80
FR.iSK T. LINDGRES, HAROLD .4. ELLIOTT 4ND JOHN W. GOFMAN
T H E ULTRACEKTRIFUGAL CHARACTERIZATIOK A S D ISOLATION OF HVMAK BLOOD LIPIDS AND LIPOPROTEISS, WITH APPLICATIOSS TO T H E STUDY OF ATHEROSCLEROSISL~2 F R A S K T. L I S D G R E S
Division uf Medical Physics, Deparlmenl of Physics, Cniversily of California, Berkeley, California
HAROLD A . E L L I O T T Rudiation Laboratory, Deparlment of Physics, University of California, Berkeley, California ASD
J O H S W . GOFMAN
Oivisiun uj’ Medical Phtqsics, Department of Physics, Uniuersily of California, Berkeley, California Received August 10, 1960
As a group of biologically occurring substances, lipids play a role of major importance in the life process. Much attention in recent years has been focused upon blood lipids, yet no satisfactory description or methods for study of these lipids in their native state have been given. The major serum lipids consisting of neutral fats, fatty acids, cholesterol and its esters, and phospholipids are present in total concentration of approximately 500 mg. per cent in the normal adult human. However, essentially none of these lipids circulate in the blood stream individually, but are present in the form of complex giant molecules ranging in molecular weight from approximately 200,000 to many million. In most chemical extraction techniques the identity of each of these molecular species is destroyed and the resulting analysis is a mere tabulation of the various chemically defined lipids that are present. For several years the existence in human sera of lipid-bearing macromolecules has been indicated by such workers as McFarlane (5) and Pedersen (7). In their ultracentrifugal studies of serum and serum fractions the presence in human sera of a low-density lipoprotein (the so-called X protein) was found to depend upon the concentration of both the serum proteins and the salt. These data led to a postulation of labile complexes of serum proteins with serum lipids. Additional information concerning serum lipoproteins was forthcoming during the war, when Cohn and coworkers (l), using low-salt, low-temperature, ethanol fractionation techniques were able to isolate two distinct lipoproteins from pooled human plasma. These were the al-and P1-lipoproteins(6). The P I lipoprotein weighed over one million molecular weight units and consisted of 75 Presented a t t h e Twenty-Fourth National Colloid Symposium, which was held under the auspices of the Division of Colloid Chemistry of t h e American Chemical Society a t St. Louis, Missouri, J u n e 15-17, 1850. This work was supported (in p a r t ) 1)) the Atomic Energy Commission and the United States Public Health Service.
HCM.%K BLOOD LIPIDS .%KD LIPOPROTEINS
81
per cent lipid. Being a low-density molecule having a density of approximately 1.04 g./cc., it was thought to be the X protein. The al-lipoprotein, on the other hand, was found to be a much smaller molecule of molecular weight about 200,000 and consisted of about 35 per cent lipid. The work of the Harvard group indicated these lipoproteins to be stable to precipitation and re-solution at low temperature. Our ultracentrifugal studies ( 2 , 4) have indicated the serum lipids and lipoproteins to be reasonably stable molecules \\hose behavior is esplainahle ivithout assuming the existence of labile lipid-protein compleses. Moreover, under certain conditions the preparative and analytical ultracentrifuge may he used to isolate and study these molecules in more nearly their native state. FLOTATIOK O F LO\V.DESSITY SEKl-M LIPIDS . \ S D 1,Il'OPROTIZISS
The ultracentrifugation of large molecules has generally iwen confined i o studies in which the molecules have undergone sedimentat ion. This has lwm the case of viruses and most proteins, whose hydrated densities have ranged from 1.1 to 1.3 g.,/cc. In dealing with lipids and 1ipoprot)einsof hydrated density close to unity it is convenient to cause the molecules to float against the centrifugal field in the ultracentrifuge. This is done by making the solvent dcnsity greatrr than the density of the molecules under study. From the theory of the cylindrical lens modified schlieren optical system, which directly records dnidz as ii function of z (x being the distance from the axis of the rotor), such floating Iioundaries will be recorded as inverse peaks. The rate of migration of such a boundary can be expressed as a flotation rate which, like a sedimentation rate, is a physical constant under specified conditions3 of temperature, solution composition, and centrifugal force. I n expressing migration rates the Svedberg unit is retained. Flotation rates are expressed in terms of Sc values, that is, Svedbergs of flot,ation.
