x^ 1 T'//. Division of Agricultural Sciences UNIVERSITY OF CALIFORNIA ^hiiQ t ' uL ^ I WESTERN x REGIONAL^ ^RESEARCH "^ PUBLICATION Editorial Committee: Ethefwyn Wilcox, Betty Hawthorne, Ruth Okey, Chairman CALIFORNIA AGRICULTURAL EXPERIMENT STATI O N BULLETIN 785 This is a short technical summary of cooperative re- search, regional project W-44, conducted under the aus- pices of the Agricultural Experiment Stations of the west- ern states — Arizona, California, Colorado, Hawaii, Idaho, Montana, New Mexico, Oregon, Utah, Washing- ton, and Wyoming — designed to develop additional in- formation on cholesterol metabolism. Specific contribu- tions are designated by name of the state only. The Cali- fornia Agricultural Experiment Station was selected to issue this publication which becomes also a publication of the other western Experiment Stations for distribution under the indicia of each. JUNE, 1962 Prepared by the Editorial Committee Betty E. Hawthorne, Oregon State College Ethelwyn Wilcox, Utah State College Ruth Okey, University of California, Berkeley, Chairman Western Regional Project W-44: The role of diet in cholesterol metabolism, and biological interrelationships in lipid metabolism of importance to man Administrative Adviser : » Dr. S. S. Wheeler, Director, Agricultural Experiment Station Colorado State University, Fort Collins, Colorado TECHNICAL COMMITTEE Dr. Ethel M. Thompson School of Home Economics Agricultural Experiment Station University of Arizona Tucson, Arizona Dr. Ruth Okey and Dr. R. L. Lyman Department of Nutritional Sciences Agricultural Experiment Station University of California Berkeley, California Dr. Adelia E. Weis Department of Home Economics Agricultural Experiment Station Colorado State University Fort Collins, Colorado Dr. Bluebelle R. Standahl Department of Home Economics and Nutrition Agricultural Experiment Station University of Hawaii Honolulu, Hawaii Miss Shirley V. Bring Home Economics Research Agricultural Experiment Station University of Idaho Moscow, Idaho Mrs. Helen L. Mayfield Department of Home Economics Agricultural Experiment Station Montana State College Bozeman, Montana Miss Edith M. Lantz Department of Home Economics Agricultural Experiment Station New Mexico State University University Park, New Mexico Dr. Betty E. Hawthorne Department of Home Economics Research Agricultural Experiment Station Oregon State University Corvallis, Oregon Dr. Ethelwyn B. Wilcox Department of Food and Nutrition Agricultural Experiment Station Utah State University Logan, Utah Dr. Mary Rose Gram Home Economics Research Agricultural Experiment Station Washington State University Pullman, Washington Dr. Callie Mae Coons Human Nutrition Research Division U. S. Department of Agriculture Washington, D.C. former members who contributed to this bulletin Dr. Irene Payne Division of Home Economics Agricultural Experiment Station University of Wyoming Laramie, Wyoming Dr. Lucile Adamson Department of Home Economics and Nutrition Agricultural Experiment Station University of Hawaii Honolulu, Hawaii A list of publications and of papers in preparation from the western laboratories is available from members of the W-44 Technical Committee in the various experiment stations. [3] CHOLESTEROL PROBLEMS A brie) review of the present concepts of cholesterol metabolism and an outline report of the W-44 projects of the Western Regional Experiment Stations and related cholesterol research elsewhere Cholesterol research has had the ad- vantage of continuing financial support because of wide publicity concerning the relation of cholesterol accumulation to the hardening and thickening of ath- erosclerotic arteries. The high proportion of deaths attributed to coronary occlusion and atherosclerotic changes in other blood vessels has nevertheless tended to result in pressure to develop practical means for preventing accumulation of cholesterol in blood and tissue, often at the expense of investigations of normal cholesterol metabolism or of the basic mechanisms involved in that accumulation. Research on cholesterol metabolism during the past fifteen years has been tremendous. A short report such as the one presented here can cover only a small part of it. Good reviews of some of the clinical problems may be found in recent literature, notably the symposia reported in the American Journal of Clinical Nu- trition (1960, 1961). The monograph edited by R. P. Cook (New York: Aca- demic Press, 1958) deals with the bio- chemistry of cholesterol. A brief outline of the comparatively well-established data pertinent to the W-44 project on cholesterol What cholesterol is Cholesterol is a white, waxy solid, sol- uble in fat and fat solvents. Chemically, it is a cyclic alcohol of relatively complex structure and high molecular weight (C 27 H 46 0). Where it is found Cholesterol has attracted the patholo- gist's attention because it is a constituent of the plaques, or thickened areas, found in the walls of atherosclerotic arteries. It is, however, also found in all tissues of vertebrates. The concentration in muscle is about 0.06 per cent; in liver it varies from about 0.2 to 0.5 per cent when an animal is fed a cholesterol-low diet, but the concentration may increase 10 to 20 times when cholesterol is fed. Human plasma averages between 160 and 250 