Prticle-size distriution of very low density plsm lipoproteins during ft sorption in mn EDWIN L. BIERMAN, THOMAS L. HAYES, JAMES N. HAWKINS, ALICIA M. EWING, nd FRANK T. LINDGREN Deprtment of Medicine, University of Wshington School of Medicine nd Veterns Administrtion Hospitl, Settle, Wshington, nd the Dormer Lortory, Lwrence Rdition Lortory, University of Cliforni, Berkeley, Cliforni ABSTRACT The size distriutions of electrophoreticlly isolted sufrctions of the very low density humn plsm lipoproteins hve een determined using electron microscopy. The primry nd secondry prticles oserved in plsm of norml sujects fter ft ingestion pper to hve similr size distriutions. Prticles produced y corn oil feeding cn e fixed y the osmium tetroxide rection while those produced y utter ft feeding could not e fixed or mde visile y this technique. Good greement etween prticle size s mesured y electron microscopy nd prticle size s predicted y ultrcentrifugl nlysis ws otined. KEY WORDS sorption. ft. primry. secondry. prticles. size. plsm very low density lipoproteins. chylomicrons electron microscopy. corn oil. utter ft. osmium fixtion. ultrcentrifugtion 9 mn 2-15) re importnt in the trnsport of lipids, especilly newly sored ft, in mn. They rnge in size from pproximtely 25 to 1, A nd include the lrge ft prticles (1, 2) (pproximtely 75 A nd ove; Sf > 4), tht produce turid or lctescent queous solutions. The electron microscope is prticulrly suitle for study of lipoproteins in this size rnge (3-7). Lipoproteins cn e frctionted ccording to differences in properties other thn prticle size, e.g., zone electrophoresis on strch lock (8, 9). Very low density lipoproteins migrte with oth z- nd @-gloulins. The cyz-lipoproteins (8) re present normlly in smll quntities in postsorptive plsm nd re considered to hve flottion rte in the Sf 2-4 rnge (pproximtely 25-75 A). Ultrcentrifugl sufrctiontion of this group of lipoproteins hs een descried (2). Their plsm concentrtion nd estimted size my e gretly incresed in some ptients with hyperlipemi mintined on ft-free diets (1). After ft feeding, plsm my contin lrge primry prticles which pper to e identicl with prticles in lymph (chylomicrons), nd lso migrte in the z-gloulin region. They cn e redily isolted from the smller Sf 2-4 lipoproteins y rief ultrcentrifugtion in sline (d < 1.6; lo6 g-min). In ddition, vrile quntities of secondry prticles lso my pper in the circultion fter ft mels. Presumly, they re derived from primry prticles (ll), ut, in contrst, migrte in the @-gloulin region (9). Susequent ultrcentrifugl seprtion from @-lipoproteins (Sf -2) presents little difficulty. On pper electrophoresis, oth primry nd secondry prticles dhere to the origin nd do not migrte (9, 13). Frctiontion of prticles lso hs een chieved y differentil floccultion in polyvinylpyrrolidone solutions (12). Preliminry estimtes (9) of the physicl properties for oth types of prticle ppering in plsm during ft sorption hve suggested tht they re in the size rnge of 7-2 A. In the present study, electron microscopy, coupled with density grdient centrifugtion nd nlyticl ultrcentrifugtion, hs een used to otin more definitive nlysis of oth prticlesize nd flottion-rte distriution of these two electrophoreticlly distinct kinds of lrge, very low density lipoprotein. METHODS Postprndil humn plsm ws otined from seven norml mle sujects 3-4 hr fter the feeding of 25 JOURNAL OF LIPID RESEARCH VOLUME 7,1966 65
