f7.8 55 (10.2) r+ 7.5 48 (18.7) 2 10.3 56 (14.4) f10.8 66.6 (11.5) 29.1 83 (13) f8.3 100 72 f6.1 40 (6.8) 25 22 (7.4) -4.7 22 (6.7) 25.6 14 (4.1) 16 - 9.8 2 5.1 6 (5) 16 .- 16.0 0 0 100 No survivors 0 100 N o survivors 2 8.1 - 8.4 59 tt 10.0 41 -8.0 2 8.2 29 -- 12.1 51 (7.9) f6.1 73 (13.8) -9.0 73 (13) 28.6 86 (14.1) f7.7 94 (11) 5 100 I- 7.9 14 .- 18.0 7.2 -+: 8.5 (5.0) 92.8 (20.0) f9.9 No survivors - Weights are those of all mice living at the stated intervals. *Numbers in parentheses are means of weight changes ( % ) in animals ( % of total in group) listed directly above. 3Standard deviations are those of changes in body weight (i.e., the mean listed directly above) and were not determined for groups smaller than 5 mice. TABLE 2 Changes in hearts and livers of mice fed choline-deficient diets conttaining 40% fat as lard (L) M as saturated triglycerides (TG) Per cent of mice showing Weeks fed diet No. of mice killed Ll 1 4 5-8 9-15 16-21 22-27 28-33 34-39 40 64 17 7 8 21 8 7 6 TG* 41 14 28 16 14 NS3 NS NS Changes in livers Myocardial lesions Complete lobular liposis L TG L TG 0 0 0 37 24 13 29 33 0 43 32 56 71 1004 100 100 100 100 100 100 100 0 57 29 37 71 NS NS NS NS NS NS Fatty cysts L 0 100 100 100 100 100 100 100 Reticulinosis and nodular parenchytqal hyperplasia TG L TG L TG 0 29 15 31 14 0 0 14 100 100 100 100 100 0 0 0 19 14 0 0 0 0 33 100 100 100 0 0 0 0 0 NS NS NS NS NS NS 'L, diet containing lard as fat. * TG, Ceroid pigment diet containing three triglycerides as fat. 3NS, no survivors. 4Complete lobular liposis within three days in all mice fed lard. NS NS NS W. 100 LANE WILLIAMS AND ROBERT I. OLIVER OBSERVATIONS dial fibrosis in which there was no eviAt autopsy the significant findings in dence of inflammation, active necrosis, or mice killed after more than 8 weeks of of deposition of ceroid pigment or of calcirestriction to the diet were depletion of the fication. The total incidence was approxiusual fat depots, pneumonia, and obvi- mately 10% and no lesions were observed ously fatty livers in the mice fed lard. prior to 17 weeks of feeding the lard diet. Other tissues and organs, including the Maximal fibrosis was less than that shown alimentary tract, appeared normal. in figure 13. Livers. The results are summarized in As early as 10 weeks (tables 2 and 4) table 2. In chronologic order the livers of of feeding triglycerides myocardial lesions mice fed the lard-containing diet showed : were present (figs. 6-13). The total inci(1) complete lobular liposis within 72 hours; ( 2 ) formation of fatty cysts within dence was 30% and the most common le5 weeks; ( 3 ) ceroid pigment within 9 sion contained necrotic myocardial fibers, weeks; and ( 4 ) within 22 weeks a nodular collagenous fibers and large amounts of hyperplasia of the parenchyma and an ap- calcium and of ceroid pigment (figs. 6-9). parent increase of reticular fibers between One-half of the damaged hearts contained these nodules. Some of these changes are such lesions (table 3 ) . Other lesions were shown in figures 1-5, and all have been of two types (table 3 ) : necrosis and fidescribed in detail previously (Meader and brosis without calcium and ceroid (figs. 10-13), and small areas of myocarditis. Williams, '57; Williams, '60). Hepatic liposis and development of In some instances the necrosis seemed very ceroid pigment were retarded and limited recent in that there was little or no leucoin livers of mice fed triglycerides as fat cytic, histiocytic or fibroblastic response; (table 2, figs. 1-5). The level of maximal and the damaged fibers were strongly PASliposis was approximately one-half of that positive (fig. 12) subsequent to diastaseseen in the mice fed lard. Ceroid pigment hydrolysis to remove glycogen (Kent and was present in only trace amounts (figs. 4 Diseker, '55). As well as could be deterand 5). The number of triglycerides-fed mined from the small number of sections mice surviving until the period in which stained to show fat and from the numerous reticulinosis and nodular parenchymal hy- sections stained by other methods there perplasia usually develop in mice receiving was no myocardial liposis in either dietary lard (Meader and Williams, '57) was so group. The blood vessels of the hearts and small (table 2) that the data are not sig- of the other organs showed no