Separation of Serum Lipoproteins of Japanese Quail by Disc Polyacrylamide Gel Electrophoresis and Single Discontinuous Density Gradient Ultracentrifugation1

Separation of Serum Lipoproteins of Japanese Quail by Disc Polyacrylamide Gel Electrophoresis and Single Discontinuous Density Gradient Ultracentrifugation1 TA-CHUNG WU2 and W. E. DONALDSON3 Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27650 (Received for publication March 1, 1982) ABSTRACT A method based on disc polacrylamide gel electrophoresis (disc PAGE) for the separation of serum lipoproteins of Japanese quail (Cotumix cotumix japonica) prestained with Sudan Black B is described and evaluated. Good separation was obtained by using 3% separating gel and decreasing the concentration of buffer solution A used for preparation of separating gel solution to one-half that recommended by Narayan (1975). Best resolution of the high density lipoproteins was achieved by using 7 to 10% of separating gel, which separated the high density lipoproteins into three bands. Clear lipoprotein bands were obtained after ultracentrifugation of prestained serum. For corresponding positions, the bands of unstained serum after staining had the same mobility in the disc (PAGE) as prestained lipoprotein bands. In quail, density smaller than 1.006 g/ml contains chylomicrons and very low density lipoproteins (VLDL); density from 1.018 to 1.05 g/ml contains the low density lipoprotein (LDL); and density more than 1.05 g/ml and less than 1.16 g/ml contains the high density lipoproteins (HDL). In the profile of serum lipoproteins after ultracentrifugation, two peaks were observed in the density range of HDL. There was one small peak with density 1.05 to 1.09 g/ml and one large peak with density 1.09 to 1.16 g/ml. Based on the lipoprotein profiles from both disc PAGE and ultracentrifugation, HDL is the predominant form, LDL is intermediate, and VLDL and chylomicrons are smallest in amount. (Key words: Japanese quail, serum lipoproteins, electrophoresis, ultracentrifugation) 1982 Poultry Science 61:2398-2406 INTRODUCTION Lipids are n o t sufficiently polar t o circulate freely in t h e serum of animals. Therefore, t h e y occur with p r o t e i n s in soluble complexes k n o w n as lipoproteins (Alfin-Slater, 1 9 7 4 ; White et al, 1 9 7 8 ) . Various m e t h o d s can b e used in t h e analysis of serum lipoproteins (see t h e review b y H a t c h and Lees, 1 9 6 8 ) . T h e m o s t c o m m o n l y used m e t h o d s are electrophoresis and ultracentrifugation (Hatch and Lees, 1 9 6 8 ; Houtsmuller, 1 9 6 9 ; Lindgren et al, 1 9 7 2 ) . Electrophoresis allows fication of t h e various quantitative analysis is inconsistent u p t a k e of

t h e qualitative identilipoproteins, b u t t h e difficult because of staining d y e b y t h e

'Paper No. 8195 of the Journal Series of the North Carolina Agricultural Research Service. The use of trade names in this publication implies neither endorsement by the North Carolina Agricultural Research Service of the products named nor criticism of similar products not mentioned. 2 Submitted to the Graduate School of North Carolina State University in partial fulfillment of the requirements for the Master of Science degree by T.C. Wu. 3 To whom reprint requests should be addressed.

lipoproteins (Hatch a n d Lees, 1 9 6 8 ; Lindgren et al, 1 9 7 2 ; Narayan, 1975). T h e media m o s t c o m m o n l y used in electrophoresis are paper, cellulose acetate, agarose gel, and polyacrylam i d e gel. Best results are o b t a i n e d b y using p o l y a c r y l a m i d e gel, because excellent separation into multiple discrete bands is achieved, and optically t h e m e d i u m is completely clear and devoid of color (Hatch and Lees, 1 9 6 8 ; Houtsmuller, 1 9 6 9 ; Narayan, 1 9 7 5 ) . T h e results of p a p e r electrophoresis of serum lipoproteins enabled Fredrickson et al. ( 1 9 6 7 ) t o classify t h e familial h y p e r l i p o p r o t e i n e m i a into five distinct t y p e s . T h e profiles of serum lipoproteins on polyacrylamide gel p e r m i t t e d R o b i e et al. ( 1 9 7 5 ) t o d e m o n s t r a t e t h e differences b e t w e e n t w o horse species and p e r m i t t e d J e n s e n et al. ( 1 9 7 8 ) t o show t h e differences d u e to age in pigeons. Ultracentrifugation allows t h e precise quant i t a t i o n of t h e various lipoproteins. T h e conventional t e c h n i q u e for separating lipoproteins is preparative ultracentrifugation, which employs cumulative floatation. T h e lipoproteins are separated sequentially according to their h y d r a t e d density b y a p p r o p r i a t e a d j u s t m e n t of

