930
F. T. LINDGREN, A. V. NICHOLS AND N. K. FREEMAN
VOl. 59
PHYSICAL AND CHEMICAL COMPOSITION STUDIES ON THE LIPOPROTEINS OF FASTING AND HEPARINIZED HUMAN SERA’ BY FRANK T. LINDGREN, ALEXV. NICHOLS^
AND
NORMAN K. FREEMAN
Donner Laboratory of Medical Physica, Division of Medical Physics, Department of Physics and the Radiation Laboratory, University of California, Berkeley, California Received February 66,1966
Chemical studies of broad band lipoproteins isolated from fasting sera show wide ranges of lipid composition for different classes of lipoproteins, but establish relative constancy of lipid composition for lipoproteins of the same class obtained from different individuals in both health and certain disease states. Moreover, chemical studies of narrow band Sf4-8 lipoproteins show no significant differences in lipid content when compared with the composition of broad band Sf0-20 lipoproteins. Following intravenous heparin injection, lipid compositions of the various lipoprotein classes show some changes in unesterified cholesterol and unesterified fatty acid content. Also, nearly one-fifth of the total serum lipids are shifted, after heparin injection, into associations with proteins present in the total ultracentrifugal “residue.” Studies involving the in vitro incubation of Sf 20-400 lipoproteins with “active fractions” obtained from heparinized plasma show similar, if not identical, lipoprotein transformations as those found to occur in vivo.
Introduction Although not directly achievable, it would be desirable to know the complete chemical composition of each lipoprotein as it exists in the circulating blood stream. However, even though some alterations in the lipoproteins may occur as a consequence to the processes of blood withdrawal, serum preparation, salt solution addition and prolonged ultracentrifugation, at present no method has been found superior to the study of in vitro lipoproteins isolated by the ultracentrifuge. Ultracentrifugally, the serum lipoproteins can.be isolated into narrow flotation or hydrated density bands. However, the more detailed the isolation procedure employed, the lower the yield of lipoproteins for chemical analysis. It is primarily for this consideration of yield that most of the present lipoprotein chemical composition studies have been confined to broad bands of ultracentrifugally isolated lipoproteins. Previous chemical studies4 of ultracentrifugally isolated lipoproteins have established rough constituent lipid and protein composition values for the major lipoprotein classes present in human serum. However, in these studies the various lipoproteins analyzed were obtained from many different individuals, not allowing evaluation of variability of composition of particular lipoproteins from person to person. A further limitation of these studies was the unavailability of analysis for unesterified fatty acid. Also, one highly important chemical analysis was of limited reliability, namely, the glyceride analysis, which was calculated by difference of the fatty acids present as cholesteryl ester and phospholipid from the total fatty acids. Although the present study is restricted to the lipid composition of certain lipoprotein classes, it nevertheless represents an improvement over the previous study in that such highly important lipid components of lipoproteins as glycerides and unesterified fatty acids are reliably analyzed. (1) This work was supported (in part) by the United States Atomic Energy Coinmisoion, The National Heart Institute of the U. S. Public Health Service and the Life Insiirancn Medical Research Fnnd (2) This work was done during the tennre of a Research Fellowship of the San Joaquin County Heart Association, a n affiliate of the American Heart Association. (3) E’. T. Lindgren, H. A. Elliott and J. W. Gofman, THISJ O U R N A L , 56, 80 (1951). (4) €1.Jones, J. Gofinan, F. Lindgren, T. Lyon, D. Graham and B. Strisower, Am. J . M e d . , 11, 358 (1951).
Intravenous sodium heparin exerts profound effects upon serum lipoproteins, particularly the low density lipoprotein^.^ Because the changes brought about by heparin strikingly resemble those transformations observed during physiologic fat absorption, there is great potential importance in understanding the basic features of the heparin transformation mechanism. For this reason, the present study includes a section dealing with physical and chemical changes in certain of the lipoprotein classes brought about; by the action of heparin. Methods A. Isolation of Broad Band Lipoprotein Fractions.-For this fractionation all the lipids and lipoproteins present in human serum or plasma are ultracentrifugally separated into the following four density classes: 1. Lipoproteins of Density6 Less than 1.006 g./ml.Fasting serum or plasma run unaltered a t 104,000 X g for 24 hours in a 6-ml. preparative tube (contained in a S inco 40.3 rotor) effects flotation of all the lipoproteins of 20 and higher Sfrates into the top 3-ml. fraction (Sr 20-400 low density lipoproteins7). The lower fraction (bottom fraction of the 1.006 g./ml. run) contains all the serum lipoproteins of density greater than 1.006 g./ml. as well as all the serum proteins. 2. Lipoproteins of Density Higher than 1.006 g./ml. but Lower than 1.063 g./ml. (S,0-20 Low Density Lipoproteins Plus the HDL18Lipoprotein).-To the bottom 3-ml. fraction from the above (1.006 g./ml.) run is added 3 ml. of a NaCl solution of density 1.115 g./ml. which is sufficient to bring the density of the protein and lipoprotein-free solution before ultracentrifugation to 1.063 g./ml. Following ultracentrifugation a t 104,000 X g for 24 hours the Sf0-20 and the HDLI lipoproteins are quantitat,ively isolated in the 3-ml. top fraction of the preparative tube. The 3-ml. bottom fraction contains the two high density lipoproteins as well as the other serum proteins. 3. Major High Density Lipoproteins.-To the 3-ml. bottom fraction of the revious (1.063 g./ml.) run is added 3 ml. of a DgO-NaN& solution of density 1.328 g./ml. which raises the small molecule solution density in the top milliliter of the preparative tube before ultracentrifugation t o 1.200 g./ml. After ultracentrifugation of this mixture a t 104,000 X g for 26 hours, practically all the major high density lipoproteins (HDLZ and HDLa) are quantitatively floated to the top fraction consisting of 1.5 ml.
kf
(5) D. Graham, T. Lyon, J. Gofman, H. Jones, A. Yankley, J. Simonton and S. White, Circulation, 4, 666 (1951). (6) All density values are given for 26’. (7) I n fasting sertim no significant amounts of Sf400-40,000 lipoproteins generally are present in the low density fraction, limiting this lipo2rotein fraction almost entirely to lipoproteins of the Sf 20-400 class. (8) H D L is a n abbreviation f o r high density lipoprotein. Thus, HDLI, H D L i and HDLa are abbreviations for the high density s e r u n lipoproteins of approximate hydrated density 1.05, 1.075 and 1.145 g./ml., respectively.
Sept., 1955
931
COMPOSITION OF LIPOPROTEINS OF FASTING AND HEPARINIZED HUMANSERA
TABLE I THELIPID CHEMICAL COMPOSITION OF ISOLATED LIPOPROTEIN FRACTIONS OBTAINED FROM FIVEFASTINQ HUMANS~ Case
Cholesteryl ester
1 2 3 4 5 Mean value
13 13 9 32 7 15
1 2 3 4 5 Mean value
51 45 47 45 45 47
Unesterified cholesterol
Glyceride
The Sr 20-400 class* 10 5 6 5 12 8 The Sr 0-20 class 10 11 12 12 16 12
Phospholipid
58 61 64 46 53 56
18 21 20 16 23 20
14 17 16 20 11 16
24 26 24 22 26 24
Unesterified fatty acids
1