significant nificant. In the triglyceride group liposis changes from normal. There were no lewas insufficient to produce extensive dis- sions of epicardium, valves or endocartortion of parenchyma or stroma, (figs. 1, dium. When mice of the stock used here are fed a completely adequate commercial 2, 5). Hearts. In table 2, 3 and 4 the types diet, the incidence of myocardial lesions is and incidence of myocardial damage are approximately 1% in animals more than presented. The lesions in the lard diet 6 months older than those used here. Such group consisted of small areas of myocar- lesions consist of small areas of fibrosis TABLE 3 Types o f myocardial lesions i n mice f e d choline-deficient, low protein-high f a t diets 138 Mice fed lard as fat (13 lesions) 113 Mice fed fat as three saturated triglycerides (34 lesions) Restricted myocardial fibrosis Extensiye necrosis and fibrosis Calcivm, ceroid pigment and fibrosis Diffuse myocarditis 100% of lesions (185 days)' 4 1% of lesions (101 days)' 50% of lesions (133 days)' 9% of lesions (79 days)' Mean. 101 HEPATIC FAT AND CARDIAC DAMAGE: TABLE 4 Myocardial lesions ~Lard as fat Weeks fed &et No: of mice killed Per cent with lesions Triglycerides as fat Weight loss With lesions Without lesions No. of mice killed Per cent with lesions Weight loss l ~ ~Without lesions ~ ~ ~- ~ % 1-4 5-8 9-15 16-21 64 17 7 8 0 0 0 30 - 35 k 4.1 22-27 28-33 21 8 24 12.5 16 * 8.1 18 2 34-39 7 28.6 22 2 40 6 33 26 2 % 1.21 f6.5 4.9 f4.3 33 C 5.7 34 f7.9 19 f9.1 16 f8.1 20 f7.7 23 C 4.8 % 41 0 - % 1.3' f8.1 14 43 33 f8.3 28 16 14 32 56 71 23 17 27.3 2 11.7 19.7 f8.9 35 * 10 10 f10.3 24 f5.2 17 2 4.8 No survivors No survi~vors No surv..vors __ 1Av. wt. changes in all mice surviving at 4 weeks. 2 N o standard deviation determined for groups smaller than 4 m ~ c e . with minimal evidence of active necrosis figs. 1-5). These hepatic changes were (Williams and Aronsohn, ' 5 6 ) . retarded and restricted in the triglycerideOther organs and tissues. The kidney fed mice (table 2; figs. 1-5). This dewere normal. Pneumonia was common in creased liposis was apparent in the triglycall mice fed either diet (lard or triglyceride) eride group prior to any significant loss in for more than three months. The skeletal body weight (ta.bles 1 and 2). The greatest muscles of the triglyceride group usually incidence of complete lobular liposis was contained calcium and ceroid when such in the mice that survived longest (table 2). materials were present in myocardium. No Here it is not possible to present the significant changes were observed in other complete history of weight changes of inorgans. Ceroid pigment was not increased dividual mice. There was no instance of in adrenals or gonads. rapid weight loss followed by significant gain of weight. Frequently there was rapid Relation of general health and of weight loss of weight during the two weeks imloss to changes in livers and hearts mediately preceding the time at which the After 4 weeks weight loss was sign& animal was killed. A great majority of the heart lesions cantly greater in mice fed the triglyceride diet (table 1). At all intervals the range were in mice with extreme decreases in in weight loss was broad in both dietary body weight. However, only at 9-15 weeks groups (tables 1 and 4). After the first 8 were the weight losses of animals killed weeks, all mice killed for study represented within a specific chronologic interval sigthe less healthy members of the dietary nificantly greater in those with cardiac legroups and the majority had lost consider- sions (table 4). Despite this, the situation able amounts of weight (tables 1 and 4). responsible for the early and rapid weight The changes in the livers showed no rela- loss of the triglyceride group must be contion to amount of loss in body weight. sidered as contributory to the production of Very fatty livers were present in all of the myocardial lesions (Williams, '60). The lard-fed mice and the other hepatic mani- constant finding of pneumonia in mice festations of the deficiency ensued in the killed after 8 weeks on the diets was menexpected relation to its duration (table 2; tioned earlier. Several experimental proce- 102 W. LANE WILLIAMS AND ROBERT I. OLIVER dures seem to precipitate this disease in mice (Williams and Davis, '59; Williams, '60). It