-*.- - L.- s 6 $ 4 I- Secondry Prticles Primry Prticles Alumin 1 2 Cthode.2.4.6.8 1. Anode (-1 (+I Reltive Migrtion FIG. 1. Strch lock electrophoresis. Cse 2. Electrophoretic seprtion of ft prticles from plsm otined 4 hr fter corn oil feeding (2 g/kg). Electrophoretic conditions: ritl uffer, ph 8.6,.1 p; 5 v/cm for 26 hr t 25 '. Migrtion rte of turid pek reltive to tht of lumin pek: primry prticles,.62; secondry prticles,.16. Stippled res indicte frctions pooled for susequent nlysis. ml of corn oil lended with skim milk. In ddition, seven sujects were given utter ft in plce of corn oil; two of these sujects lso hd received corn oil in nother experiment. Smples (8-17 ml) of fresh, unrefrigerted plsm were frctionted y strch lock electrophoresis under the conditions previously descried (9). The turidity peks of primry nd secondry prticles, s determined y nephelometry (Colemn Model 14) of eluted strch lock segments, migrted (reltive to lumin, 1.) within the rnge previously reported (men f SD for 23 oservtions =.6 f.1 nd.23 f.8 respectively) (9). Lrge strch segments contining primry prticle nd secondry prticle frctions (Fig. 1) were eluted with.9% sline y spirtion through corse sintered glss filter or y vortex mixing, centrifugtion, nd decnttion. Elutes were concentrted nd wshed twice y ultrcentrifugl flottion through.9% sline for pproximtely 3 X 16g-min (35, rpm; 2') in Spinco SW 39 rotor. In most experiments, to reduce convection, the concentrted frctions were mixed with sucrose solutions to finl sucrose concentrtion of 5y prior to ultrcentrifugtion through sline. The frctions were stored t 4-1' until nlysis. Flottion rtes of these primry nd secondry prticle frctions were determined y nlyticl ultrcentrifugtion nd prticle-size distriution y electron microscopy. Totl lipid content of ech frction ws determined from grvimetric ssy of n extrct ptterned fter the method of Sperry nd Brnd (14). One primry nd three secondry prticle frctions were studied in the nlyticl ultrcentrifuge to deter- mine, if possile, the lower limit of the Sf rte distriution. For this purpose, 87.1 mg of solid NCl ws dded to 1 ml of ech frction to ring the solvent density t 26' to 1.61 3 g/ml (1.7 moll NCl). Although diffuse turidity oundry rpidly migrted through ech frction during ccelertion of the ultrcentrifuge, no resolvle concentrtion of lipoproteins less thn Sf 4 ws oserved. Unfortuntely, the turidity of prticles of 75 A nd lrger (corresponding to Sf vlues of 4 nd greter) effectively interferes with resolution in the schlieren opticl system. Nevertheless, these ultrcentrifugl findings definitely indicte tht the Sf rtes of the primry nd secondry prticle frctions studied re essentilly > 4. In order to evlute further the prticle-size distriution s well s lipoprotein stility to preprtive ultrcentrifugtion, secondry-prticle frction ws sufrctionted in density grdient (1 5). A secondryprticle frction (cse 3) in 1.7 moll NCl (.5 ml) ws introduced t the ottom of previously formed nonliner NCl grdient y using spinl needle nd syringe. The conditions of low-speed centrifugtion (38 g t the midpoint of the tue for 23 min t 2') would llow recovery of lipoproteins Sf > 2,2 in the top milliliter of the preprtive tue. In ll, 17 frctions (1 X 1 ml nd 16 X.5 ml frctions) were tken y seril spirtion from the.5 X 3.5 inch preprtive tue. Precision refrctometry of control NCl grdient fter ultrcentrifugtion ws used to evlute the density' nd viscosity in ech region of the preprtive Unless otherwise indicted, ll densities re given t 2' 66 JOURNAL OF LIPID RESEARCH VOLUME 7,1966
