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Fat transport by lipoproteins in health and disease
Journal of A therosclerosis Research Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Editorial FAT T R A N S P O R T BY L I P O P R O T E I N S IN H E A L T H AND DISEASE Fats are ubiquitous components of cells and tissues. But they are only sparingly, if at all, soluble in aqueous media. Their transport and transfer between sites in the body are therefore dependent upon combination with carrier proteins which confer the necessary solubility to allow their distribution in body fluids. The varying proportion of lipid to protein in the major forms allows their separation and characterization in terms of density, and therefore of flotation characteristics, in media of defined density, especially by ultracentrifugation. In the analytical ultracentrifuge, these characteristics can be given expression as sedimentationflotation (Ss) values. The term "chylomicron" was originally coined to designate the visible lipid particle transporting triglyceride from the gut to the blood via the chyle. The expression came to be widened to include all visible particulate lipid in the blood. In recent years it has come to be recognized that endogenous lipid is also transported in particulate form. It has been shown that these "secondary particles" can be separated from true chylomicra containing exogenous lipid, and from low-density lipoproteins, by the respective behaviour of each in a polyvinyl pyrrolidone gradient 1. Both kinds of visible particle contain relatively small amounts of protein (about 1-2 % by weight). Their relatively high lipid content results in a density of about 0.93 g/ml and fast sedimentation-flotation characteristics. Secondary particles have S! values of about 400, while chylomicra have values from 1,000 to 10,000 (ref.2). Low-density lipoproteins vary in density between 0.96 and 1.03 g/ml and in protein content between 8 and 21 per cent 3. They show a continuum of SI values between 3 and about 100 in health, extending perhaps to 200 or more in some hyperlipidaemias. But there is unequal distribution of S! values over the continuum with maxima in the region of S! 3-9 and S! > 20. The g(S) distribution within these limits has been examined and the varying composition of the lipids associated with broad sub-classes (S! 3-9, 10-20 and 20-100) has been described s. Many authors refer to the S! 20-100 sub-class separately as "very low-density lipoproteins". But it has been shown that the protein moiety is antigenically identical throughout the L.D.L. 4. High-density lipoproteins contain about 50 % protein or more and vary in density between 1.08 and 1.21. It seems probable that there is again a continuum varying in lipid composition but characterised by a single protein moiety which is different from that of L.D.L. 5. In addition to triglycerides, cholesterol and phospholipids, the low-density and high-density lipoproteins are responsible for the transport of fat-soluble provitamins and vitamins such as the carotenoids and tocopherols 4.
j. Atheroscler. Res., 7 (1967) 533-536
534
K.W. WALTON
Information about the functional significance of lipoproteins has accumulated in recent years with studies of (a) rare individuals showing congenital deficiencies of these proteins; (b) variations in lipoprotein serum concentrations and turnover in a variety of pathological conditions; and (c) careful metabolic and genetic studies of essential hyperlipidaemias. In some of these studies crucial information has been obtained by the use of isotopic tracer techniques. For example, the use of radioactive isotopes has shown that the fatty-acids, cholesterol and phosphatides exchange between L.D.L. and H.D.L. and also between these lipoproteins and cells (for references see ref.2). The turnover of these components is thus independent of that of the carrier proteins. Cholesterol, for instance, has been shown to have a biological half-life in man of 53-62 days 6 while that of L.D.L. is only 2-3.5 days 7. The relatively constant lipid composition of the lipoproteins in health, therefore, presumably reflects an equilibrium