Size and shape determination of fixed chylomicrons and emulsions with fluid or solid surfaces by three-dimensional analysis of shadows

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1 Size and shape determination of fixed hylomirons and emulsions with fluid or solid surfaes by three-dimensional analysis of shadows Donald Gantz, Susanne Bennett Clark, Arie Derksen, and Donald M. Small Departments of Biophysis, Mediine, Biohemisty, and Physiology, Boston University Shool of Mediine, Housman Mddial Researh Center, 80 East Conord Street, Boston, MA Summary Chylomirons and hylomiron-sized emulsions are spherial partiles in suspension but their shape and apparent size may be distorted by eletron mirosopy proessing. To assess adsorption to grids, flattening, and shrinkage, hylomirons and emulsions were fixed with osmium tetroxide and together with polystyrene beads were shadowed with platinum. Vertial profiles projeted from partile shadows indiated that the hylomirons and emulsions were slightly shrunken, slightly trunated, oblate spheroids while the polystyrene beads were spheres. Partile diameters were orreted by assuming that volumes of oblate spheroids on the grid surfae were equal to volumes of spheres in the original lipid suspension. Beause of the ompensating effets of shrinkage (dereases diameter) and flattening (inreases diameter) the differenes between the means of measured diameters and orreted diameters were 55%. - Gantz, D., S. Bennett Clark, A. Derksen, and D. M. Small. Size and shape determination of fixed hylomirons and emulsions with fluid or solid surfaes by three-dimensional analysis of shadows. J. L+id Res : Supplementary key words eletron mirosopy triolein unesterified holesterol osmium tetroxide fixation unidiretional platinum shadowing phosphatidylholines Eletron mirosopi (EM) tehniques have been used for more than 30 years to determine the sizes of hylomirons (1-5), smaller lipoproteins (6-8), and syntheti emulsions of hylomiron size (2, 9-11). A omprehensive review of early work involving various EM tehniques has been prepared by Forte and Nihols (12). One of the frequently used tehniques has been fixation with osmium tetroxide and subsequent shadowing with metals suh as hromium or platinum (13-17). n general, onlusions about the spheriity of partiles have been based on the assumption that an osmium-fixed partile is rigid. Partile diameters have been obtained by diret measurement of the fixed or fixed and shadowed partile. Some workers have expressed onern about partile ollapsdflattening and the resulant effet on apparent partile diameter (5-7, 14, 18). t is possible that the partile surfae adsorbs to and spreads on the grid surfae ausing an asymmetri flattening of the bottom of the partile. Also, irradiation of the partile by the eletron beam may alter partile size. n the present study, we have quantitated partile flattening and orreted the apparent diameter by using a three-dimensional analysis of shadowed hylomirons and hylomiron-sized emulsions with fluid or solid surfaes. MATERALS AND METHODS Preparation of emulsions The two emulsions prepared for this study differed only in phosphatidylholine (PC) speies. At ambient temperatures, emulsions made with egg yolk PC (EYPC) have a fluid surfae while those made with distearoyl PC (DSPC) have a solid surfae (19). Stok solutions of trioleoylglyerol (melting point 4OC) and unesterified holesterol in hloroform, and EYPC or DSPC in hloroform-methanol 2:l (v/v) were mixed in a glass vial, yielding lipid mixtures, after solvent evaporation, of 80% triolein, 1.5% holesterol and 18.5% (w/w) EYPC or DSPC (20). Emulsifiation of the lipid mixtures and isolation of the partiles were performed as desribed previously (21). The isolated emulsions ontained 88 wt% triolein, 1.5 wt% holesterol, and 10.5 wt% EYPC or DSPC. Preparation of hylomirons Cannulas for gastri infusion and mesenteri lymph olletion were surgially implanted into male Sprague- Dawley rats weighing g under pentobarbital anaesthesia as previouslv desribed (22). A gastri saline infusion (0.15 M NaCl) was