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the density of the medium with KBr or NaBr and NaCl (Havel et al, 1955; Hatch and Lees, 1968; Lindgren et al, 1972; Lindgren, 1975). However, those methods are time-consuming and laborious. Recently, more rapid and simpler techniques have been developed by using discontinuous density gradients with lipoproteins separated in a single run (Redgrave et al, 1975; Chung et al, 1980) or by using an airfuge (Bronzert and Brewer, 1977) in which a small amount of plasma (175 Jill) is applied and separated within a few hours. McDonald and Ribeiro (1959) introduced a procedure for prestaining serum with Sudan Black B before application of paper electrophoresis. This dye stains the lipid portion of lipoproteins, and clear bands of lipoproteins can be detected after electrophoresis. The prestaining with Sudan Black B has also been used with polyacrylamide gel electrophoresis for the separation of serum lipoproteins of humans (Frings et al, 1971; Hall et al, 1972; Ultermann, 1972; Allen, 1972; Mead and Dangerfield, 1974; Muniz, 1977) and other animals (Narayan, 1975; Narayan and Calhoun, 1975; Robie et al, 1975; Jensen et al, 1978; Yashiro and Kirmura, 1980). The prestaining technique has been applied also to ultracentrifugation. Cornwell and Kruger (1961) correlated the intensity of each prestained lipoprotein with its lipid content after ultracentrifugation. Bronzert and Brewer (1977) demonstrated the elevation of different lipoproteins after ultracentrifugation with Fat Red 7B prestaining. Advances have been made in research on serum lipoproteins both in human and other animal species (Mills and Taylaur, 1971;Lasser et al, 1973; Mahley, 1978; Mahley and Holcomb, 1977; Mahley and Weisgraber, 1974a,b; Mahley et al, 1976; Scanu et al, 1975; Narayan, 1975; Shore and Shore, 1976; Rudel et al, 1977). These advances were due to a substantial increase in general interest in the study of atherosclerosis associated with an abnormalities in circulating lipoproteins. Japanese quail proved to be a good experimental model for the study of atherosclerosis (Smith and Hilker, 1973; Day and Stafford, 1975), but their lipoprotein profiles have not been elucidated. The purpose of this study was to separate the serum lipoproteins of Japanese quail (Coturnix coturnix japonica) by using both disc PAGE and single discontinuous density gradient

ultracentrifugation profile.

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MATERIALS AND METHODS

Animals. Adult, male, Japanese quail (Coturnix coturnix japonica) were housed in wire cages (2 per cage) with raised floors and kept in an air conditioned room starting at 5 weeks of age. The birds were fed a corn-soybean meal layer diet (24% protein, 3% fat and 2805 kcal ME/kg). Feed and water were available ad libitum. At approximately 12-weeks of age, birds were decapitated after overnight fasting (2100 to 0900 hr), and blood was collected. Serum was obtained by low-speed centrifugation after the blood had clotted for 1 hr at room temperature as described by Frings et al. (1971). Part of the serum of each bird was prestained with Sudan Black B solution in the ratio of 5:1 (serum:dye) as suggested by McDonald and Ribeiro (1959). The mixture was allowed to stand at room temperature in the dark for 1 hr and then the excess dye was removed by centrifugation. Both serum and prestained serum were maintained at 4 C until needed. Sudan Black B solution was prepared by the method of Narayan (1975). Disc Polyacrylamide Gel Electrophoresis. The solution identification system and solution compositions were those used by Frings et al. (1971). The reservoir buffer (pH 8.3 to 8.4) was 3 g Tris base (Sigma, St. Louis, MO) and 14.4 g glycine (Fisher, Fair Lawn, NJ) in 1 liter of deionized water. Three gel solutions were prepared. The concentrating gel solution was obtained as described by Frings etal. (1971). The separating gel solutions, ranging from 2 to 10% of monomer concentration of polyacrylamide in .5% increments, were made according to Narayan (1975), except that the buffer solution A contained 1.5 M Tris (hydroxymethyl) amino methane, ImM N.N.N 1 .N 1 tetramethyl-ethylenediamine in .24N HC1 (one-half the recommended concentrations). The sample gel solution was prepared by mixing .2 ml of concentrating gel solution with 25 fil of prestained serum or 20 jul of unstained serum. In both cases the sera were placed on the concentrating gel and mixed by inversion of the gel tube. The gel solution were added to each gel tube (i.d. 5.8 mm; length 75 mm) and polymerized as described by Frings et al (1971) with the