between the affinities of the carrier-proteins for the various lipids. This equilibrium can evidently be displaced, as, for example, in biliary cirrhosis in which the characteristically abnormal lipid compositions of L.D.L. and FLD.L. may reflect a disequilibrium brought about by the "dammingback" in the blood of cholesterol and phospholipid because of blocking of their final excretory pathway in the bile. The serum concentration of L.D.L., even in health, varies with age and sex, and in females is affected by pregnancy, suggesting unusual sensibility to hormonal influences. This is also reflected in differences between the sexes in the catabolism of L.D.L. and more marked sensitivity than other serum proteins to the output of thyroid hormone 8. In disease, altered concentration of serum lipids is almost invariably reflected in alterations of L.D.L. levels, but only rarely in alterations of H.D.L. levels. A valuable review of the significance of these variations in serum lipoproteins has been presented in a recent series of papers by FREDERICKSON et al. 9. The first part outlines the normal tasks of fat transport, supplementing information from orthodox physiological sources about the role of lipoproteins, with that obtained by this group's unique access to examples of "Nature's experiments" (i.e., cases of inherited lipoprotein deficiency states). For example, a study of cases of abetalipoproteinaemia (congenital deficiency of L.D.L.) makes it clear that the absorption of exogenous lipid in the form of chylomicra is dependent upon an ability to synthesize L.D.L.; whereas individuals with Tangier Disease (congenital deficiency of H.D.L.) can absorb exogenous lipid normally but, in the absence of H.D.L., develop a curious form of thesaurosis with storage of lipid in reticulo-endothelial tissues. The second part of the review, dealing with the immunochemistry of the lipoproteins, is less satisfactory and may prove confusing to the general reader. In particular, the acceptance of a "new" lipoprotein characterized in terms of its apoprotein, C, overlooks the possibility that this protein may originate from "platelet dust ''1~ rather than be a true circulating transport protein. The concept of a "pre-fllipoprotein complex", consisting of both L.D.L. and H.D.L., is also one which would not be universally acceptable. j . Atheroscler. Res., 7 (1967) 533-536
FAT TRANSPORT BY LIPOPROTEINS
IN H E A L T H A N D D I S E A S E
535
Arising from this work, the authors have proposed a system for the reclassification of the hyperlipidaemias. The system is based primarily upon patterns obtained by paper-electrophoresis of serum and staining of the strips with Oil-Red O, but is supplemented by lipid analysis, ultracentrifugation and other techniques. It is suggested that the patterns obtained allow differentiation into five Types, each of which may occur as a Primary form (inheritable) or as a Secondary form (as an expression of altered metabolism due to some other recognizable disease). Type I in its primary form corresponds to the rare "familial fat-induced hyperlipaemia" or "persistent exogenous hyperlipaemia" of other authors. As the older terms imply this Type describes individuals who show a prolonged hyperchylomicronaemia on diets containing fat. Whether Type I occurs in secondary form is uncertain. Type II (primary) corresponds to "essential hypercholesterolaemia", with predominant increase of S 13-9 L.D.L. Type II (secondary) is the similar pattern, commonly encountered in hypothyroidism, the nephrotic syndrome and certain other conditions. Type III is characterized by an increase of S I 12-200 L.D.L. without accompanying chylomicronaemia; Type IV by a similar alteration of L.D.L. accompanied by the presence of "secondary particles" (endogenous hyperlipaemia) and Type V by the additional presence of exogenous chylomicra. These types therefore represent new subdivisions of what have hitherto been referred to as essential, or secondary, hyperlipaemias. Although this represents a praiseworthy attempt to rationalize the investigation and understanding of this group of diseases, a word of caution is necessary to those intending