interrupted after 12 h to administer a gastri bolus of 300 pl triolein. Chylous lymph was olleted for 10 h at room temperature in the presene of 1 mm EmA and 1 mm phenylmethylsulfonyl fluoride to inhibit proteolysis. Chylomirons were separated from the lymph by ultraentrifugation in a swinging buket rotor (SW 41, 30,000 rpm, 30 min. at 10 C) and were harvested from the tops of the tubes by tube sliing. Polystyrene beads Three polystyrene bead samples were used to onfirm the geometri arrangement of shadowing. Polystyrene samples of diameters 109 nm and 204 nm were obtained from Seragen Diagnostis, n., ndianapolis, N (23) and a 364-nm sample omposed of styrene-butadiene 95:5 (wt%) was furnished by Duke Sientifi Corp., Palo Alto, CA (24). Fixation Emulsions and hylomirons were diluted to 1 mg/d (total lipids) with HEPES-NaCl buffer or 0.15 M NaCl, ph 7.4, respetively, and then fixed as desribed by Forte and Nordhausen (6). A 0.5-pl aliquot was dialyzed against 0.15 M aodylate, ph 7.4, buffer ontaining 2% os01 for at least 4 h at mom temperature (12-14 kda moleular weight ut 06 Spetrum Medial ndustries, n., Los Angeles, CA) and then dialyzed overnight against distilled water. Abbreviations: EM, eletron mirosopy; EYPC, egg yolk phosphatidylholine; DSPC, distearoyl phosphatidylholine. Journal of Lipid Researh Volume 31, 1991 Note on Methodology 163

2 Grid and sample preparation Copper grids of 300 mesh were oated with Formvar and arbon and the surfae was rendered hydrophili by a 5-min glow disharge (25, 26). A 1-pl droplet of fixed emulsions or hylomirons was plaed on a oated grid and allowed to air-dry. Shadowing Prepared grids of hylomirons, emulsions, and polystyrene beads were plaed in a Denton 502A vauum evaporator (Denton Vauum, n., Cherry Hill, NJ) for unidiretional platinum shadowing, using a modifiation of the method of Hall (27). Grids and platinum wire were preisely arranged at a geometri shadow ratio (28) of 2:l (distaneheight) or a shadowing angle of 30. The grids were plaed in an ar so that the horizontal distanes from the grid enters to the metal soure were equal. Care was taken to ensure that the grids lay flat. Viewing mages were examined and reorded in a Hitahi HU- 11C Eletron Mirosope with a 50-p objetive aperture. A arbon grating replia with 2160 rossed lined" (Ernest F. Fullam, n., Latham, NY) was used for alibration. Measurements Partile and shadow measurements were taken diretly from eletron image film using a Peak 7 x measuring magnifier fitted with a gratiule with 0.1-mm divisions (Polysiene, n., Warrington, PA). Partiles from at least four different fields near a grid enter were measured. All partiles were measured exept those that were lumped or had overlapping shadows. The total number of EYPC emulsion, DSPC emulsion, and hylomiron partiles measured were 88, 268, and 213, respetively. Partiles with idential shadow diameters were grouped; groups ontained between 1 to 36 partiles depending upon the lipid preparation. We assumed that shadow diameter is the most aurate representation of original partile diameter beause partile dimensions an be altered by irradiation. Exessive metal deposition an alter the partile diameter and hange the apparent partile enter (29). The best measure of the total shadow length of a partile is the distane from shadow tip to the edge of the partile plus onehalf of the shadow diameter (Fig. 1). Partile height was obtained by dividing the total shadow length by 2. Vertial profiles The vertial profile of a partile was onstruted from its shadow (refer to Fig. 1). An enlarged image of a sha- - +' ~LXalptr B! Partkk H.lght 1 Fig. 1. Traing of an enlarged image of a rigid partile shadowed at a distane:height ratio of 2:l or an angle of 30' (A) and its vertial profile onstruted by projetion (B). A horizontal segment on the shadow is 2 x a segment on the projetion. The dashed lines represent an interpolation of unseen shadow. See Methods for details on vertial profile onstrution. dowed partile was overlayered with thin graph paper and positioned so that the partile and its shadow were biseted