tue. Initilly, the top of the grdient tue hd density of 1.65 g/ml nd the ottom 1.65 g/ml. Prticle-size distriutions for ech frction were determined using model HU-11 Hitchi electron microscope. The fixtion process for electron microscopy utilized 1% Os4 in uffer t ph 7.4 fter the prticle frctions hd come to room temperture. After exposure for 3 min, the fixed lipoproteins were plced on the electron microscope grid for viewing. Mesurements of prticle dimeter were mde from prints w th totl mgnifiction of out 15, X. Dt otined in this wy were nlyzed in order to yield prticle-size distriution, mss distriution, nd sedimenttion-rte distriution s well s verge vlues of these prmeters. Prticle-dimeter distriutions were converted to mss nd flottion-rte distriutions y mens of computer. The experimentl dt, consisting of mesurements of ech individul prticle dimeter, were punched on Stndrd IBM crds nd nlyzed y progrm for the IBM 794 computer. The computer procedure ws pproximtely s follows: (u) reding the dt for single electron microscope plte; () performing desired clcultion on ech dtum, e.g., conversion of mesured dimeter to ctul dimeter in ngstroms, clcultion of moleculr weight nd flottion rte; (c) I I I I I I I 1 4c I I I I I I I CASE 14 (Direct screen count) 3 Prticle dimeter, Angstroms I l l I I I I I.41 4 1 4 1 Sf rte (thousnds) CASE 16 PRIMARY PARTICLES (n = 196) 3c.- 2c - io 3 - E : 2. Prticle dimeter, Angstroms L 1 1 I I I 1.4 1 4 1 4 Sf rte (thousnds) (Direct screen count) PRIMARY PARTICLES (n = 5) (n IS3 IO ECONDARY PARTICLES 51 - I 8( Prticle diometer, Angstroms 2 4 6 Prticle diometer, Angstroms - BO.4 1 4 1 4 Sf rte (thousnds).4 1 4 IO 4 Sf rte (thousnds] FIG. 2. Prticle-size distriutions of primry nd secondry prticles electrophoreticlly isolted from plsm fter corn oil feeding, s determined y electron microscopy. Direct screen counts were done in the electron microscope utilizing scle in the inoculr eyepiece to mesure the imge formed on the fluorescent screen. All other counts were otined y mesuring prticle dimeter from photogrphs. BIERMAN, HAYES, HAWKINS, EWING, AND LINDGREN Prticle Size of Plsm VLDL 67
u, 1.. ' FIG. 3. Typicl electron microgrph of primry-prticle frction (cse 2). X 11,4. A similr preprtion of the secondry-prticle frction from the sme cse ppered identicl. i % FIG. 4. Chromium shdowed preprtion of secondry-prticle frction (cse 2). >; 11,4. generting histogrm for ech series of newly clculted vlues. The size of the histogrm intervls ws determined y n input crd t the eginning of ech use of the progrm. The progrm ws written in Fortrn nd ws esily modified to clculte ny vlues for which histogrm distriutions were desired. 68 JOURNAL OF LIPID RESEARCH VOLUME 7,1966
RESULTS A comprison (Fig. 2) of the prticle-size distriution of primry- nd secondry-prticle frctions otined from the sme plsm (cse 2; corn oil) shows rnge of prticle dimeters of 75-6 A in ech frction. Two dditionl pirs of primry- nd secondry-prticle frctions otined from other sujects fter corn oil feeding were nlyzed ; the prticle-size distriution for ll these frctions is similr (Fig. 2). In ech of the pirs, secondry prticles pper to hve somewht lrger medin prticle dimeter thn primry prticles. Typicl electron microgrphs used in the study of primryprticle frction (Fig. 3) nd secondry-prticle frction (Fig. 4) from the sme suject (cse 2) re shown. In none of the cses of utter ft feeding could the ft prticles isolted from plsm e mde sufficiently visile for prticle-size mesurements in the electron I I I I I EXPERIMENTAL (MASS AVERAGES) A CALCULATED THRESHOLD RECOVERY CALCULATED 1% RECOVERY CALCULATED LARGFST DlAMETER I I I I I 1 2 3 4 5 Prticle dimeter, Angstroms FIG. 5. Comprison of the recovery of prticles experimentlly otined from electron microgrphs with vrious recoveries clculted from ultrcentrifuge theory for the density-grdient flottion experiment..4.3 u 6-2 2.2 Q).- c - Q) nc.1 I I I I I I I I 1 Prticle dimeter, Angstroms FIG. 6. Distriution of prticle dimeters for the 3rd, 7th, llth, nd 15th sufrctions otined y electron microscopy From the density-grdient flottion of secondry-prticle frction (cse 3; Tle 1). For clrity of presenttion, the histogrms hve een converted to continuous prticledimeter distriution. BIERMAN, HAYES, HAWKINS, EWING, AND LINDGREN Prticle Size of Plsm VLDL 69