to apply the scheme. This is to re-emphasize a point made by these authors-namely, that no single test or manoeuvre is sufficient, by itself, to sort out these cases. While paper electrophoresis may serve as a useful preliminary screening-procedure, it is not a complete substitute for other analytical and physico-chemical procedures in the categorization of the hyperlipidaemias. Another point worth stressing, in relation to the special interest of readers of this journal, is that the altered patterns of lipid-staining upon which these Types are primarily based evidently reflect other examples of disequilibria of distribution of lipids among their carrier proteins. But, in many cases, regardless of the pattern of lipid alteration, there is a marked association between high total serum concentrations of L.D.L., the occurrence of xanthomata and an unusually high incidence of atherosclerosis in almost all varieties of hyperlipidaemia. Metabolic studies in the hyperlipidaemias (whether primary or secondary) have shown an increase of the total exchangeable pool of L.D.L., reflected in increases of both intravascular and extravascular pools, regardless of the mechanism of alteration of metabolism of the protein moiety. Both indirect and direct evidence suggest that the increased incidence of atherosclerosis and the occurrence of xanthomatosis are expressions of this enlarged extravascular distribution in the form of accumulation of L.D.L. in arterial walls and subcutaneous tissues respectivelyll, 12. If this is so, there is added point to the need for accurate delineation of the particular variety of hyperlipidaemia from which a given patient is suffering, since J. Atheroscler. Res., 7 (1967) 533-536
536
K . W . WALTON
t h i s will d e t e r m i n e t h e m o s t a p p r o p r i a t e i o r m of t r e a t m e n t . R e d u c t i o n of s e r u m l i p i d s and lipoproteins must therefore not be looked upon as an academic exercise in rectif y i n g a b i o c h e m i c a l a b n o r m a l i t y , b u t r a t h e r a s a m e a n s of p r o p h y l a x i s a n d t r e a t m e n t of a t h e r o s c l e r o s i s .
Department of Experimental Pathology, University of Birmingham, Birmingham 15 (Groat Britain)
K. W. W A L T O N
REFERENCES 1 BIERMAN, E. L., D. PORTE, D. D. O'HARA, M. SCHWARTZ AND F. C. WOOD, Characterization
of fat particles in plasma of hyperlipaemic subjects maintained on fat-free high-carbohydrate diets. J. din. Invest., 1965, 44: 261. 2 CORNWELL, D. G., Lipoproteins. In G. SCHETTLER (Ed.), Lipids and Lipidoses, SpringerVerlag, Berlin, 1967, p. 168. 30NCLEY, J. L., K. W. WALTON AND D. G. CORNWELL, A rapid method for the bulk isolation of /~-lipoproteins from h u m a n plasma, J. amer. chem. Soc., 1957, 79: 4666. 4 WALTON, K. W. AND S. J. DARKE, Immunochemical characteristics of h u m a n low-density lipoproteins, Immunoehem., 1964, 1: 267. 5 LEVY, R. I. AND n . S. FREDERICKSON, Heterogeneity of plasma high density lipoproteins, J. clin. Invest., 1965, 44: 426. 6 LEWIS, B. AND N. B. MYANT, Studies in t h e metabolism of cholesterol in subjects with normal cholesterol levels and in patients with essential hypercholesterolaemia, Clin. Sci., 1967, 32:201. 7 WALTON, K. W., P. J. SCOTT, J. VERRIER JONES, R. F. FLETCHER AND T. P. WHITEHEAD, Studies on low-density lipoprotein turnover in relation to Atromid therapy, J. Atheroscler. Res., 1963, 3: 396. 8 WALTON, K. W., P. J. SCOTT, P. W. DYKES AND J. W. L. DAVIES, Alterations of metabolism and turnover of 131I-low-density lipoproteins in hypothyroidism and thyrotoxicosis, Clin. Sci., 1965, 29: 217. 9 Fm~DERICKSON, D. S., R. I. LEVY AND R. S. LEES, F a t transport in lipoproteins. An integrated. aplSroach to mechanisms and disorders, New Engl. J. Med., 1967, 276: 32, 94, 148, 215, 273. x0 WOLF, P., The nature and significance of platelet products in h u m a n plasma, Brit. J. Haematol., 1967, 13: 269. 11 SCOTT, P. J. AND C. WINTERBOURN, Low-density lipoprotein accumulation in actively growing xanthomas, J. Atheroscler. Res., 1967, 7: 207. in WAr,TON, K. W., Lipoproteins in t h e vessel wall, Arch. Mal. Cwur, 1966, 8: Suppl. 2 (Revue de l'Ath~roscl~rose), p. 41.