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10% FIG. 1. Separation of serum lipoproteins of pooled, prestained sera by disc PAGE on different percent separating gels in the order of 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, and 10%.

following modifications: a) 1.2 ml of separating gel solution and .2 ml of concentrating gel solution per gel tube were used; and b) the sample gel solution was layered with reservoir buffer and polymerized for 20 min. Then the gel tubes were inserted in the electrophoresis cell in which the upper and lower parts were filled with buffer at 4 C as recommended by Ha.Uet al. (1972). Electrophoresis was performed for 30 min at 5mA per gel tube at 4 C. Densitometric tracings were obtained by scanning the prestained serum after electrophoresis on 3% gel at 620 ran (Muniz, 1977) with a Model UA-5 Absorbance Monitor equipped with a type 6 optical unit (ISCO, Lincoln, NE). The results were compared and recorded from both prestained and unstained serum after electrophoresis on 3% gel. Gels of unstained serum were removed from the gel tubes and immersed in Sudan Black B solution for 3 hr and then rinsed with water. The results of the poststaining method were compared with the prestaining method. Discontinuous Density Gradient Ultracentrifugation. One milliliter of pooled unstained sera or .6 ml of pooled prestained sera and .1 ml of dye solution were pipetted into thin-wall polyallomer centrifuge tubes (o.d., 14.5 mm; length, 96 mm; capacity, 12 ml). All samples were adjusted to density 1.2163 g/ml with solid

NaBr (Fisher) calculated by the method of Lindgren (1975). The density of the dye solution was considered as 1.1088 g/ml, the specific gravities of ethylene glycol at 20 C, and the dye solutions were adjusted in the same manner as serum. The ultracentrifugation was performed as described by Redgrave et al. (1975). The adjusted sample solutions were added to a NaBr solution with a density of 1.2163 g/ml so that the total volume was 3.0 ml. The discontinuous density gradients were then formed by layering the top of the sample with 3 ml each of the following densities of NaBr solutions: 1.063 g/ml, 1.019 g/ml, and 1.006 g/ml. To minimize mixing at the density junctions, the solutions were added by a density gradient former (ISCO, Model 570). The samples were centrifuged in a B-60 ultracentrifuge equipped with a SB-283 swinging bucket rotor (I.E.C., Needham Heights, MA) for 48 hr at 20 C and 41,000 rpm (283,200 X g). Fractions of .6 ml each were collected with a density- gradient fractionator (ISCO, Model 640) by puncturing the bottom of the centrifuge tube and using a chase solution of NaBr (density 1.308 g/ml). Densitometry at 620 nm was performed during collection using the same equipment as for disc PAGE. The final density gradient shape at the end of the run was determined by forming a discon-

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FIG. 2. Separation of serum lipoproteins by disc PAGE on 3% gel. Tubes 1 to 3 were bird 1, Tubes 4 to 6 were bird 2, Tubes 7 to 9 were bird 3. Twenty-five microliters of prestained serum was applied in Tubes 1, 4, and 7; 20 ul of unstained serum in Tubes 2, 3, 5, 6, 8, and 9.

tinuous density gradient without sample solution. The refractive index of each fraction was measured with an Abbe Refractometer (Bausch and Lomb Co., Rochester, NY) at 20 C and the values converted to density using standard tables. The purity of the lipoprotein from ultracentrifugation was checked by disc PAGE. Fractions from the same peak by densitometry were pooled as follows (see results): fraction 1, VLDL and chylomicrons; fractions 2 to 4 (no peak); fractions 5 to 9, LDL; fractions 10 to 12, first HDL peak; and fractions 14 to 17, second HDL peak. Each pooled lipoprotein was electrophoresed on 3% gel. Pooled fractions from unstained sera were stained prior to electrophoresis.