TABLE 1 SUBFRACTIONATION OF SECONDARY-PARTICLE SAMPLE. PHYSICAL DATA DERIVED FROM ELECTRON MICROSCOPY Frction 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 * No. of Prticles Counted Prticle Dimeter Moleculr Weight X 1-6 52 8 58 36 68 52 3 62 151 85 94 83 12 54 67 63 5 46 78 143 12 139 9-3 349 12 339 15 12 15 15 249 235 A 4,24 f 1,5t 4,15 f 1,42 3,87 f 897 3,51 f 959 4,1 f 984 4,1 f 1,11 3,48 f 66 3,55 f 966 3,44 f 864 3,69 f 674 3,19 f 667 3,6 f 824 2,93 f 698 2,94 f 453 2,86 + 77 3,84 + 577 2,72 f 747 2,71 f 521 2,22 f 387 2,43 f 482 1,95 z!= 376 2,1 f 33 1,98 + 37 - - 1,7 f 324 1,85 f 323 1,72 f 27 1,64 f 315 1,61 f 174 1,27 f 194 1,16 f 28 1,16 f 25 1,15 f 215 1,132 f 214 * Q nd represent duplicte electron microscopic determintions. t Mens f SD. microscope y using the usul techniques of osmium fixtion. The clculted recovery of sufrctions from the density grdient flottion experiment nd the recovery determined experimentlly from electron microgrphs re shown in Fig. 5. From prticle-size mesurements otined from this density grdient seprtion y electron microscopy, the verge prticle size, moleculr weight, nd Sf rte for the 17 frctions were clculted y mens of the computer progrm (Tle 1). The prticle-dimeter distriutions otined for the 3rd, 7th, llth, nd 15th frctions re given in Fig. 6. Figure 7 shows typicl electron microgrph for frction 13. The comprison etween prticle sizes otined experimentlly y mens of electron microscopy nd those otined y clcultion from ultrcentrifugl flottion ws mde with the simplifying ssumptions tht 3,5 f 25,7t 28,1 f 23,4 19,7 f 12,6 15,4 f 1,1 22,4 f 19,6 23,7 f 26,8 13,4 f 5,77 16,5 f 19,9 14,3 f 13,5 16,3 f 9,98 1,99 + 8,69 1,1 f 6,44 8,79 f 8,95 7,99 f 3,29 8,72 f 12,9 7,7 f 8,6 7,69 f 12,7 6,48 f 4,18 3,51 f 1,86 4,82 f 5,93 2,42 f 1,35 2,55 f 1,36 2,45 f 1,9 - - 1,63 f 1,45 2,1 f 966 1,61 f 712 1,45 f 828 1,26 f 395 65 f 293 54 f 28 523 f 313 489 f 266 472 f 267 15,2 f 9,441. 14,4 f 8,75 11,8 f 5,18 9,85 f 4,74 12,7 f 6,65 12,9 f 8,22 9,27 f 2,93 1,1 f 6,56 9,35 f 5,4 1,5 f 4,7 7,88 f 3,63 7,44 f 3,53 6,71 f 3,76 6,55 f 1,91 6,48 f 4,78 6,2 f 3,27 5,89 f 4,71 5,5 f 2,27 3,72 f 1,32 4,51 f 2,48 2,88 f 1,1 3,1 f 975 2,94 f 91 - - 2,19 f 978 2,56 f 861 2,21 f 673 2,3 f 778 1,9 f 47 1,19 f 367 997 f 365 1,1 f 425 975 f 364 95 f 365 continuous size distriution of lipoprotein prticles of constnt hydrted density (.93 g/ml) existed nd tht ech of the 17 regions of the grdient ws homogeneous medium. Although there is considerle overlp in size distriution for djcent frctions, nd somewht smller prticle sizes were otined experimentlly, the results re consistent with nticipted recovery. These results suggest tht preprtive centrifugtion does not significntly lter the prticle-size distriution. DISCUSSION The prticle-size distriutions of electrophoreticlly nd ultrcentrifuglly frctionted very low density plsm lipoproteins from sujects fed corn oil hve een determined using the electron microscope. Results otined on four secondry-prticle frctions nd three primry- 7 JOURNAL OF LIPID RESEARCH VOLUME 7, 1966