Isolation of the low-density lipid a d lipoprotein group Since the low-density lipids and lipoproteins are present in serum at, low concentrations, their concentration and isolation from the other serum protein molecules are necessary before study in the analytical ultracentrifuge. This is accomplished using the angle preparative ultracentrifuge. Serum is first given a density increment with sodium chloride such that a solvent density of l.O(i3 g./cc. is achieved (this density is calculated independent of any contribution from either proteins or lipoproteins to density). Upon ultracentrifugation of this solution all lipids and lipoproteins of hydrated density significantly less than the solvent are floated t o the top of the preparatiTre tube, while the more dense serum proteins undergo sedimentation toward the bottom of thc prepslative tube. Ultracentrifugation a t approximately 81,000 X g for 13 hr. in a 12' angle centrifuge results in the distribution of serum molecules approximately as in figure 1. The lipids and lipoproteins of density significantly less than the Unless otherwise stated, all runs reported were made a t a temperature of 27°C. rt 2" in u sodium chloride solution of density 1.063 g./cc.
H l . M . \ S BLOOD LIPIDS .\SD LIPOPHOTEIKS
S3
tion of this class of lipids increases greatly following fat-containing meals :iii(I therefore represents part of the alimentary lipemia. The chylomiri,ons :ire 01)servable as a turbidity boundary as the ultracentrifuge accelerates to full sptwl ( 3 , 6 4 0 R.P.M.). B . Species of S,30-70: This class of molecules comprises a major fraction of alimentary lipemia and is modified in concentration \\-ith relationship t o me:ils. C. Species of S,. 20-50: Molecules of this class are under study, hut as yet no significance can be attributed to their presence. 11. Species of Sf10-20; These components of molecular \\.eights in the neigliIjoi,hood of lo6 are present in the sera of many individuals studied. They i i w of special interest, since they appear to be related in some way to the presencr of atherosclerosis. This disease and our experimental findings \vi11 be discussed in a later section. E. Specics which migrate with rates between 2 and 8 S,. itmiis: These lipoprotrin molecules contain a major fraction of the serum cholesterol and phospholipid and may correspond vlosely to the so-called P-lipoprotein. -4 given individual may hiive one or se\,eral of these components. This component or set of components carrying a major fraction of the serum lipids is present in every one ~f some (io00 intlividuuls studied, at concentrations varying from individual to iiidividual hut at essentially :I constant level for a given individual from time 1 0 time. Scriirn lipoprolciiis o,f dcnsil,y less fhari 1.d.i g. cc.
IIy c~om1)inings d t addition to the serum a n d dilution \vith c*onccnti~atr(l I),O ~oliition,i i solvrnt tlensity of 1.24 g . ( Y ' . can be avhieveti. If this serum soliit i o i l (0.2 (',,I is ventiifugetl a t 81,000 X g f(ii, approximately 40 hr. in a 12' iinglc. ulti~;icc~nti~ii'~igc, tlic top fi,iic*tionof t h e pwpaixtive tube \\-ill contiiin neai+y dl t l i v lipids and lipoproteins ~f clensity less than 1.24 g. cc. C'hole~tet~ol analyaih ( i f tlir top and bottom fixrtions of sc\.ei,al samples indicated the top friictioiih to contain over 95 per c8entof the cholesterol originally present in the preparntivcl tulle. .I typical analytical ultracentrifuge pattei,n for this total lipid ancl lipoprotein fraction is sho\vn in figure 3. I n addition to the lox-density 1ipopi.ot rins 11-hirhno\\-eshihit murh greatel. flotation rates, t \ v o lipoproteins of approxim i i t r hydi,:ited densities 1 .OT3 g, (T, iintl I . I % g. 'cc, ai'e observed Thew tlensc lipcipi~oteinsproliably correspond to the a-1ipoproti.h i,eferred t o in the 1iteratui.e. In genrral the role of these high-density lipopi,oteins remains to be esplainetl. Ilo~vev(~i,, preliminary findings indicate that some differences in concent ration of thehe wmponents exist ivith i,espect to sex. c:dt
I'l~l.:OI'I~:R.~TIVI~ l.LTR.\CI.:STRIFL-G.~L
IHOLh'TIOS O F SE1tt-M LII'IDS
Ah-D I.IPOI'ROTEISS
. I . 12alr sqmrutioii oJ aygregatrs and inolc'ctrlrs o,f tlrrasity l c s s thntl 1.006 g. cc. ( I ) ('hylorriicroris: l'he chylomicrons which xeigh in the neighborhood of 10" molrciilai. ivright units :ire c~hai~acterizetl by a flotation rate of iiljoiit i o 4 S, iiiiits i n :i stilt, solution of Ilenhity 1 .OOfi g.,'cc. Isolation can readily be mnde on
HUMAA- BLOOD LIPIDS .4ND LIPOPROTEINS
85
(2) Lipids and lipoproteins of S , values 17-70: Molecules of this class have hydrated densities less than that of the serum supernatant (1.006 g./cc.). I t is therefore not possible in aqueous salt solutions to separate these molecules by density differential ultracentrifugation. Their separation can be achieved in a manner analogous to the chylomicron isolation, that is, by differential rate separation. A small volume of concentrated chylomicron-free Sf 17-70 fraction is layered in a preparative tube beneath a salt solution of density 1.006 g./cc. After critical ultracentrifugation partial recovery of the fastest and slowest component present in the layered fraction can be made analogous to preparative electrophoretic isolation technique. For intermediate components Dresent in this class, isolation may be achieved by successive rate fractionations, the conditions for each of which must be empirically determined.