by a single grid mark. The outline of the partile and its shadow was traed. The shadow outline was divided into equal horizontal segments. The unseen portion of the shadow was interpolated and is represented by dashed lines. As a result of 2:l geometry, one segment of shadow length was twie that of a orresponding segment of height on the projetion. Proeeding from the shadow tip to enter of partile, a vertial profile of the partile was obtained. The typial vertial profile of an emulsion or hylomiron partile on a grid surfae is a slightly trunated oblate spheroid. Assuming that the volume of the oblate spheroid on a grid is equal to the volume of the spherial partile in suspension, then a orreted diameter may be alulated in the following way. 164 Journal of Lipid Researh Volume 31, 1990 Note on Methodology

3 - = Volume of oblate spheroid = volume of sphere 41 3aa2b = 4 3ar3 a2b = r3 r (radius) 2 ( -) = 2r (sphere diameter) where a = shadow diameter and b = partile height + 2. RESULTS AND DSCUSSON Eletron mirographs of shadowed 109-nm polystyrene beads, EYPC and DSPC emulsions, and hylomirons are shown in Fig. 2. Shadow margins are sharp and distint indiating partile stability during platinum shadowing. Partile lumping ourred in eah of the four preparations, and was more prevalent in the DSPC emulsion and hylomirons (up to 30% of partiles) than in the EYPC emulsion and polystyrene beads (up to 15%). Size distribution histograms, omposed of all partiles analyzed in this study, are presented in Fig. 3. Eah polystyrene bead sample is quite homogeneous. The size ranges and modal diameters of the three lipid preparations are similar. The skewed hylomiron population is similar to that obtained by Fraser, Cliff, and Courtie (17), whereas the pop- Fig. 2. Eletron mirographs of platinum shadowed A) polystyrene beads (109 nm), B) EYPC emulsion, C) DSPC emulsion, and D) hylomirons. Emulsions and hylomirons were ked with osmium and dialyzed against water before shadowing. flistograms in Fig. 3 inlude paniles shown here ( x 21,400). Journal of Lipid Researh Volume 31, 1990 Note on Methodology 165

4 ' F g 20 k n T r roo 2-3!9 h A Beads C DSPC Emulsion i n B EYPC Emulsion 1 L > D Chylomirons Fig. 3. Size distribution histograms ontaining all partiles analyzed in this study; A) polystyrene beads (inludes three samples), B) EYPC emulsion, C) DSPC emulsion, and D) hylomirons. Eletron mirographs of some of these partiles appear in Fig. 2. See Methods for details on measurements. ulations of EYPC and DSPC emulsions are more symmetrial. The relationships between shadow diameter and partile height for polystyrene beads, emulsions, and hylomirons are illustrated in Fig. 4. For polystyrene beads, shadow diameter was the same as partile height (Fig. 4A) indiating that the beads, whih are rigid, did not flatten. However, the heights of the emulsion and hylomiron partiles were redued by approximately nm (P < 0.05) (Fig. 4B-D). Sine the slopes of these lines did not differ signifiantly from 1.00, the observed flattening appears to be independent both of partile size and of surfae phospholipid hain saturation (physial state). t is likely that ore fluidity is a determining fator in the flattening of these partiles. Vertial profiles of seleted polystyrene and EYPC emulsion partiles shown in Fig. 5 illustrate the nature of the flattening. The rigid polystyrene bead (Fig. 5A) has minimal surfae ontat and is spherial. The emulsion partile in Fig. 5B shows typial flattening; partiles in Fig. 5C and 5D represent extremes in surfae ontat and shape. All three EYPC partiles are oblate spheroids slightly trunated where they ontat the arbon surfae. We have quantitated differenes in surfae ontat and expressed them as a perentage of total partile surfae area (Table 1). A omparison of two of the emulsion partiles (B and C of Fig. 5; 1 and 2 of Table 1) is partiularly interesting. Aording to projetions, these partiles have unequal apparent diameters and unequal areas of surfae to grid ontat beause of different degrees of flattening. However, their orreted diameters (spherial