With only dye solution, the dye remained at the origin, and this led to difficulty in observing the chylomicron band. Whether the chylomicron band exists or is an artifact of dye solution is not certain. With gels below 3.5% concentration, the LDL band migrated deeper into the separating gel, but diffusion occurred at less than 2.5% separating gel. The LDL band was completely blocked by separating gel concentrations greater than 5%. The HDL bands appeared as two sharp, close bands in the a region with separating gel concentrations below 3.5%. The HDL bands began to diffuse when the gel concentration was increased, and were separated to three bands between 7 to 10% gel. The mobilities of serum lipoproteins with and without prestaining are compared in Figure RESULTS 2. Yellow bands of HDL without prestaining Disc Polyacrylamide Gel Electrophoresis. were observed, and in all cases these bands Figure 1 shows the serum lipoproteins prestained migrated slightly faster than HDL bands with with Sudan Black B after separation with prestaining. This indicates that the dye solution different percentages of gels. In all cases, VLDL slowed the migration of the HDL bands. was trapped just after entering the separating Prestained LDL with more intensive color gel whereas the chylomicrons remained between (birds 2 and 3) migrated more slowly than LDL the sample gel and concentrating gel. These two from bird 1. This implies that LDL with high bands were difficult to observe visually. lipid content migrates more slowly, because the

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dye is specific for the lipid portion of the lipoprotein. The corresponding position of the LDL bands without prestaining from birds 2 and 3 showed a faint yellow band that indicated that the dye solution did not affect the mobility of the LDL. The effects of prestaining and poststaining of gels are compared in Figure 3. A marked background was apparent in poststaining gels, which made densitometry difficult. Diffusion of bands of prestained lipoproteins occurred increasingly during storage after separation, especially in the HDL bands. This is one defect of prestaining and suggests that densitometry should be performed as soon as possible after separation. Figure 4 demonstrates diagramatically the typical serum lipoprotein pattern and the profile from densitometry after disc PAGE on 3% gel. Although the HDL showed two bands in the a region, only one peak appeared on the profile owing to the proximity and diffusion of these two bands. Based on the color intensity and profile from densitometry, HDL is predominant, LDL is intermediate, and VLDL and chylomicrons are smallest in amount. Discontinuous Density Gradient Ultracentrifugation. Figure 5 shows the position of the major serum lipoprotein bands separated by discontinuous density gradient ultracentrifugation. There were three blue regions observed in

0

1

2

3

4

FIG. 3. Separation of serum lipoproteins by disc PAGE on 3% gel. Tubes 1 and 2 were bird 1, Tubes 3 and 4 were bird 2. Gel in Tubes 2 and 4 were poststained with Sudan Black B solution.

the prestained serum after ultracentrifugation. Chylomicrons and VLDL were present together in the top region, LDL appeared in the middle region, and HDL appeared in the lowest region.

B -Chylomicrons -

"=+*- V L D L (post-B) ~-LDL(B)

—HDL ( « ) -

J

© 1 -

Sample gel

2 -

Concentrating gel

3 - Separating gel

FIG. 4. Diagram showing the serum lipoprotein pattern after separation by disc PAGE on 3% separating gel (A), and a typical profile presented by their densitometric plots (B). All lipoproteins were scanned at an absorbance range of 1 with chart speed 30 cm/hr except the HDL, which was scanned at an absorbancc range of 2.

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Chylomicrons

FIG. 5. Diagram showing: (A) the visual observation of major serum lipoproteins of .5 ml serum plus .1 ml dye after ultracentrifugation; and (B) the lipoprotein profile by densitometry at 620 nm; and their correlation to (C) each fraction (.6 ml/tube) and (D) density at 20 C. • = Distinct band, heavily stained; » = distinct band, lightly stained; ss = area of diffuse staining.

Protein debris and excess dye appeared at the bottom of the centrifuge tube. In the corresponding positions of unstained lipoproteins, a white cloudy band floated at the top. A band of clear visible yellow color was located at the position corresponding to HDL. Nothing was observed in the LDL region through the transparent centrifuge tube. Pure dye solution without serum after ultracentrifugation resulted in the precipitation of the dye on the bottom of the tube and the adherence of some dye to the tube. The lipoprotein profile obtained from ultracentrifugation is shown diagramatically in Figure 5. Quantitation of VLDL plus chylo-

microns was achieved, but VLDL and chylomicrons were not separated as compared with disc PAGE. Again, HDL appeared to be the predominant lipoprotein, LDL was intermediate, and VLDL plus chylomicrons were smallest in amount. During the course of centrifugation, there was a smoothing of the discontinuous density gradient of NaBr without serum sample. The relationship between each peak of lipoprotein and density after ultracentrifugation, as shown in Figure 5, was corrected for changes in the density gradient centrifuged without sample. Peak one contained VLDL and chylomicrons and floated at the top of the solution with a