seems highly probable that pneumonia contributed significantly to the poor health including the weight loss observed in all animals, those fed lard and those fed triglycerides. DISCUSSION Hepatic liposis. In choline deficiency maximal liposis occurs in livers of rats or mice fed diets containing natural fats (lard or butter) composed of a broad range (as to chain length and degree of saturation) of lipids including unsaturated compounds (Williams, Cardle and Meader, '59). In the triglyceride diet used here the 40% level of fat consisted of 50% of a Cs compound (trihexanoin), 25% of a CM compound (trimyristin) and 25% of a CI6 compound (tripalmitin). This diet lacked many of the saturated compounds and all of the unsaturated ones present in lard and butter (Bailey, '51). In choline deficiency the quantity of the hepatic liposis is controlled to some extent by the chemical composition of the dietary lipids (Stetten and Salcedo, '45; Hartroft, '55; Williams, Cardle and Meader, '59). Ceroid pigment. In mice fed the triglycerides the rarity of ceroid in livers and its frequency in myocardium seem to present a contradiction as to the specific relation of diet to the production of this pigment. The small amount of hepatic ceroid is explained by the absence of unsaturated fat in the diet (Endicott, '44; Dam, '49; Hartroft, '51; Alpert, '53). In the absence of unsaturated dietary fats the presence of small amounts of ceroid in these livers is probably the result of local synthesis of unsaturated compounds (Stetten and Salcedo, '45; Gurin, '53) which would be available for atypical oxidation or polymerization that may produce the pigment (see Alpert, '53). In livers the usual oxidative processes related to fatty acids may be absent or severely restricted or altered in choline deficiency (Artom, '53, '58, '60). The small amount of unsaturated fat available from the cod liver oil mixture used as the source of vitamins A and D may have been used in ceroidogenesis in mice fed the triglyceride diet. However, the low incidence and delayed appearance of this pigment would not seem to substantiate such a conclusion. In this same group of mice (those fed triglycerides) material staining as does ceroid was common in areas of myocardial necrosis. This ceroid is probably part of a local response to tissue injury and necrosis (see Alpert, '53; and Hartroft, '53b) and in no way related to a high intake of fat (Williams and Aronsohn, '56). Calcium-ceroid in myocardial lesions. The occurrence of the mineral or the pigment, or both, has a broad background as to contributory nutritional deficiencies. Included are low protein, vitamin E deficiency, and cystine supplementation (Mason and Emmel, '45; Moore, '49; Highman and Daft, '51; Alpert, '53; Williams and Aronsohn, '56); fatty acid deficiencies (Hill, Warmanen, Hayes and Holman, '57); and non-specific relations to feeding of highly purified diets (Constant, Phillips and Angevine, '52; Constant and Phillips, '54). In some species deficiencies in essential fatty acids seem to have a direct relationship to calcification in the cardiovascular system (Hill, Warmanen, Hayes and Holman, '57). The lesions containing ceroid and calcium occurred in both types of striated muscle, skeletal and cardiac. Deposition of ceroid in skeletal muscle has been associated with deficiencies of protein and of vitamin E (Mason and Emmel, '45; Williams and Aronsohn, '56). Neither ceroid nor calcium, nor the combination, was observed in striated (including cardiac) muscle of mice fed diets low in protein, not supplemented with vitamin E, but high in content of saturated and unsaturated fats (Williams, '60). The livers of these mice contained large amounts of ceroid when the diets, as above, were deficient in choline. Other types of myocardial lesions. The low incidence of myocarditis (table 3) does not seem significant. Necrosis and fibrosis (without calcium and ceroid) represented 41% of the lesions (table 3 ) . Such lesions were similar if not identical with those previously observed in mice fed choline-supplemented diets that were high in fat (lard) and low in protein (Williams, '60). The incidence and relatively prolonged latent period of all cardiac lesions HEPATIC FAT AND CARDIAC DAMAGE observed here do not suggest the type of myocardial injury