diameters) are almost the same. Sine these two partiles are presumably idential in omposition (30) and fixation, it is probable that the extent of partile flattening is related to differenes in adhesion to the grid surfae. n previous work, no attention was direted to possible irradiational effets of the eletron beam on fixed partiles; stability was assumed. Our data suggest that emulsion and hylomiron partiles are redued in size during eletron mirosopy (Fig. 6). n the absene of shrinkage the partile diameter should be equal to the shadow diameter as shown for the polystyrene beads (Fig. 6A). However, 166 Journal of Lipid Researh Volume 31, 1990 Note on MethodoloQ

5 400 h Y r m 300 ' a.- 0 r P a a ' 200 ' 300 Fig. 4. Relationships between shadow diameters and partile heights (.) of A) polystyrene beads, H) EYE emulsion, C) DSPC emulsion, and D) hylomirons. The linear repsion equation SD - PH * X + R giw the relation betwxn shadow diameter (SD) and panile height (PH). Equations are a5 follows: polystyrene beads: SD PH ; EYPC emulsion: SD PH ; DSPC emulsion: SD PH ; hylomirons: SD PH The dashed lines represent the 95% onfidene limits (P < 0.05). ' % Fig. 5. Vertial profiles of four partile3 and the shad& pakick from whih eah was projeted; A) polystyrene bead and R-D) EYPC emulsion paniles. R is an averaqe aw and C and > are extreme ases. An analysis of surfae arra ontat between eah pakile and the support film appan in Table 1 ( x 28,900). A B C D Journal of Lipid Researh Volume 31, 1990 Note on Methodolo,v 167

6 TABLE 1. Radii of Oblate Spheroidb Partiles a b Comparison of surfae area ontat between partiles and grid support film Radius of Surfae Contat Radius of Volume of Surfae Area Contat Area of as % of Total Sphere Sphere of Sphered Cirle* Contat Panile Surfae nm Polystyrene bead nla nla EYPC emulsion nm x 106nm2 x 105nm nm x i03nmz These four partiles are shown in Fig. 5 as follows: polystyrene bead (from 5A); EYPC emulsion partiles 1-3 (5B-5D). *Radius an equals one-half shadow diameter; radius b equals one-half partile height. Values for sphere radii of emulsions were obtained by assuming that volumes of oblate spheroids (413xa b) and spheres (4/37rr3) were equal; see Methods. dsurfae area equals 47rr. These values are estimates beause the projetions of partile bases in Fig. 5 have been derived from interpolated shadow ontours. Values were obtained by dividing the area of ontat ( d) by the total partide surfae area (47rr2) and multiplying by 100. the partile diameters of emulsions and hylomirons is loaded into the vauum evaporator it is subjeted to a larger than approximately 150 nm are systematially vauum of the same order of magnitude as in the eletron smaller than the shadow dmters (Fig. 6B, C, D). We ex- mirosope torr). Hene any volume hanges aused plain this phenomenon as follows. When a fixed partile by drying or redued pressure should our prior to metal l h 300 w Fig. 6. Relationships between shadow diameters and partile diameters (0) of A) polystyrene bertds, B) EYPC emulsion, C) DSPC emulsion, and D) hylomirons. The linear mgmsion equation SD = PD. X + B gives the dation between shadow diameter (SD) and partide diameter (PD). Equations are as follows: polystyrene beads: SD = 1.01 PD EYPC emulsion: SD = ; DSPC emulsion: SD = 0.88 PD , hylomirons: SD = 0.89 PD The dashed lines represent the 95% onfidene limits (P < 0.05). 168 Journal of Lipid Researh Volume 31, 1990 Note on Methodology

7 n f 300 Y 5 g 200 m a L m n L Correted Diameter nm! Fig. 7. Relationships between partile diameters and orreted diameters (A) of A) EYPC emulsion, B) DSPC emulsion, and C) hylomimns. The linear regression equation CD = PD. X + B giw the dation between orned diameter (CD) and partile diameter (PD). Equations are as follaws: EYPC emulsion: CD = 0.91 PD ; DSPC emulsion: CD = 0.86 PD ; hylomimns: CD = 0.89 PD The dashed lines represent the 95% onfidene limits (P < 0.05). shadowing. Further, if any distortion, deformation, or shrinkage ours during the evaporation of platinum, the resultant shadows should be indistint and diffiult to