Coronary Arterial Lesions in Swine Fed Supplemental Vitamin D with Skimmed Milk Powder*

Takayoshi TODA**,Akihiko MIZOE***,Kiyosumi OHISHI***, Yumiko TODA****and Fred KUMMEROW*****

levelsof lipidand calcium (Table 1).Group I was Introduction fed the basal ration containing 200,000 IU of Using swine as an experimental animal model, vitamin D per ton of diet.GroUp II received9.1% we previously demonstrated that administration of skimmed milk powder for 4 months. excessive amounts of vitamin D can produce The basal diet contained 3% fat and 14.3% atherosclerotic coronary arterial lesions1). We protein as furnished by 1.745 pounds of ground also previously reported that the potent angio- yellow corn, 200 pounds of pressure-cooked and toxic erects of vitamin D may be due to the solvent extracted soybean meal and 55 pounds of deterioration of membrane permeability to cal- a premix of multiple vitamins and minerals per ton cium2). of basal ration. Several authors3,4) have reported vitamin D in In the second experiment, 66 two month old- combination with milk powder which contains swines were divided into 3 groups as shown in abundant calcium is more biologically active than Table 2. Group I recieved 4.8% skimmed milk when combined with oil. powder and group II,9.1% skimmed milk powder Since vitamin D is widely used to fortify milk5), for 4 months. Subgroups A, B and C were fed and vitamin D and calcium are closely linked 100,000 IU, 200,000 IU or 300,000 IU of vitamin metabolically, it is important to determine the D per ton of diet respectively. level at which vitamin D, in combination with All swines were sacrificed at 6 months of age at milk, produces an atherogenic effect. In the which time blood was collected and stabilized in present study, we undertook a microscopic and heparinized tubes for biochemical studies. Plasma ultramicroscopic survey of the coronary arteries total cholesterol levels were determined according from swine fed diets containing several different to the method of Allain et al.6) Plasma triglyceride levels of vitamin D in combination with skimmed was determined by the method of Foster and milk powder. Dunn7). The concentration of plasma phospho- lipids was measured according to the method of Materials and Methods Eng and Noble8). Plasma calcium levels were In the first experiment, a total of 31 two month- measured by the method of Sarkar and Chauham9). old Yorkshire barrow and gilts were used to For morphological studies, the proximal 5cm of monitor the effects of milk powder on serum the left anterior interventricular coronary artery was dissected and immersed in phosphate buffered *This paper was presented at the winter meeting 3% glutaraldehyde (7.4) and later cross-sectioned of the Japan Atherosclerosis Society in Japan to obtain 10 tissue blocks from each animal. The on January 1985 tissue blocks were post-fixed in phosphate buffered **Department of Pathology , Ryukyu University 1% osmium tetroxide. The tissues were dehy- School of Medicine, Okinawa ***Department of Internal Medicine drated with increasing concentrations of ethanol, , Kenyukai rinsed with propylene oxide, embedded in EM-bed Kamito-machi Hospital, Nagasaki ****Department of Pathology 812 epoxy resin and sectioned with glass knives. , Institute for Tropical Medicine, Nagasaki University, Nagasaki Thick sections were stained with alkaline toluidine *****Burnsides Research Laboratory , Department of blue to determine the degree of intimal thickening Food Science, University of Illinois, U. S. A. and to examine arterial lesions.

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Fig. 1 Degenerate endothelial cell (D) is shown. Numerous tiny elastic fibers (arrow) are seen in the subendothelial space (×5,500).

Table 1 Plasma concentration of calcium, cholesterol, triglycerides, and phospholipid

Table 2 Average thickness of coronary arterial intima Table 3 Frequency and percentage of degenerate in experimental groups cells without stainable lipid and lipid-con- taining cells in coronary arteries of experi- mental group

Ultrathin sections were stained with uranyl acetate and lead citrate. These stained thin sections blocks per animal were selected for counts and were examined with a Hitachi HU-12 electron computation of the frequency of degenerate cells microscope. Two epoxy resin embedded tissue without stainable lipid and lipid-containing cells.

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Fig. 2 Lipid-containing cells (LC) in the thickened intima (×2,000). E:endothelial cell.

a greater degree of intimal thickening of the Results coronary arteries than the other subgroups (t-test The values of plasma lipid and calcium did not p<0.05). differ significantly between the group fed basal Electron microscopic observation ration alone and the group fed basal plus skimmed The specimens from the second experiment were milk powder (Table 1). subjected to the electron microscopic observation. As shown in Table 2, subgroup II-C displayed The coronary arteries from the group I had mild

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Fig. 3 Degenerate lipid-containing cells (LC) are shown (×3 ,200).