TABLE 1 Co rresponden ce of lip ^proteins separated by ultra centrifugation and disc PAGE

Classificat on Chylomicron + VLDL LDL HDL HDLa HDLb

Disc PAGE

Ultracentrifugal fractions

3% Separating gel

1 2- 4 5- 9 10-17 10-12 14-17

Origin and post-/ No band 0 a (two bands) a ( o n e band) a (two bands)

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density less than 1.006 g/ml. There was no peak between density 1.006 and 1.018 g/ml. Peak two represents LDL that started at density 1.018 g/ml and ended at density 1.05 g/ml. Peaks three and four were HDLa with a density of 1.05 to 1.09 g/ml and HDLb with a density of 1.09 g/ml to 1.16 g/ml, respectively. The last small peak was due to protein debris and excess dye. Each lipoprotein band obtained after ultracentrifugation was subjected to disc PAGE and the results are shown in Table 1. Each lipoprotein behaved in the same manner as the corresponding lipoprotein of whole serum. This results indicates comparable separation of serum lipoproteins by both methods. DISCUSSION

Allen (1972) indicated that gel concentration and buffer ions play important roles in the separation of lipoproteins by the gel techniques. Three solutions are required for preparing separation gel solution: polyacrylamide gel solution, buffer solution, and ammonium persulfate solution. Narayan (1975) reported there is no influence of ammonium persulfate. Robie et al. (1975) claimed that the optimum resolution of serum lipoproteins in all species will not necessarily be obtained by the same type of electrophoretic method. It is rational to adjust both the buffer solution and polyacrylamide gel solution to obtain the condition for separating serum lipoproteins of Japanese quail by disc PAGE. Reduction of the concentration of buffer solution A to one-half in Narayan's method (1975) resulted in less diffuse and sharper HDL bands. Because discrete, clear HDL bands can be achieved in this way, no attempt was made to study this buffer solution further. Separation of lipoproteins (or proteins) by PAGE depends on both electrophoretic mobility and molecular size (Frings et al., 1971). With a low percentage of gel the molecular sieving effect on LDL became less and, thus, the mobility of LDL into separating gel was increased. The LDL migrated further in 2 and 2.5% gel than 3% gel, yet 2 and 2.5% gel were too soft to be handled if further study of lipoproteins was necessary, e.g., extracting the lipoproteins from the gel. A 3% gel is suggested and has been used by many groups (Allen, 1972; Hall et al, 1972; Sezille et al, 1975). A 3.125% gel was used by Muniz (1977). With higher percentage of gel, HDL tends to separate

further. Ultermann (1972) observed four bands of HDL in human serum, and we observed three bands of HDL in Japanese quail serum using 7.5% gel. Quantitation of the chylomicron band by disc PAGE Was difficult because excess dye remained at the origin. This problem might.be solved by using a large pore size of concentrating gel as described by Hall et al. (1972) and allowing the chylomicrons to migrate into the concentrating gel. The most important defect of Sudan Black B prestaining (true of all lipid stains) is that the intensity of the bands does not reflect absolute quantities of lipid (Hatch and Lees, 1968; Narayan, 1975). However, Muniz (1977) was able to demonstrate a linear response between the relative amount of Sudan Black B bound to the different lipoproteins and the amount of bound cholesterol in his PAGE study. Cornwell and Kruger (1961) found that total cholesterol, as well as chylomicron and Sf 10-400 lipoprotein cholesterol, correlate well with staining intensity if Sudan Black B whereas Sf 0-10 and HDL cholesterol do not. Different preparations of dye solution and different methods and ratios of mixing serum and dye could no doubt lead to different qualitative and quantitative interpretation. Whether the serum lipoprotein profile of Japanese quail in the present experiment is really a measure of cholesterol distribution or not remains to be studied. Different animal species have different density ranges for their lipoproteins (Mahley, 1978). The density ranges may or may not be the same as human density ranges. Applying the density ranges of humans to animals might lead to a false result, and this situation should only be used for comparative purposes (Mills and Taylaur, 1971). Obtaining the density ranges of lipoproteins for various animals is a tedious job that can be accomplished only by ultracentrifugation and subjecting the resulting fractions to electrophoresis or some other technique (Lasser et al., 1973; Mahley and Weisgraber, 1974a,b; Rudel et al, 1977). In this study, by application of prestaining technique to discontinuous density gradient, the density can be measured directly by a single ultracentrifugation, which with subsequent electrophoresis, provides a simple method to determine the density range of serum lipoproteins. REFERENCES Alfin-Slater, R. B., 1974. The chemistry of lipids. Page