produced in young rats fed a choline deficient-diet containing ethyl laurate as the lipid component (Kesten, Salcedo and Stetten, '45). Myocardial necrosis, fibrosis and liposis, with and without arterial lesions, have been described in young rats fed high fat diets (Hartroft, '53a; Wilgram and Hartroft, '55; Thomas and Hartroft, '59). It should be emphasized that in mice, the heart, rather than the liver seems to be the more common target for necrotizing actions of several diets which have one feature in common-low protein, but vary considerably as to content of fat and of choline (Highman and Daft, '51; Williams and Aronsohn, '56; Meader and Williams, '57; Williams, '60). In mice fed low protein diets the incidence of extensive myocardial necrosis is significant in the absence of a high fat intake (Highman and Daft, '51; Williams and Aronsohn, '56). When the dietary level of fat is very high, an adequate supplementation with choline increases the amount of cardiovascular injury over that observed in choline deficiency (Wissler, Eilert, Schroeder and Cohen, '54; Williams, '60). The composition of the lipid component of the triglyceride diet used here was so atypical (i.e,, completely lacking in unsaturated fat) that on a nutritional basis it could be considered as deficient in fat (Hill, Warmanen, Hayes and Holman, '57). This may explain the similarity of the my+ cardial lesions to those previously described in mice fed diets low in protein, but not high in fat (Highman and Daft, '51; Williams and Aronsohn, '56). SUMMARY 1. In mice fed choline-deficient diets for as long as 7 months hepatic liposis was extremely limited when dietary fat consisted of three saturated triglycerides. In these mice significant production or deposition of ceroid pigment did not occur in livers. In livers of mice fed lard the liposis was rapid, massive, and progressive; and the expected pattern of abundant ceroide genesis, parenchymal hyperplasia and reticulinosis ensued. 2. In the mice fed triglycerides 30% showed extensive myocardial lesions that 103 included necrosis, calcinosis and deposition of ceroid pigment. In lard-fed mice cardiac damage consisted of a very limited amount of myocardial fibrosis and the total incidence of such lesions was 10%. 3. It is Concluded that a broad range (with reference to saturation and chain length) of dietary lipids is essential for production of the massive hepatic liposis and of ceroid pigment characteristic of choline deficiency. The absence of unsaturated compounds from the diet seems to afford considerable protection (from liposis) to the liver, but renders the myocardium susceptible to extensive damage. LITEIRATURE CITED Alpert, M. 1953 Hormonal induction of deposition of ceroid pigment in the mouse. Anat. Rec., 116: 469-494. Artom, C. 1953 Role of choline in the oxidation of fatty acids by the liver. J. Biol. Chem., 205: 101-111. 1958 Rolle of choline in the hepatic oxidation of fat. Am. J. Clin. Nutrition, 6: 22 1-234. 1960 Mechanism of action of choline. Ibid., 8: 303-3051. Bailey, A. E., (editor) 1951 Industrial Oil and Fat Products. Interscience Publishers, New York, chap. 11, pp. 39-73. Buckley, G. F., and W. S. Hartroft 1955 Pathology of choline deficiency in the mouse. Arch. Path., 59: 185-197. Constant, M. A., and P. H. Phillips 1954 The occurrence of a calcinosis syndrome in cotton rats. IV. The effect of diet and the age of the animals on the development of the disease and on the urinary excretion of various metabolites. J. Nutrition, 5 2 . 165-186. Constant, M. A., P. H. Phillips and D. M. Angevine 1952 The occurrence of a calcinosis syndrome in the cotton iat. 11. Pathology. Ibid., 47: 327-339. Croxton, F. E. 1933 Elementary Statistics with Application in Medicine and the Biological Sciences. Dover Publications, New York. Dam, H. 1949 Relationship of vitamin E deficiency to tissue peroxides. Ann. N. Y. Acad. Sci., 52: 195-19!3. Endicott, K. M. 1944 Similarity of the acid fast pigment ceroid and oxidized unsaturated fat. Arch. Path., 37: 49-53. Gurin, S. 1953 The liver and fat metabolism. Transactions of the Twelfth Conference on Liver Injury. Josiah Macy, Jr. Foundation, pp. 67-97. Hartroft, W. S. 1951 In vitro and in n v o production of a ceroid-like substance from erythrocytes and certain lipids. Science, 113: 158-166. 