mg per 100 ml, or approximately 0.16 to 0.25 per cent. Values are usually in- creased in hypothyroidism, diabetes, and other metabolic disorders. Brain and spinal cord contain about 3 per cent cho- lesterol; egg yolk, approximately 2 per cent; and butter, 0.3 per cent. Strangely enough, adipose tissue and animal fats, other than wool fat, contain very little. Lard contains less than 0.1 per cent. Cho- lesterol does not occur in plant tissues nor is it the chief sterol in those of in- vertebrates, such as crab, shrimp, oyster, etc. Sterols of similar, but not necessarily identical, structure have been found in all species investigated. Some of the related compounds found in plants have been shown to interfere with the absorp- tion of cholesterol from foods, and are, themselves, relatively poorly absorbed. Its functions The functions of cholesterol in the ani- mal body are not fully known. Evidence indicates that it is a precursor of steroid hormones elaborated in the adrenals and [4] the ovaries, and of vitamin D in the skin. As a constituent of cell membranes, cho- lesterol may affect the ease with which different nutrients enter cells. In nerve tissue it may limit the path of a given impulse. Recent findings indicate that plasma cholesterol may serve as a carrier of essential fatty acids. Plasma and liver cholesterol values drop sharply during some acute infections. White blood cells are very rich in cholesterol (Oregon). The above suggest a function in relation to resistance to infection. Apparently, therefore, cholesterol fulfills some useful purposes in the body. Where it comes from Cholesterol in animal tissues is derived either from diet or from tissue synthesis. Acetic acid, as well as other fatty acids of low molecular weight, serves as raw material. In other words, it can be made from metabolic breakdown products of carbohydrate, fat, or protein. The syn- thesis is a complex one which takes place in stages. Some of the more important in- termediaries are mevalonic acid, far- nesoic acid, squalene, and desmosterol. The liver both synthesizes cholesterol and breaks it down to bile acids, which it ex- cretes. However, most tissues apparently share the liver's capacity to synthesize cholesterol. Cholesterol synthesis has been studied with labeled isotopes, both in the intact animal and with surviving tissue slices. One particularly interesting find- ing is that liver slices taken following a fast have lost their capacity to make cho- lesterol from acetate. How it is absorbed and transported Cholesterol is fat-soluble and water- insoluble. It is more readily absorbed from the digestive tract when it is fed with fat. It is absorbed via the lymph, DEFINITION OF TERMS Atherosclerosis. Thickening of the walls of arteries with accumulation of lipid or fatty material, usually in definite areas or plaques. Sterol. A general term applied to solid alcohols having a ring structure similar to that of cholesterol. Characteristic sterols are found in the tissues of plants as well as animals. Cholesterol is the important sterol in tissues of vertebrates. Steroid. Having a structure similar to (but not identical with) a sterol. The term is applied to an important group of hormones secreted by the adrenals, the ovaries, the testes ; for example, cortisone, estrogens. The term is also sometimes applied to oxidation products of sterols, such as bile acids. Isotopes. Different forms of chemical elements. The term is often applied to a form of the element having special properties, such as radioactivity, which permit its measurement when only traces are present. Coronary occlusion. Blocking of circulation in the coronary artery. Often caused by impaction of a blood clot in an atherosclerotic blood vessel. Essential fatty acid. One required for normal nutrition and not synthesized in the body. The two most important are linoleic (furnished by vegetable oils) and arachidonic, which is found in lipids from animal sources. [5] rather than the portal circulation, that is, it is shunted around the liver, as are long-chain fatty acids. Nevertheless, the liver removes excess dietary cholesterol from the bloodstream. Feeding cholesterol, except in very large amounts, is more likely to result in increased cholesterol of the liver than of plasma. Different species of animals show great differences, however, in their ca- pacity to maintain plasma cholesterol within normal ranges. For example, it is more difficult to increase plasma choles- terol in a rat, by feeding it cholesterol, than in a rabbit. Except during the im- mediate period of absorption (2 to 4 hours) of a fat- and cholesterol- rich meal (scrambled eggs and brains, for in- stance), the plasma cholesterol level in man is altered comparatively little as a result of moderate increases in choles- terol intake. It has been shown that cholesterol synthesis, especially in liver slices, is decreased by cholesterol feeding; that is, rate of synthesis is affected by sup- ply. Changes in cholesterol values of any single individual must be interpreted in terms of starting values, time, and age of subject. Cholesterol in the animal body exists in two forms — "free," and "esterified" (combined with fatty acids). Rapidity of exchange in tissues is apparently in- creased by esterification. Only in plasma leucocytes (Oregon), in adrenal cortex, and in the livers of cholesterol-fed ani- mals, do we find a considerable percent- age of the cholesterol esterified. Both free and esterified cholesterol are usually in more or less loose combination with protein — such as the lipoproteins of plasma. In general, a large proportion of the plasma cholesterol is to be found in the heavier lipoprotein aggregates — those containing a higher percentage of protein — while the larger proportion of the true fat or triglyceride is in the lighter lipoprotein aggregates. The pres- ence of a high proportion of certain types of lipoproteins has been considered evi- dence of susceptibility to atherosclerosis by Gofman and his associates. Cholesterol and phospholipids Cholesterol in tissue is nearly always accompanied by phospholipids. These are compounds that can be synthesized in the body. They contain one or two fatty acid units in combination with phosphoric acid and, usually, a nitrogenous base. They are, essentially, molecules which, unlike cholesterol, tend to orient them- selves in oil-water boundaries, with the fatty acids toward the oil and the highly polar base toward the aqueous solution. Lecithin, the chief phospholipid of liver, contains the base choline, which is rated as lipotropic, that is, when fed it pre- vents the accumulation of excess liver fat and cholesterol. Consequently, a great deal of attention has been devoted to cho- lesterol :lecithin ratios in assessing the effects of diet on tissue composition. Gen- erally, the ratio increases with age or in- activity of a tissue. Phospholipids as well as cholesterol are found in lipoprotein aggregates. Cholesterol and essential fatty acids Fatty acids are classified as "satu- rated," or filled to capacity with hydro- gen, and "unsaturated," or having the capacity to take up 2 or more additional hydrogen atoms per molecule. A mono- unsaturated acid, such as palmitoleic or oleic, has one carbon-carbon double bond (C = C), and can take up 2 more hydrogens; a dienoic, such as linoleic, has two carbon-carbon double bonds (-C = C-C-C = C), and can take up 4 atoms of hydrogen, and so forth. The term "polyunsaturated" is used popu- larly to designate any fatty acid that has more than one "double bond," or can take up 4 or more hydrogen atoms per molecule. Certain unsaturated fatty acids cannot be made in the body and must be sup- [6] plied by diet. Phospholipids and choles- teryl esters carry a large proportion of the essential fatty acids in tissue, espe- cially when the supply in the diet is lim- ited. Linoleic acid (a C 18 acid with two double bonds) is the chief dietary source of essential fatty acid. There is reason to believe, however, that the "working" es- sential fatty acid is arachidonic (a C 20 fatty acid with 4 double bonds) , and that it is elaborated, in tissue, from linoleic acid. It is interesting that linoleic acid occurs in comparatively high percent- ages in vegetable oils and in the adipose tissue fat of animals fed linoleate-rich fat, while arachidonic acid is found only in trace amounts except in cholesteryl ester and phospholipid of animal tissue (California) . Arachidonic acid is easily oxidized, and foods containing it deterio- rate rapidly. The percentages of arachi- donic acid in cholesteryl ester vary both with diet and with species of animal. Swell and Treadwell, at George Washing- ton University Medical School, have pos- tulated that an animal's tendency to develop atherosclerosis is inversely pro- portional to the usual percentage of ar- achidonic acid in that animal's plasma cholesteryl ester. On the other hand, high percentages of saturated and moneonoic acids in cholesteryl ester have been taken as indications of ease of development of atherosclerotic lesions. Work partially supported by Western Regional Project W-44 This work has been directed primarily toward increased understanding of the factors influencing metabolism of choles- terol, rather than the pathology of athero- sclerosis. While studies with human sub- jects have been emphasized, certain aspects of the problems, for example, changes in tissue composition, have ob- viously had to be studied with experi- mental animals. Cost of human studies has also been a serious limiting factor. Some of the findings from the more pro- ductive areas of investigation, both in the Western Region and elsewhere, are outlined below. Stations contributing spe- cific research are indicated in paren- theses. Effect of ethnic origin, climate, and altitude The Western Region includes peoples of many ethnic groups. Its areas of des- ert, mountains, and coastal plains were considered to offer excellent opportuni- ties for study of the effects of climate and altitude. Ethnic origin, per se, did not appear to influence serum cholesterol values significantly (subjects studied in Hawaii, Thailand, and Manila [Ha- waii] ) . The low values observed in Navajo and Papago Indian and Spanish- American children (Utah, Arizona, and New Mexico) may very well have been related to diet. It was interesting that no correlation of serum cholesterol with changes in temperature was found in studies with nurses and postmen in desert heat (Arizona). Older women living at altitudes of 7,000 feet (Wyoming) were found to have higher serum cholesterol values than