ULTRACENTRIFUGAL 4LBUMlN ULTR4CENTRlFUGAL GLOBULIN
I
LiWPROTEINS ~ U ~ ~ l 0 6 3 p m l c c l CONCENTRATION OF MOLECULES IN PERCENT
FIG.5 . Distribution of serum molecules following a 24-hr. preparative r u n of unaltered serum at an average centrifugal force of 104,ooO X g.
R. Two-stage differential density preparative isolation of serum lipoproteins of hydrated density greater than 1.006 g./cc. ( 1 ) Sf 13 molecules: If serum is run unaltered in the preparative ultracentrifuge for a sufficient time, the distribution of serum molecules (when present as is indicated in figure 5 . This distribution is obtained by running in the analytic ultracentrifuge under reference conditions successive layers pipetted from the preparative tube. By carefully pipetting fluid from the preparative tube it is possible to obtain a fraction free of serum lipids and lipoprotein molecules of Sr rates 17 and higher. Such a fraction will contain variable amounts of S,2-13 class lipoproteins, some of the more dense lipoproteins of u’s > 1.063 g./cc., and variable amounts of albumin. In order to free the Sf13 molecules from lower Sr class lipoproteins, another preparative run is necessary. Prior to the second run an appropriate density increment is given the fraction containing Sr 13, which is then layered beneath a salt solution in a preparative tube (see figure 6). I t is necessary for hydrodynamic stability that this density increment be sufficient to bring the solvent density to the same or slightly greater density than the salt solution that is layered above. This salt solution (of density 1.019 g./cc.) is appropriate to float the Sr 13 molecules to the top of the preparative tube in a
HUMAN BLOOD LIPIDS AND .LIPOPROTEINS
87
(8) Lipoproteins of hydrated density greater than the S, 13 (u = 1.016 g./cc.): Molecules of hydrated density greater than 1.015 g./cc. may also be isolated by density differential ultracentrifugation. In each case, the first ultracentrifugation involves the addition of an appropriate density increment to the serum to cause all lipoproteins of lower hydrated density than the molecules desired to float and a t the same time to allow sedimentation of the molecular species desired. In particular, it is desirable to allow the molecular species desired to pile up in the region of the albumin boundary, allowing removal in a fraction of small volume. To this fraction is added another appropriate density increment, after which this fraction is layered beneath a proper salt solution in a preparative tube. This salt solution in the top layer is appropriate to float the species desired but not species of higher hydrated density. Ultracentrifugation of this layered preparative tube permits separation of the molecular species desired from lipoproteins of higher density and the other serum proteins. It is worthy of note that the layering technique used in the second stage minimizes contamination of small molecules present in the serum fraction which do not undergo appreciable sedimentation or flotation during the ultracentrifugation. This is important in establishing accurate chemical composition of these molecules and also in large-molecule radioactive tracer work. Optimum conditions for preparative isolation work include careful temperature control and a sufficiently high vacuum to reduce thermal gradients within the rotor to a minimum. Also it is advisable to incorporate a small amount of appropriate buffer in the salt solutions used in the layering process. PHYSICAL A S D CHEMICAL PROPERTIES OF CENTRIFUGALLY ISOLATED SERUM LIPOPROTEIXS
Minimum hydrated molecular weights calculated on the basis of flotation alone, assuming Stokes’ frictional forces for spheres. give the following values : COMFnNESI
Lipoprotein lipoprotein Lipoprotein Lipoprotein Lipoprotein
of c = 1075 of Sr 1
1
YOLECULAP WEICET
I
of Sr 6 of Sr 13 of Sr 10
Flotation rates of these molecules in a medium of defined density and composition a t present seem to be the most convenient method of identification for those components of hydrated density less than 1.063 g./cc. Another method of classification is the hydrated density of the molecules, which may be more useful with reference to the denser lipoproteins. Studies of variation in migration rate can be made in media of different densities and an estimate of the hydrated density obtained by extrapolation to zero migration rate. A summary of flotation properties for most of the serum lipoproteins is given in figure 7. We are aware of the potential error in using sodium chloride or other small molecules to provide