Fig. 4 Degenerate smooth muscle cells without stainable lipid (D)(×4 ,200). intimal lesions which were seen in those in our structural changes. Degenerate endothelial cells previous experiment10). were sometimes observed:they were characterized The thickened intima of the coronary arteries by an increase in the electron density of the nuclei from a subgroup II-C included various ultra- and cytoplasm and the presence of obscure limiting

-1478- Coronary Arterial Lesions in Swine Fed Supplemental Vitamin D with Skimmed Milk Powder membranes of organelles (Fig. 1). Smooth muscle whole and skimmed milk both caused a significant cells in the thickened intima had more abundant decrease in human serum cholesterol levels, admin- rough endoplasmic reticulum and incomplete base- istration of an equivalent amount of calcium was ment membranes compared to medial smooth without effect. Segall16) indicated that the fat muscle cells. Occasional macrophages, mast cells, content of milk is at least partly responsible for plasma cells and leucocytes were seen in the sub- the association between milk and the incidence of endothelial space. Lipid-containing cells were also ischemic heart disease. present in the thickened intima (Fig. 2). The most To eliminate the factor of milk fat, we used prevalently observed type of lipid-containing cells, skimmed milk powder in the present experiment. which appeared to have originated from macro- No significant decrease in plasma lipid levels phages, contained small lipid droplets, lysosomal were observed in any of the present experimental dense bodies and cytoplasmic processes without groups, which is in accordance with the findings basement membranes. The other type of lipid- of Hussi et al.17) However, the present morpho- contining cell, which apparently originated from logical results indicate that dietary vitamin D smooth muscle cells, had various sized lipid drop- plus skimmed milk powder have an additive syner- lets, fusiform densities and basement membranes. gic effect upon the production of coronary arterial Some of these lipid-containing cells displayed lesions with activated and degenerate smooth pyknosis (Fig. 3). The stroma contained some cell muscle cells, and lipid-containing cells. Our quanti- debris accompanied by calcium deposits. Focal tative data suggests that degenerate smooth fragmentation of the internal elastic lamina were muscle cells without stainable lipid represent more frequently noted. severe type of injuried cells in the developing Two categories of degenerate smooth muscle arteriosclerotic lesions than focal cytoplasmic cells without stainable lipid, which have been necrosis of smooth muscle cells observed in the reported in the aortas of swine fed excess vitamin arteries of aged animals18). D,11) were observed in this study;the first included Since the diets used in this study contained only rarefied smooth muscle cells with electron-lucent a small amount of fat, lipid-containing cells may cytoplasm, disarrayed fusiform densities and have been produced by a mechanism other than the vesiculated organelles. The other category con- one responsible for lipid-containing cell formation tained condensed smooth muscle cells with py- in hyperlipidemic animals19). knotic nuclei and increased cytoplasmic density In view of a recent report by Kramsch et al.20) (Fig. 4). which stated that calcium influx into the arterial Table 3 presents counts and computations of wall plays as important role in the development of degenerate cells without stainable lipid and lipid- atherosclerosis, excessive dietary supplementation containing cells. There was no significant differ- of vitamin D may be potentially cytotoxic and ences in the frequency of the degenerated cells mitogenic to arterial cells. without stainable lipid and lipid-containing cells between subgroups I-A, I-B, I-C and II-A. (χ2-test Acknowledgement p>0.05). Just as the degree of intimal thickening We acknowledge gratefully Mrs. Virginia was greatest in the coronary arteries of the animals Yamamoto for her skillfull technical and editorial from subgroup II-C, so was the frequency of assistance. degenerate cells without stainable lipid and lipid- containing cells. References

Discussion 1) Taura, S., Taura, M., Tmai, H. and Kummerow, F. A.:Coronary atherosclerosis in normocholes- Various milk products, such as whole milk, terolemic swine. Artery, 4:395(1978). yogurt, butter-milk and skimmed milk have been 2) Toda, T., Leszczynski, D. E. and Kummerow, reported to lower plasma cholesterol levels in F. A.:The role of 25-hydroxy-vitamin D3 in the people11) and in animals13). Howard14) suggested induction of atherosclerosis in swine and rabbit by that the calcium content of milk products might hypervitaminosis D. Acta Pathol. Jpn., 33:37 be a hypocholesterolemic factor. However, his (1983). subsequent experiment15) showed that whereas 3) Lewis, J. M.:Clinical experience with crystaline