did a group of older women at sea level (California, W-4) . Regulation of total food intake The literature indicates that continued gross overfeeding may be conducive to development of coronary lesions, but this result may have been due indirectly to lack of muscular development and the burden of carrying excess fat. Increased caloric intakes compensated for by exer- cise did not significantly affect the serum cholesterol values in university athletes [7] (Utah) and in postmen (Arizona). Nu- merous data indicate that moderate limi- tation of food intake and moderate exer- cise may be expected to be conducive to normal cholesterol metabolism. Overnight fasting lowered serum cho- lesterol values in rats. While liver size was decreased, the percentages of liver lipid and cholesterol often increased, pos- sibly because the loss of weight was due to a loss of water and glycogen (Mon- tana, California) . A number of investigations have shown that time limitation of access to food ("meal feeding") in experimental ani- mals may, in female rats fed cholesterol, increase the level of plasma cholesterol (California) . Investigators elsewhere have suggested that "meal feeding" stim- ulates cholesterol synthesis. The possi- bility that ingestion of a large meal merely prolongs post-absorptive increases in the serum cholesterol values cannot, however, be ignored. The beneficial ef- fects of more uniform distribution of total food intake throughout the whole day have been emphasized by investi- gators at Chicago. Feeding of specific substances The influence of protein intake on cho- lesterol metabolism has been widely in- vestigated. The histo-pathologists have found evidence that the initial lesions in arteriosclerosis are in the protein walls of the arteries, and that these lesions may be found even in very young children. These workers have suggested that the subsequent deposition of fatty material is secondary to those early lesions. Ac- cumulating evidence indicates that the quality of protein eaten may be im- portant at all stages of development of lesions. Investigations point to the im- portance of amino acid balance. High intakes of certain amino acids, such as cystine (California), or low intakes of some amino acids, such as threonine (work in other regions), have been shown to produce fatty livers in experi- mental animals. On the other hand, methionine, probably because it is a source of labile methyl, tends to prevent lipid accumulation in liver (Arizona, California) . Protein intakes that are too low to support normal growth have been 4 shown to produce fatty livers in young rats (California) . Results favor an ade- ■ quate, but not excessively high intake of protein of good quality. Feeding cholesterol has resulted in a decrease in the amount of cholesterol synthesized by experimental animals. * Normally, excess cholesterol absorbed from the digestive tract leads to accumu- lation of cholesteryl ester in the livers of animals rather than to any large in- crease in plasma cholesterol. However, different species show different responses to cholesterol feeding. Many laboratories elsewhere have fed excess cholesterol with i thiouracil (which interferes with thyroid function), and with cholic acid (which increases cholesterol absorption), and have so produced high serum cholesterol values in even the most resistant species of animal, such as the rat. There is some question whether results secured by this procedure are comparable with those ob- served when cholesterol is fed to man. The effects of feeding fats of different composition at various levels have been investigated. Fats rich in linoleic acid, such as safflower, corn, cottonseed, soy, \ and peanut oils, evidently have some ef- fect in lowering serum cholesterol levels in human subjects (California, Utah) . Feeding these same fats in excessive quantities to laboratory animals resulted in the development of fatty livers (Utah, Idaho, Montana, California) . When mod- erate amounts of the highly unsaturated fats were fed with cholesterol, the amounts of cholesteryl ester in the result- ing fatty livers were significantly greater than in livers of animals fed otherwise identical diets, which contained butter or hydrogenated shortening or coconut oil (California) . Coconut oil consists largely of saturated fat, chiefly laurate [8] and myristate, while butter is rich in sat- urated acids of very low molecular weight, but also contains a large percent- age of oleic acid. Species and age differ- ences in the effects of feeding butter are especially marked. The polyunsaturated fats combine very readily with oxygen and thus become rancid. Oxidative deterioration may take place during storage, in the digestive tract, or in tissue, following absorption. The presence of such oxidation products is characteristic of aging and atheroma- tous tissue. The quantities and kinds of natural antioxidants in food fats are con- sequently important. It is especially in- teresting that substances closely resem- bling vitamin E, but not identical with it, have proved the more potent antioxidants outside the body and in the digestive tract, while vitamin E per se (a-toco- pherol) is the better antioxidant in tis- sue. The effectiveness of both types of an- tioxidant may be greatly altered by other substances