measure; this was not the ase. We onlude, therefore, that the redution in partile size ourred subsequent to shadowing and was probably due to an effet of irradiation on the triolein-rih ore. This effet (shrinkage) would be expeted to be most apparent in larger partiles, whih have larger ore:surfae ratios. Overall, the relationships between measured partile diameters and the orreted diameters were similar for both emulsions and hylomirons (Fig. 7). Measured diameters of partiles < 100 nm overestimate the orreted diameters, beause the partiles have flattened and widened. However, the measured diameters of partiles > 175 nm underestimate orreted diameters, probably beause the derease in diameter aused by partile shrinkage during irradiation is greater than the inrease in diameter due to TABLE 2. Comparison of mean shadow, partile and orreted diameters among hylomirons, EYPC and DSPC triolein emulsions Chylomirons EYPC Emulsion DSPC Emulsion Diameter (n - 213) (n - 88) (n - 268) nm Shadow Partile Correted f f f f f f * f * 29.7 Mean f SD. Eletron mirographs and size distribution histograms of these partiles appear in Fig. 2 and 3, respetively. Comparisons of mean differenes between partile and orreted diameters, partide and shadow diameters, or shadow and orreted diameters, by paired f-tests indiated signifiant differenes (P < 0.001). Journal of Lipid Researh Volume 31, 1990 Note on Methodology 169

8 flattening. Mean differenes between partile diameters and orreted diameters of EYPC and DSPC emulsions and hylomirons were statistially signifiant (P < 0.001) as determined by paired t-tests (Table 2). However, omparison of the mean partile and mean orreted diameters within eah preparation indiates that the opposing effets of shrinkage and flattening over the whole population are approximately equal (Table 2). Relatively small errors in radius measurements represent larger errors in alulated partile areas (proportional to r ) and partile volumes (proportional to r3). For example, four hylomiron partiles had partile diameters of 191 nm and orreted diameters of 198 nm, a differene of 3.5%. Differenes in surfae areas and volumes would be 6.9% and 10.2%, respetively. n summary, osmium-fixed EYPC and DSPC emulsions and hylomions are not rigid spheres when resting on grid surfaes. nstead, these partiles flatten and widen to beome slightly trunated oblate spheroids. The partile flattening is independent of partile size and of surfae phospholipid physial state. Differenes in the amount of flattening of two emulsion partiles of idential omposition and similar size an be explained by variation in the adhesion of these partiles to the grid. Shrinkage of hylomirons and EYPC and DSPC emulsions is attributed to irradiational effets on the triolein ores. Diret partile measurement of a population of fixed hylomirons or DSPC or EYPC emulsions is a reasonably aurate method to determine the mean of that population. However, measurements of subpopulations of very small or very large partiles are less aurate. We thank Dr. T. E. Felker for ritial disussion of the manusript. The assistane with statistial analysis by Suzette Levenson, Data Coordinating Center, -Shool of Publi Health, BUMC, was appreiated. Exellent tehnial help with preparation of figures was kindly provided by John Steiner, Dept. of Biophysis, BUSM. We also thank rene L. Miller for assistane with preparation of the manusript. This work was supported by National nstitutes of Health researh grant HL and training grant HL Manusript reeiued 9 June 1989 and in revised form 19 September REFERENCES 1. Salpeter, M. M., and D. B. Zilversmit The surfae oat of hylomirons: eletron mirosopy. J. Lipid Res. 9: Shoefl, G The ultrastruture of hylomira and of the partiles in an artifiial fat emulsion. Pm. R. SOC. Lond. B. Biol. Si. 169: Fraser, R Size and lipid omposition of hylomirons of different Svedberg units of fl0tation.j. Lipid Res. 11: Blanhette-Makie, E. J., and R. 0. Sow Retention of lipolyti produts in hylomirons inubated with lipoprotein lipase: eletron mirosope study. J. Lipid Res. 17: Bhattaharya, S., and T. G. Redgrave The ontent of apolipoprotein B in hylomiron partiles. J. Lipid Res. 22: Forte, T. M., and R. W. Noranausen Eletron mirosopy of negatively stained lipoproteins. Methodr Enzymol. 