QUAIL SERUM LIPOPROTEINS 20—23 in Atherosclerosis. F. J. Stave, ed., Medcom, New York, NY. Allen, R. C., 1972. Electrophoresis separation of pre-stained lipoproteins on polyacrylamide gel slabs and their relationship to other plasma proteins. Pages 287—297 in Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel. R. C. Allen, and H. R. Maurer, ed., Walter de Gruyter, New York, NY. Bronzert, T. J., and B. Brewer, Jr., 1977. New micromethod for measuring cholesterol in plasma lipoprotein fractions. Clin. Chem. 23:2089-2098. Chung, B. H., T. Wilkinson, J. C. Geer, and J. P. Segret, 1980. Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor. J. Lipid Res. 2 1 : 2 8 4 - 2 9 1 . Cornwell, D. G., and F. A. Kruger, 1961. Lipoprotein pre-staining and ultracentrifugation analysis in a density gradient. Proc. Soc. Exp. Biol. Med. 107:296-299. Day, C. E., and W. W. Stafford, 1975. New animal model for atherosclerosis research. Pages 339—347 in Lipids, Lipoproteins, and Drugs. D. Kritchevsky, R. Paolette, and W. L. Holmes, ed. Plenum Press, New York, NY. Fredrickson, D. S., R. I. Levy, and R. S. Lees, 1967. Fat transport in lipoproteins. An integrated approach to mechanisms and disorders. New England J. Med. 276:33-34, 9 4 - 1 0 3 , 148-156, 215-225, 2 7 3 - 2 8 1 . Frings, C. S., L. B. Foster, and P. S. Cohen, 1971. Electrophoresis separation of serum lipoproteins in polyacrylamide gel. Clin. Chem. 17:111—114. Hall, F. F., C. R. Ratliff, C. L. Westfall, and T. W. Culp, 1972. Serum lipoprotein electrophoresis: An improved polyacrylamide procedure. Biochem. Med. 6:464-470. Hatch, F. T., and R. S. Lees, 1968. Practical method for plasma lipoprotein analysis. Adv. Lipid Res. 6:1-68. Havel, R. J., H. A. Eder, and J. H. Bragdon, 1955. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34:1345-1353. Houtsmuller, A. J., 1969. A survey of the literature. Pages 1—8 in Agarose-Gel Electrophoresis of Lipoproteins. A. J. Houtsmuller, ed. Royal VanGorcum, Assen, Netherland. Jensen, P. F., G. L. Jensen, and S. C. Smith, 1978. Serum lipoprotein profiles of young atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons. Comp. Biochem. Physiol. 60B:67-69. Lasser, N. L., P. S. Roheim, D. Edelstein, and H. A. Eder, 1973. Serum lipoproteins of normal and cholesterol-fed rats. J. Lipid Res. 1 4 : 1 - 8 . Lindgren, F. T., 1975. Preparative ultracentrifugal laboratory procedures and suggestions for lipoprotein analysis. Pages 204—224 in Analysis of Lipids and Lipoproteins. E. G. Perkins, ed., Am. Oil Chem. Soc, Champaign, IL. Lindgren, F. T., L. C. Jensen, and F. T. Hatch, 1972. The isolation and quantitative analysis of serum lipoproteins. Pages 182—270 in Blood Lipids and Lipoproteins: Quantitation, Composition, and Metabolism. G. J. Nelson, ed. Wiley-Interscience,