1953a Cardiovascular lesions in choline-deficient rats. Transactions of the Twelfth - 104 W . LANE WILLIAMS AND ROBERT I . OLIVER Conference on Liver Injury. Josiash Macy, Jr. Foundation, pp. 98-100. 1953b Pathogenesis and significance of hemoceroid and hyaloceroid, two types of ceroidlike pigment found in human atheromatous lesions. J. Gerontology, 8: 158-166. Hartroft, W. S. 1955 Effects of various types of lipids in experimental hypolipotropic diets. Fed. Proc., 14: 655-660. Highman, B., and F. S. Daft 1951 Calcified lesions in C,H mice given purified low-protein diets. Arch. Path., 52: 221-229. Hill, E. G.,E. L. Warmanen, H. Hayes and R. T. Holman 1957 Effects of essential fatty acid deficiency in young swine. Proc. SOC.Exp. Biol. Med., 95: 274-278. Kent, S. P., and M. Diseker 1955 Early myocardial ischemia. Lab. Invest., 4: 398405. Kesten, H.D., J. Salcedo, Jr. and Dew. Stetten, Jr. 1945 Fatal myocarditis in choline-deficient rats fed ethyl laurate. J. Nutrition, 29: 171177. Mason, K. E., and A. F. Emmel 1945 Vitamin E and muscle pigment in the rat. Anat. Rec., 92: 33-60. Moore, T. 1949 The significance of protein in vitamin E deficiency. Ann. N. Y. Acad. Sci., 52: 206-216. Meader, R. D.,and W. L. Williams 1957 Choline deficiency in the mouse. Am. J. Anat., 100: 167-204. Stetten, Dew., Jr., and J. Salcedo, Jr. 1945 The effect of chain length of the dietary fatty acid upon the fatty liver of choline deficiency. J. Nutrition, 28: 167-170. Thomas, W. A., and W. S. Hartroft 1959 Myocardial infarction in rats fed diets containing high fat, cholesterol, and sodium cholate. Circulation, 19: 65-73. Wilgram, G. F., and W. S. Hartroft 1955 Pathogenesis of fatty and sclerotic lesions in the cardiovascular system of choline-deficient rats. Brit. J. Exp. Path., 36: 298-305. Williams, W. L. 1960 Hepatic liposis and myocardial damage in mice fed choline-deficient or choline-supplemented diets. Yale J. Biol. Med., 33: 1-14. Williams, W. L., and R. B. Aronsohn 1956 Cardiac and hepatic lesions in mice fed yeastprotein diets. I. Diets containing British bakers yeast. Ibid., 28: 515-524. Williams, W. L., J. B. Cardle and R. D. Meader 1959 The nature of dietary fat and the pattern of hepatic liposis in choline-deficient mice. Ibid., 31: 263-270. Williams, W. L., and R. L. Davis 1959 Effects of cortisone and vitamin Bia on starved mice. Anat. Rec., 134: 7-23. Wissler, R. W., M. D. Eilert, M. A. Schroeder and L. Cohen 1954 Production of lipomatous and antheromatous arterial lesions in the albino rat. Arch. Path., 57: 333-351. PLATE 1 EXPLANATION OF FIGURES All figures show livers ( X 100) of mice fed the lard or triglyceride diet for 16 weeks (figs. I, 2, 3) or 27 weeks (figs. 4 and 5). 1 Maximal liposis produced by triglyceride diet. Frozen section, oil red 0 stain. 2 Minimal liposis produced by triglyceride diet. Frozen section, oil red 0. 3 Usual amount of liposis seen in mice fed the lard diet. Frozen section, oil red 0. 4 Usual amount of ceroid pigment in mice fed lard diet. Paraffin section, Sudan black. 5 Maximal amount of ceroid pigment seen in mice fed triglyceride diet. Paraffin section, Sudan black. HEPATIC FAT AND CARDIAC DAMAGE W. Lane Williams and Robert I. Oliver PLATE 1 105 HEPATIC FAT AND CARDIAC DAMAGE PLATE 2 Mi". Lane Williams and Robert I. Oliver All figures show hearts of mice fed triglyceride diet for 16 weeks. Positive reaction for calcium. Alizarin stain. X 68. 7 Several areas showing myocardial necrosis and deposition of calcium and ceroid pigment. H. and E. X 100. 8 Positive reaction for ceroid pigment. Paraffin section decalcified with nitric acid and stained with oil red 0. X 500. 9 Lesions similar to those i n figure 7. PAS with hematoxylin counterstain. X 100. 6 106 HEPATIC FAT AND CARDIAC DAMAGE W. Lane Williams and Robert I. Oliver PLATE 3 All figures show hearts of mice fed triglyceride diet for 12 1.0 26 weeks as stated. 10 Extensive myocardial fibrosis at 12 weeks. H. and E. X 80. 11 Small area within same section as shown in figure 10. The fibers shown in cross section are shrunken. X 475. 12 Intense PAS positivity (following diastase-hydrolysis ) indiczding very early necrosis of myocardial fibers at 16 weeks. Hematoxylin counterstain. > 200. 13 Minimal necrosis and fibrosis as seen in a triglyceride-fed (2.6-week) mouse. Masson stain, x 200.