88
FRANK T . LINDGREN, HAROLD A . ELLIOTT AND JOHS V’. GOFMAS
the density increment. Currently studies with serum albumin are being performed which will allow the estimation of hydrated densities in the absence of effects that might accompany the use of salt or DzO. Chemical analysis of various isolated components indicated that all of the lowdensity group of lipoproteins (Sf 2 4 0 ) contain phospholipid, cholesterol, and protein. Unfortunately the low concentration of many of these molecules in serum make the task of obtaining adequate quantities of these molecules for chemical analysis extremely difficult. Precise studies cf chemical composition must await sufficient quantities of isolated components. At present it would be extremely hazardous to speculate as to details of structure of these molecules until accurate chemical composition has been determined. THE
Sr 10-20
MOLECULES AND ATHEROSCLEROSIS
Atherosclerosis is one of the major vascular diseases. In this country atherosclerosis and its consequences result in greater death and disability than any other disease, exceeding by a factor of three in death alone all forms of cancer combined. The disease involves the deposition of lipid material into the inner wall (or intima) of the arteries such that the intima thickens, the result of progression of the lesion. If this process continues blood flow may be impaired and eventually occlusion of the vessel may occur. Unfortunately, atherosclerosis is particularly prone to attack the vital coronary and cerebral arteries, where an occlusion may result in either sudden death or sustained disability. Most “heart attacks” and “strokes” are dramatic consequences of atherosclerosis long after the disease itself began. I t has long been suspected that blood lipids might in some way he related to atherosclerosis. Cholesterol has received much attention, in part because many disease states associated with an elevated blood cholesterol predispose to early and severe atherosclerosis. However, by far the largest class numerically affected by atherosclerosis are the presumed normal individuals, whose total serum cholesterols are in the range of 125-260 mg. per cent as measured by the Schoenheimeraperry method. Moreover, efforts to correlate the extent of atherosclerosis with elevated serum cholesterols within the normal range have not been conclusive. Experimental hypercholesterolemia and atherosclerosis may be induced in the rabbit by a diet high in cholesterol. Our early ultracentrifugal studies (3) of rabbit lipids and lipoproteins provided pertinent clues to the analogous situation in the human. Normal rabbit sera when studied under our conditions show at least one lipoprotein of 5-9 Sfunits. However, after a short period of cholesterol feeding most rabbits develop additional components in the range of 10-30 Sf units. Despite continued cholesterol feeding some rabbits failed to develop both the hypercholesterolemia and the higher S f components. Autopsy after 15 weeks of cholesterol feeding revealed the degree of atherosclerosis to be well correlated with the terminal concentration of the St 10-30 molecules. Rabbits that failed to develop the higher S f components exhibited no gross atherosclerosis, whereas those
HCMAN BLOOD LIPIDS AND LIPOPROTEINS
89
rabbits that developed high concentrations of the Sr 10-30 class molecules did develop the disease. I t was of immediate interest to see if any analogous class of molecules existed in humans. In many of 6000 people studied, of both normal and many disease
FIG 8. Variations in flotation rate with solvent density for the major lipids and lipoproteins of serum. Density variations were achieved by t h e addition of sodium chloride and by variation in the D20content of the solvent.
states, a similar class of molecules of Sr values between 10-20 units was found. Analysis of the incidence and concentrations of these molecules among the various categories studied supports the hypothesis that the occurrence of the S, 10-20 class molecules in the sera of humans is related to the presencp of atherosclerosis. The findings are best presented in the form of incidence (figure 8) and average concentration (figure 9) of the S r 10-20 molecules for each category.