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vitamin D:The influence of the menstruum on the 12) Marks, J. and Howard, A. N.:The hypocholes- effectiveness of the antirachitic factor. J. Pediatr., terolemic effect of milk. Lancet, 2:763(1977). 362(1935). 13) Thakur, C. P. and Jha, A. N.:Influence of milk, 4) Supplee, G. C., Ansbacher, S., Bender, R. C. and yogurt and calcium on cholesterol-induced athero- Flanigen, G. E.:The influence of milk constituents sclerosis in rabbits. Atherosclerosis, 39:211(1981). on the effectiveness of vitamin D. J. Biol. Chem., 14) Howard, A. N.:The Masai, milk and the yogurt 114:95(1936). factor- an alternative explanations. Atherosclerosis, 5) Arnaud, S. B., Matthusen, M., Gilinson, J. B. and 27:383(1977). Goldsmith, R. S.:Components of 25-hydroxy- 15) Howard, A. N. and Marks, J.:Hypocholesterolae- vitamin D in serum of young children in upper mic effect of milk. Lancet, 2:255(1977). mid-western United States. Am. J. Clin. Nutr., 16) Segall, J. J.:Is milk a coronary health hazard? 30:1082(1977). Br. J. Prey. Soc. Med., 31:81(1977). 6) Allain, C. C., Poon, L. S., Ghan, S. G., Richmond, 17) Hussi, E., Miettinen, A., Ollus, A., Kostiainen, E., W. and Fu, P.:Enzymatic determination of total Ehnholm, C., Haglund, B., Huttunen, J. K. and serum cholesterol. Clin. Chem., 20:470(1974). Manninen, V.:Lack of serum Cholesterol-lowering 7) Foster, L. B. and Dunn, R. T.:Stable reagents for effect of skimmed milk and butter milk under the determination of serum triglycerides by a col- controlled conditions. Atherosclerosis, 39:267 orimetric Hantzsch condensarion method. Clin. (1981). Chem., 19:338(1973). 18) Joris, I. and Majno, G.:Cellular breakdown within 8) Eng, L. F. and Noble, E. P.:The maturation of the arterial wall:An ultrastructural study of the rat brain myelin. Lipid, 3:157(1968). coronary artery in young and aging rats. Virchow 9) Sarkar, B. and Chauham, U.:A new method for Archiv. A Path. Anat. and Histol., 364:111(1974). determination microquantities of calcium in bio- 19) Toda, T., Leszczynski, D. E., McGibbon, W. H. chemical materials. Anal. Biochem., 20:155(1967). and Kummerow, F. A.:Coronary arterial lesins 10) Toda, T., Ito, M., Toda, Y., Smith, T. and in sexually mature non-layers, layers, and roosters. Kummerow, F.:Angiotoxicity of a moderate Virchow Arch. A Path. Anat. and Histol. 388: excess of dietary vitamin D3 in swine. Fd. Chem. 123(1980). Toxic., 23:585(1985). 20) Kramsch, D. M., Aspen, A. J. and Aspen, C. S.: 11) Kamio, A., Kummerow, F. A. and Imai, H.: Suppression of experimental atherosclerosis by the Degeneration of aortic smooth muscle cells in swine Ca++-antagonist lanthanum. Possible role of cal- fed excess vitamin D3. Arch. Pathol. Lab. Med., cium in atherosclerosis. J. Clin. Invest. 65:967 101:378(1977). (1980).

Summary

Ninety seven 2 month-old wealing swine fed IU of vitamin D plus 100kg skimmed milk powder either a basal ration alone or the basal ration per ton of diet. The intima of these coronary containing different levels of vitamin D (100,000, arterial lesions contained activated smooth mus- 200,000 or 300,000 IU per ton diet) plus skimmed cle cells, degenerate smooth muscle cells without milk powder (50 or 100kg per ton diet) for 4 stainable lipid, and lipid-containing cells derived months. No significant changes were observed in from macrophages and smooth muscle cells. These plasma calcium, cholesterol, triglycerides or phos- results suggest that excess dietary vitamin D plus pholipid levels between the experimental groups skimmed milk powder has a synergistic effect on fed diets containing 200,000 IU of vitamin D per the development of coronary atherosclerosis. ton diet with or without skimmed milk powder. Marked coronary arterial lesions were found in the Key words:coronary artery, vitamin D, milk group which was fed the diet containing 300,000 powder, swine.

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