normally present in foods or tissues, for example, amino acids, en- zymes, etc. It is therefore not easy to pre- dict the stability of a polyunsaturated fat. Caution in advising the use of large amounts of highly unsaturated fat in the diet therefore seems desirable. (See re- view of M. K. Horwitt, Amer. Jour. Clin. Nutrition, 8:451, 1960). Effect of vitamins and minerals The effects of vitamin A on serum cho- lesterol in human subjects have not been consistent, probably because of differ- ences in experimental procedure. In rats given very high dosages of vitamin A with cholesterol, lowering of serum and liver cholesterol was observed (Idaho) . Vitamin D effects on cholesterol me- tabolism were of special interest because of the wide differences in altitude and ultraviolet radiation in the areas covered by the states participating in the W-44 project. However, no significant effects could be demonstrated in the animal study carried out in Colorado. A negative correlation between ascor- bic acid intake and mortality from heart disease shown in nutritional status studies with older people (W-4) pointed to the desirability of further study of ascorbic acid in relation to fat and cho- lesterol metabolism. No significant cor- relations between serum ascorbic acid and cholesterol concentrations were found for six women on a constant diet [Oregon) ; lowering the ascorbic acid intake resulted in a significant decrease in serum cholesterol concentration for one adult woman (Arizona), but no con- sistent change among twelve adolescent girls (Washington) . Attention has been drawn to riboflavin since the flavin-containing enzyme sys- tems are important in the intermediary metabolism of fat. Supplementary ribo- flavin reduced the usual effect of a high fat intake in producing an increased con- centration of liver cholesterol in rats, but resulted in higher concentrations of serum cholesterol; increasing fat intakes also tended to decrease the concentration of riboflavin-containing coenzymes in both serum and liver of rats (Colorado) . No correlation of fasting serum choles- terol to serum riboflavin concentrations was found for six women on controlled diets (Oregon) . A test dose of riboflavin, which increased concentrations of ribo- flavin-containing coenzymes in serum, red cells, and white cell-platelet fractions of blood, resulted in no change in serum or red cell cholesterol, and in inconsistent changes in the white cells and platelets of human subjects (Oregon) . Rats on biotin-deficient diets lost ca- pacity to incorporate labeled acetate into liver lipids but not into liver glycogen (California) . Pyridoxine has been studied as a pos- sible requirement for synthesis of arachi- donic acid from linoleic acid {Cali- fornia) . Choline, like other sources of labile [9] methyl, tended to prevent the accumula- tion of fat and cholesterol in liver (Mon- tana, California, and elsewhere) . In lay- ing hens, choline lowered serum choles- terol concentrations and reduced choles- terol deposits in the aorta (Arizona) . Mineral metabolism, especially the me- tabolism of calcium, magnesium, and iron, has been considered as possibly in- terrelated with the functioning of choles- terol in the animal body. Higher serum cholesterols were associated with the high hemoglobin values observed at 7,200 feet (Wyoming) . Effect of hormones Deficient thyroid secretion usually re- sults in high serum cholesterol values. Administration of thyroid lowers serum cholesterol. However, studies with nor- mal student nurses, and with postmen, during a hot summer, showed no correla- tion between the lowered PBI (protein- bound iodine) and basal metabolism and serum cholesterol values (Arizona). Administration of estrogenic hormone to intact and castrate, cholesterol-fed rats increased serum and liver cholesterol values (California) . Further work needed In many cases, a study planned to deal with one problem has inevitably raised additional questions. No attempt has been made in this report to outline the work on methods, which has made it possible to carry out the studies of cho- lesterol and lipid metabolism. New Mex- ico, Oregon, Utah, and Hawaii have de- veloped and/or published micro proce- dures for cholesterol determinations for serum, blood cell fractions, and livers. California has devoted considerable time to chromatographic separation of lipids and fatty acids. Indeed, every laboratory in the Western Region has given much attention to laboratory techniques. As this report indicates, much work is still to be done before full understanding of the functions of cholesterol and their relationships to the metabolism of fats and other lipids is achieved. Research in the laboratories of the Western Region, as well as in those of other parts of the world, has produced conflicting evi- dence. Recommendations for very drastic changes in the American diet are hardly warranted by our present knowledge of cholesterol metabolism. However, evi- dence exists for encouragement of regular and moderate exercise and for the main- tenance of a desirable body weight. Also, there is evidence to indicate that mod- eration in the choice and amount of fat eaten is desirable, just as are moderation in total food intake and maintenance of an adequate and balanced diet. 101 15m-6,'62(C8533)LL