Jks P... S e ' t * c FIG.7. Electron microgrph of ultrcentrifugl sufrction 13 (Tle 1). prticle frctions indicte considerly roder prticle-size distriution thn ws previously estimted from centrifugl ehvior (9). Clumping of prticles during preprtive centrifugtion t low temperture could yield opertionl estimtes of size differing from those oserved with the electron microscope. In ddition, these results suggest tht oth primry nd secondry prticles hve size distriution tht extends over most of the known plsm lipoprotein spectrum ove Sf 4. If the cuic reltionship of volume to dimeter is considered, pproximtely two-thirds of the prticle mss (nd hence, triglyceride) my e found in the dimeter rnge of 15-4 A. Further, from the fct tht the prticle-size distriutions were similr in primry nd secondry prticles from ny one plsm smple, it is pprent tht significntly different properties my exist mong lipoproteins in the sme size rnge. Using only the mesurement of dimeter, we hve clculted moleculr-weight distriution nd flottionrte distriutions of ech lipoprotein frction nd sufrction studied with the electron microscope. In these clcultions it hs een ssumed tht no chnge in prticle size occurs s result of osmium fixtion. Since t 3 min the fixtion rection (16) with very low density lipoproteins requires pproximtely 1-2 toms of osmium per doule ond, the lipoproteins might e expected to increse their size y n mount pproximtely equl to the dded volume of osmium. Becuse of the very smll prtil specific volume of osmium, it is estimted X 11,4. tht the fixed lipoproteins re out 5% lrger thn the ntive unfixed lipoproteins. The degree of lipid unsturtion is importnt in the osmium tetroxide-lipoprotein rection (16, 17), nd the filure of osmium to fix the more sturted utter ftproduced prticles my reflect this phenomenon. Alterntively, since chylomicron triglyceride produced y feeding utter ft is semisolid t room temperture nd seprtes into n oil phse, fter eing cooled to ' 4 nd then rewrmed, more redily thn corn oil-produced chylomicron triglyceride (18), it is possile tht differences in the physicl stte of lipids in lipoproteins lso influence the fixtion rection. In contrst, Jones, Thoms, nd Scott (6) did oserve osmiophilic msses in the chyle of rts fed utter ft. Further studies re needed to determine the effect of diet nd severl hyperlipemic sttes on the prticlesize distriution of these very low density plsm lipoproteins. This work ws supported in prt y PHS Reserch Grnts AM 667-2, H E 229-9, nd H E 1882-1 from the Ntionl Institutes of Helth, U.S. Pulic Helth Service, nd y the U.S.Atomic Energy Commission. Mnusmjt received 75 July 7965; ccepted 37 August 7965. REFERENCES 1. Dole, V. P., nd J. T. Hmlin, 111. Physiol. Rev. 42: 647, 1962. BIERMAN, HAYES,HAWKINS, EWING,AND LINDGRENPurlic Size of Plsm VLDL 71 t
2. Gustfson, A., P. Alupovic, nd R. H. Furmn. Biochemistry 4: 596, 1965. 3. Hyes, T. L., nd J. D. Hewitt. J. Appl. Physiol. 11: 425, 1957. 4. Lindgren, F. T., A. V. Nichols, T. L. Hyes, N. K. Freemn, nd J. W. Gofmn. Ann. N.Y. Acd. Sci. 72: 826, 1959. 5. Hyes, T. L., F. T. Lindgren, nd J. C. Schooley. University of Cliforni Lwrence Rdition Lortory Report, No. 9897, 1961, p. 91. 6. Jones, R., W. A. Thoms, nd R. F. Scott. Exptl. Mol. Pthol. 1: 65, 1962. 7. Csley-Smith, J. R. J. Cell Biol. 15: 259, 1962. 8. Kunkel, H. G., nd R. Trutmn. J. Clin. Invest. 35: 641, 1956. 9. Biermn, E. L., E. Gordis, nd J. T. Hmlin, 111. J. Clin. Invest. 41: 2254, 1962. 1. Biermn, E. L., D. Porte, Jr., D. D. O Hr, M. Schwrtz, nd F. C. Wood, Jr. J. Clin. Inoest. 44: 261, 1965. 11. Biermn, E. L., nd D. E. Strndness, Jr. Am. J. Physiol., in press. 12. Gordis, E. Proc. SOC. Exptl. Biol. Med. 11: 657, 1962. 13. Fredrickson, D. S., nd R. S. Lees. Circultion 31: 321,1965. 14. Sperry, W. M., nd F. C. Brnd. J. Biol. Chem. 213: 69, 1955. 15. Lindgren, F. T., A. V. Nichols, F. T. Uphm, nd R. D. Wills. J. Phys. Chem. 66: 27, 1962. 16. Hyes, T. L., F. T. Lindgren, nd J. W. Gofmn. J. Cell Biol. 19: 251, 1963. 17. Hyes, T. L., nd J. N. Hwkins. University of Cliforni Lwrence Rdition Lortory Report, No. 11184, 1963, p. 11. 18. Zilversmit, D. B. J. Clin. Invest. 44: 161, 1965. 72 JOURNAL OF LIPID RESEARCH VOLUME 7, 1966