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New York, NY. Mahley, R. W., 1978. Alterations in plasma lipoproteins induced by cholesterol feeding in animals including man. Pages 181 — 197 in Distribances in Lipid and Lipoprotein Metabolism. J. M. Dietschy, A. M. Gotto, Jr., and J. A. Ontko, ed. Am. Physiol. Soc, Bethesda, MD. Mahley, R. W., and K. S. Holcombe, 1977. Alterations of the plasma lipoproteins and apoproteins followng cholesterol feeding in the rat. J. Lipid Res. 18:314-324. Mahley, R. W., and K. H. Weisgraber, 1974a. An electrophoretic method for the quantitative isolation of human and swine plasma lipoproteins. Biochem. 13:1964-1969. Mahley, R. W., and K. H. Weisgraber, 1974b. Canine lipoproteins and atherosclerosis. Isolation and characterization of plasma lipoproteins from control dogs. Circ. Res. 3 5 : 7 1 3 - 7 2 1 . Mahley, R. W., K. H. Weisgraber, T. Innerarity, and H. B. Brewer Jr., 1976. Characterization of the plasma and apoproteins of the Erythrocebus patos monkey. Biochemistry 15:1928—1933. McDonald, H. J., and L. P. Ribeiro, 1959. Ethylene and propylene glycol in the pre-staining of lipoproteins for electrophoresis. Clin. Chim. Acta 4:458-459. Mead, M. G., and W. G. Dangerfield, 1974. The investigation of "midband" lipoproteins using polyacrylamide gel electrophoresis. Clin. Chim. Acta 51:173-182. Mills, G. L., and C. E. Taylaur, 1971. The distribution and composition of serum lipoproteins in eighteen animals. Comp. Biochem. Physiol. 40B-.489-501. Muniz, N., 1977. Measurement of plasma lipoproteins by electrophoresis on polyacrylamide gel. Clin. Chem. 23:1826-1833. Narayan, K. A., 1975. Electrophoretic methods for the separation of serum lipoproteins. Pages 225—249 in Analysis of Lipids and Lipoproteins. E. G. Perkin, ed. Am. Oil Chem. Soc, Champaign, IL. Narayan, K. A., and W. K. Calhoun, 1975. The influence of some dietary factors and/or treadmill exercise on rat and chicken tissue lipids and serum lipoproteins. Pages 383—401 in Atherosclerosis Drug Discovery. C. E. Day, ed. Plenum Press, New York, NY. Redgrave, T. G., D.C.K. Roberts and C. E. West, 1975. Separation of plasma lipoproteins by densitygradient ultracentrifugation. Anal. Biochem. 65:42-49. Robie, S. M., S. C. Smith, and J. T. O'Conner Jr., 1975. Equine serum lipids: Serum lipoprotein profiles of Morgan and thoroughbred horses. Am. J. Vet. Res. 36:1709-1713. Rudel, L. L., D. G. Greene, and R. Shah, 1977. Separation and characterization of plasma lipoproteins of rhesus monkey (Macaca mulatta). J. Lipid Res. 18:734-744. Scanu, A. M., C. Edelstein, and P. Keim, 1975. Serum lipoproteins. Pages 317—391 in The Plasma Proteins. R. W. Putnam, ed. 2nd ed. Vol. 1, Academic Press, New York, NY. Sezille, G., J. C. Fruchart, J. Jaillard, P. Dewailly, and C. Desreumaux, 1975. Improved serum lipopro-

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tein electrophoresis procedure in polyacrylamide gradient gel. Biomed. 23:315-317. Shore, B., and V. Shore, 1976. Rabbits as a model for the study of hyperlipoproteinemia and atherosclerosis. Pages 123—141 in Atherosclerosis Drug Discovery. C. E. Day, ed. Plenum Press, New York, NY. Smith, R. L., and D. M. Hilker, 1973. Experimental dietary production of aorta atherosclerosis in Japanese quail. Atherosclerosis 17:63—70. Utermann, G., 1972. Human serum lipoproteins: Differentiation and characterization by disc electrophoresis. Pages 287—297 in Electro-

phoresis and Isoelectric Focusing in Polyacrylamide Gel. R. C. Allen and H. R. Maurer, ed., Walter de Gruyter, New York, NY. White, A., P. Handler, E. L. Smith, R. L. Hill, and I. R. Lehman, 1978. Page 568-606 in Lipid Metabolism. I. Principles of Biochemsitry. 6th ed. A. White, P. Handler, E. L. Smith, R. L. Hill, and I. R. Lehman, ed. McGraw-Hill, New York, NY. Yashiro, M., and S. Kirmura, 1980. Effect of voluntary exercise and dietary protein levels on serum lipoprotein distribution and lecithin cholesterol acyltransferase activity of mice. J. Nutr. Sci. Vitaminol. 26:59-69.