90
FR.4NK T. LJNDQREN, HAROLD A. ELLIOTT AND JOHN W. QOFMAN
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HUMAN BLOOD LIPIDS AND LIPOPROTEINS
91
Nomnals There is no known way to determine the presencaor absence of atherosclerosis in an individual unless clinically manifested, as for example in the event of heart attack, We may assume therefore that of the presumably normal individuals reported a large fraction are developing atherosclerosis in accord with the known incidence of atherosclerosis obtained by autopsy of normal individuals killed accidentally. In the normal females studied the occurrence of the Sf 10-20 molecules is very low in the 2 0 4 0 age group, but increases markedly in the age group above 40. This agrees with the findings (8) that atherosclerosis in normal young women is a rarity but that after 40 women lose their relative protection against the disease. In the normal males the incidence of the Sf10-20 molecules increases with age, a t least up to the age of 60. This is in accord with the known occurrence of atherosclerosis, which increases with age. The relatively high occurrence of these molecules in the younger age group is not surprising, in view of the common occurrence of atherosclerosis even among young males.
Myocardial infarction The best choice of a group of living individuals manifesting atherosclerosis is that of patients who have survived a myocardial infarction, that is, a localized death of part of the heart muscle. At least 95 per cent of this group have had such an infarction as the result of atherosclerosis involving their coronary arteries. That nearly all these patients have the Sf10-20 molecules in their blood provides evidence of the close association of these molecules with the presence of atherosclerosis.
Diabetes mellitus Though less pronounced, there is a greater incidence of the Sf 10-20 molecules in diabetics as compared with corresponding normals of the same age and sex. This agrees with the fact that diabetics are prone to develop early atherosclerosis. Of particular interest is the much higher incidence of the Sf 10-20 molecules in the diabetic females of the 2 0 4 0 age group as compared to the low values for the corresponding normal females for the same age group.
Hypertension
It has never been shown that atherosclerosis is causatively related to hypertension, though the higher occurrence of atherosclerosis among hyperteasives is well known. I t is of significance therefore that such a high incidence of the Sr 10-20 molecules is found in this group. A summary of the average concentrations of the Sf10-20 molecules in all categories studied shows the myocardial infarction group and those groups in which a high incidence of atherosclerosis is expected to exhibit significantly higher average concentrations of the Sf10-20 molecules than found in the corre-
92
FRANK T. LIliDGREN, H.IROLD A . ELLIOTT AND JOHX IT. GOFM.4K
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H \ . M \ Y B L O O D L I P I D S \ Y D LIPOPROTEISS
(33
sponding normtal groups. 'l'licse (lata further support the hypothesis that the Si 10-20 molecules are in some way related to the presence of at~her~~sclerosis. The concentrations of thc S i 10-20 molecules have been studied with refercnce to diet. It lias not, I w n possiblr to tkmonstratc any significant influence of a single meal high in fat ant1 cholesteri~l.Further, two 01' more tests taken a t intervals of a fe\v days to \reelis on numerous individuals reveal no appreciable change in mncentwtion of the S r10-20 molecules, provided no dietary changrh w e i t made. In most cases \vr have found that I.est,riction of fat and cholesteid \vi11 significantly reduce the concentration of these molecules or bring them t o :t concentration below resolution I Jour ~ present technique of study. Individunls vary c*onsiderablyin response to this dirt, requiring from 2 t 3 G weeks to rrducc. significantly the concentration of the Sf10-20 molecules. The potential implications of being able to remove these molecules from the blood are obvious. Yet it remains to be proven ivhrther elimination of these molecules from the sera will prevent the development of alheroselerosis. SL-MMARY
It is possible under certain conditions to characterize the lipids and lipoprotein, of human sera, using the prepamtive and analytical ultracentrifuge. I n addition, ultracentrifugal techniques may he employed for isolating the individual ('omponents of lipid and lipoprotein. Studies of over 6OOO individiials reveal a complex system of serum lipids and liproteins. The present study indicates certain of these molecules (the S, 10-20 class) to be intimately related to the cwnmon and highly important discasc atherosclerosis. With further study we may espect the biological role of thew complcs giant molecules of lipid and lipoprotcin to be more fully understood. REFERENCES (1) COHN, E . J., et a l . : J. Am. Chem. SOC.68, 459 (1946). (2) G O F M 4 N , J. W . , LINDGRES, F. T., ASIJ ELLIOTT, 13.: J. B i d . Chrm. 179, 973 (1949). (3) G o ~ . ~ aJ. s ,W.,et a l . : Science 111, 166 (1950). ( 4 ) LINDGRES, F.T.,l