128: Glomset, J. A., K. Applegate, T. Forte, W. C. King, C. D. Mithell, K. R. Norum, and K. Gjone Abnormalities in lipoproteins of d < g/ml in familial leithin:holesterol ayltransferase defiieny. J. Lipid Res. 21: Forte, T. M., and L. A. Carlson Eletron mirosopi struture of serum lipoproteins from patients with fish eye disease. ArteriaFhk. 4: Du Plessis, J., L. R. Tiedt, C. J. van Wyk, and C. Akermann A new transmission eletron mirosope method for the determination of partile size in parenteral fat emulsions. Znt. J. Phai t. 34.: Hamilton-Attwell, V. L., J. du Plessis, and C. J. van Wyk A new sanning eletmn mirosope (SEM) method for the determination of partile size in parenteral fat emulsions. J. M S. 145: Connelly, P. W., and A. Kuksis Effet of ore omposition and partile size of lipid emulsions on apolipoprotein transfer of plasma lipoproteins in vivo. Bwhim Bwphys. Ata Forte, T., and A. V. Nihols Appliation of eletron mirosopy to the study of plasma lipoprotein struture. Ada Li id Res. 10: Jones, 1. L., and J. M. Prie Some methods of eletron mirosopi visualization of lipoproteins in plasma and hyle. J. HirtOhem. Cytohem. 15: Sata, T., R. J. Havel, and A. L. Jones Charaterization of subfrations of triglyeride-rih lipoproteins separated by gel hromatography from blood plasma of normolipemi and hyperlipemi humans. J. Lipid Res. 13: Hayes, T. L., and J. E. Hewitt Visualization of individual lipoprotein maromoleules in the eletron mirisope. J. AHG. physwi. 11: Bierman, E. L., T. L. Hayes, J. N. Hawkins, A. M. Ewing, and F. T. Lindgren Partile-size distribution of very low density plasma lipoproteins during fat absorption in man. J. Lipid&. 7: Fraser, R., W. J. Cliff, and F. C. Courtie The effet of dietary fat load on the size and omposition of hylomirons in thorai dut lymph. J. Exp. Phyhl. 53: Okner, R. K., and A. L. Jones An eletron mirosopi and funtional study of very low density lipoproteins in intestinal lymph. J. Lipid Res. 11: Small, D. M., J. W. Steiner, A. Derksen, and S. Bennett Clark Thermal transitions of phosphatidylholines on the surfae of lipoprotein-like emulsion partiles. Biophys. J. 53: 211a. 20. Derksen, A,, and D. M. Small nteration of apoa-1 and apoe-3 with triglyeride-phospholipid emulsions ontaining inreasing holesterol onentrations. Model of triglyeride-rih nasent and remnant lipoproteins. Biohemti@! Derksen, A., S. Ekman, and D. M. Small Olei aid allows more apoprotein A- to bind with higher affinity to large emulsion partiles saturated with holesterol. J. Biol. Ch. 264: Journal of Lipid Researh Volume 31, 1990 Note on Methodoloo

9 22. Bennett Clark, S Chylomiron omposition during duodenal triglyeride and leithin infusion. Am. J Physiol. 235: E185-El Bangs, L. B Uniform Latex Partiles. 2nd ed. Seragen Diagnostis, n., ndianapolis, N Analytial Referene Partiles. Duke Sientifi Gorp., Palo Alto, CA. Bulletin Dubohet, J., and M. Groom The mounting of maromoleules for eletron mirosopy with partiular referene to surfae phenomena and the treatment of support films by glow disharge. Adv. Opt. E h n Mk. 8: Reissig, M., and S. A. Orrell A tehnique for the eletron mirosopy of protein-free partile suspensions by the negative staining method. J Ulhnrtnrt. Res. 32: Hall, C. E Measurement of globular protein moleules by eletron mirosopy. J Biophys. Bioh. Cytol. 7: Slayter, E. M Eletron mirosopy of globular proteins. Zn Physial Priniples and Tehniques of Protein Chemistly. S. G. Leah, editor. Aademi Press, New York Misra, D. N., and N. N. Das Gupta Distortion in dimensions 'produed by shadowing for eletron mirosopy. J R. M ~ SOL.. 84: Miller, K. W., and D. M. Small Struture of triglyeride-rih lipoproteins: an analysis of ore and surfae phases. n Plasma Lipoproteins. A. M. Gotto, J. editor. New Comprehensive Biohemistry, Vol. 14. Elsevier, Amsterdam, The Netherlands Journal of Lipid Researh Volume 31, 1990 Note on Methodology 171