J. Atheroscler. Res., 7 (1967) 533-536
Editorial FAT T R A N S P O R T BY L I P O P R O T E I N S IN H E A L T H AND DISEASE Fats are ubiquitous components of cells and tissues. But they are only sparingly, if at all, soluble in aqueous media. Their transport and transfer between sites in the body are therefore dependent upon combination with carrier proteins which confer the necessary solubility to allow their distribution in body fluids. The varying proportion of lipid to protein in the major forms allows their separation and characterization in terms of density, and therefore of flotation characteristics, in media of defined density, especially by ultracentrifugation. In the analytical ultracentrifuge, these characteristics can be given expression as sedimentationflotation (Ss) values. The term "chylomicron" was originally coined to designate the visible lipid particle transporting triglyceride from the gut to the blood via the chyle. The expression came to be widened to include all visible particulate lipid in the blood. In recent years it has come to be recognized that endogenous lipid is also transported in particulate form. It has been shown that these "secondary particles" can be separated from true chylomicra containing exogenous lipid, and from low-density lipoproteins, by the respective behaviour of each in a polyvinyl pyrrolidone gradient 1. Both kinds of visible particle contain relatively small amounts of protein (about 1-2 % by weight). Their relatively high lipid content results in a density of about 0.93 g/ml and fast sedimentation-flotation characteristics. Secondary particles have S! values of about 400, while chylomicra have values from 1,000 to 10,000 (ref.2). Low-density lipoproteins vary in density between 0.96 and 1.03 g/ml and in protein content between 8 and 21 per cent 3. They show a continuum of SI values between 3 and about 100 in health, extending perhaps to 200 or more in some hyperlipidaemias. But there is unequal distribution of S! values over the continuum with maxima in the region of S! 3-9 and S! > 20. The g(S) distribution within these limits has been examined and the varying composition of the lipids associated with broad sub-classes (S! 3-9, 10-20 and 20-100) has been described s. Many authors refer to the S! 20-100 sub-class separately as "very low-density lipoproteins". But it has been shown that the protein moiety is antigenically identical throughout the L.D.L. 4. High-density lipoproteins contain about 50 % protein or more and vary in density between 1.08 and 1.21. It seems probable that there is again a continuum varying in lipid composition but characterised by a single protein moiety which is different from that of L.D.L. 5. In addition to triglycerides, cholesterol and phospholipids, the low-density and high-density lipoproteins are responsible for the transport of fat-soluble provitamins and vitamins such as the carotenoids and tocopherols 4.
j. Atheroscler. Res., 7 (1967) 533-536
534
K.W. WALTON
Information about the functional significance of lipoproteins has accumulated in recent years with studies of (a) rare individuals showing congenital deficiencies of these proteins; (b) variations in lipoprotein serum concentrations and turnover in a variety of pathological conditions; and (c) careful metabolic and genetic studies of essential hyperlipidaemias. In some of these studies crucial information has been obtained by the use of isotopic tracer techniques. For example, the use of radioactive isotopes has shown that the fatty-acids, cholesterol and phosphatides exchange between L.D.L. and H.D.L. and also between these lipoproteins and cells (for references see ref.2). The turnover of these components is thus independent of that of the carrier proteins. Cholesterol, for instance, has been shown to have a biological half-life in man of 53-62 days 6 while that of L.D.L. is only 2-3.5 days 7. The relatively constant lipid composition of the lipoproteins in health, therefore, presumably reflects an equilibrium between the affinities of the carrier-proteins for the various lipids. This equilibrium can evidently be displaced, as, for example, in biliary cirrhosis in which the characteristically abnormal lipid compositions of L.D.L. and FLD.L. may reflect a disequilibrium brought about by the "dammingback" in the blood of cholesterol and phospholipid because of blocking of their final excretory pathway in the bile. The serum concentration of L.D.L., even in health, varies with age and sex, and in females is affected by pregnancy, suggesting unusual sensibility to hormonal influences. This is also reflected in differences between the sexes in the catabolism of L.D.L. and more marked sensitivity than other serum proteins to the output of thyroid hormone 8. In disease, altered concentration of serum lipids is almost invariably reflected in alterations of L.D.L. levels, but only rarely in alterations of H.D.L. levels. A valuable review of the significance of these variations in serum lipoproteins has been presented in a recent series of papers by FREDERICKSON et al. 9. The first part outlines the normal tasks of fat transport, supplementing information from orthodox physiological sources about the role of lipoproteins, with that obtained by this group's unique access to examples of "Nature's experiments" (i.e., cases of inherited lipoprotein deficiency states). For example, a study of cases of abetalipoproteinaemia (congenital deficiency of L.D.L.) makes it clear that the absorption of exogenous lipid in the form of chylomicra is dependent upon an ability to synthesize L.D.L.; whereas individuals with Tangier Disease (congenital deficiency of H.D.L.) can absorb exogenous lipid normally but, in the absence of H.D.L., develop a curious form of thesaurosis with storage of lipid in reticulo-endothelial tissues. The second part of the review, dealing with the immunochemistry of the lipoproteins, is less satisfactory and may prove confusing to the general reader. In particular, the acceptance of a "new" lipoprotein characterized in terms of its apoprotein, C, overlooks the possibility that this protein may originate from "platelet dust ''1~ rather than be a true circulating transport protein. The concept of a "pre-fllipoprotein complex", consisting of both L.D.L. and H.D.L., is also one which would not be universally acceptable. j . Atheroscler. Res., 7 (1967) 533-536
FAT TRANSPORT BY LIPOPROTEINS
IN H E A L T H A N D D I S E A S E
535
Arising from this work, the authors have proposed a system for the reclassification of the hyperlipidaemias. The system is based primarily upon patterns obtained by paper-electrophoresis of serum and staining of the strips with Oil-Red O, but is supplemented by lipid analysis, ultracentrifugation and other techniques. It is suggested that the patterns obtained allow differentiation into five Types, each of which may occur as a Primary form (inheritable) or as a Secondary form (as an expression of altered metabolism due to some other recognizable disease). Type I in its primary form corresponds to the rare "familial fat-induced hyperlipaemia" or "persistent exogenous hyperlipaemia" of other authors. As the older terms imply this Type describes individuals who show a prolonged hyperchylomicronaemia on diets containing fat. Whether Type I occurs in secondary form is uncertain. Type II (primary) corresponds to "essential hypercholesterolaemia", with predominant increase of S 13-9 L.D.L. Type II (secondary) is the similar pattern, commonly encountered in hypothyroidism, the nephrotic syndrome and certain other conditions. Type III is characterized by an increase of S I 12-200 L.D.L. without accompanying chylomicronaemia; Type IV by a similar alteration of L.D.L. accompanied by the presence of "secondary particles" (endogenous hyperlipaemia) and Type V by the additional presence of exogenous chylomicra. These types therefore represent new subdivisions of what have hitherto been referred to as essential, or secondary, hyperlipaemias. Although this represents a praiseworthy attempt to rationalize the investigation and understanding of this group of diseases, a word of caution is necessary to those intending to apply the scheme. This is to re-emphasize a point made by these authors-namely, that no single test or manoeuvre is sufficient, by itself, to sort out these cases. While paper electrophoresis may serve as a useful preliminary screening-procedure, it is not a complete substitute for other analytical and physico-chemical procedures in the categorization of the hyperlipidaemias. Another point worth stressing, in relation to the special interest of readers of this journal, is that the altered patterns of lipid-staining upon which these Types are primarily based evidently reflect other examples of disequilibria of distribution of lipids among their carrier proteins. But, in many cases, regardless of the pattern of lipid alteration, there is a marked association between high total serum concentrations of L.D.L., the occurrence of xanthomata and an unusually high incidence of atherosclerosis in almost all varieties of hyperlipidaemia. Metabolic studies in the hyperlipidaemias (whether primary or secondary) have shown an increase of the total exchangeable pool of L.D.L., reflected in increases of both intravascular and extravascular pools, regardless of the mechanism of alteration of metabolism of the protein moiety. Both indirect and direct evidence suggest that the increased incidence of atherosclerosis and the occurrence of xanthomatosis are expressions of this enlarged extravascular distribution in the form of accumulation of L.D.L. in arterial walls and subcutaneous tissues respectivelyll, 12. If this is so, there is added point to the need for accurate delineation of the particular variety of hyperlipidaemia from which a given patient is suffering, since J. Atheroscler. Res., 7 (1967) 533-536
536
K . W . WALTON
t h i s will d e t e r m i n e t h e m o s t a p p r o p r i a t e i o r m of t r e a t m e n t . R e d u c t i o n of s e r u m l i p i d s and lipoproteins must therefore not be looked upon as an academic exercise in rectif y i n g a b i o c h e m i c a l a b n o r m a l i t y , b u t r a t h e r a s a m e a n s of p r o p h y l a x i s a n d t r e a t m e n t of a t h e r o s c l e r o s i s .
Department of Experimental Pathology, University of Birmingham, Birmingham 15 (Groat Britain)
K. W. W A L T O N
REFERENCES 1 BIERMAN, E. L., D. PORTE, D. D. O'HARA, M. SCHWARTZ AND F. C. WOOD, Characterization
of fat particles in plasma of hyperlipaemic subjects maintained on fat-free high-carbohydrate diets. J. din. Invest., 1965, 44: 261. 2 CORNWELL, D. G., Lipoproteins. In G. SCHETTLER (Ed.), Lipids and Lipidoses, SpringerVerlag, Berlin, 1967, p. 168. 30NCLEY, J. L., K. W. WALTON AND D. G. CORNWELL, A rapid method for the bulk isolation of /~-lipoproteins from h u m a n plasma, J. amer. chem. Soc., 1957, 79: 4666. 4 WALTON, K. W. AND S. J. DARKE, Immunochemical characteristics of h u m a n low-density lipoproteins, Immunoehem., 1964, 1: 267. 5 LEVY, R. I. AND n . S. FREDERICKSON, Heterogeneity of plasma high density lipoproteins, J. clin. Invest., 1965, 44: 426. 6 LEWIS, B. AND N. B. MYANT, Studies in t h e metabolism of cholesterol in subjects with normal cholesterol levels and in patients with essential hypercholesterolaemia, Clin. Sci., 1967, 32:201. 7 WALTON, K. W., P. J. SCOTT, J. VERRIER JONES, R. F. FLETCHER AND T. P. WHITEHEAD, Studies on low-density lipoprotein turnover in relation to Atromid therapy, J. Atheroscler. Res., 1963, 3: 396. 8 WALTON, K. W., P. J. SCOTT, P. W. DYKES AND J. W. L. DAVIES, Alterations of metabolism and turnover of 131I-low-density lipoproteins in hypothyroidism and thyrotoxicosis, Clin. Sci., 1965, 29: 217. 9 Fm~DERICKSON, D. S., R. I. LEVY AND R. S. LEES, F a t transport in lipoproteins. An integrated. aplSroach to mechanisms and disorders, New Engl. J. Med., 1967, 276: 32, 94, 148, 215, 273. x0 WOLF, P., The nature and significance of platelet products in h u m a n plasma, Brit. J. Haematol., 1967, 13: 269. 11 SCOTT, P. J. AND C. WINTERBOURN, Low-density lipoprotein accumulation in actively growing xanthomas, J. Atheroscler. Res., 1967, 7: 207. in WAr,TON, K. W., Lipoproteins in t h e vessel wall, Arch. Mal. Cwur, 1966, 8: Suppl. 2 (Revue de l'Ath~roscl~rose), p. 41.
J. Atheroscler. Res., 7 (1967) 533-536