The lipid composition of human serum lipoproteins.

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Content THE LIPID COMPOSITION OF HUMAN SERUM LIPOPROTEINS by TAKAO USHIYAMA A Dissertation Presented to the FACULTY- OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry and Nutrition) June 1958 UMI Number: DP21586 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI DP21586 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 •ph. D 'SS U-8S T his dissertation, w ritte n by ...........Takao.Ushiyama under the direction o f his G uidance C o m m itte e , and app ro ve d by a ll its members, has been p r e sented to and accepted by the F a c u lty of the G raduate School, in p a rtia l fu lfillm e n t of re quirem ents f o r the degree of D O C T O R O F P H I L O S O P H Y Dean G u id a n c e C o m m ittee .. f Chairman ACKNOWLEDGEMENTS It is with sincere gratitude that I acknowledge the invaluable guidance, counsel, and encouragement of Dr. John W. Mehl. I would like to express my appreciation to the staff and faculty of the Department of Biochemistry and Nutrition for their generous aid and counsel during my graduate career. I am particularly grateful to the members of my guidance committee whose advice and dis cussions have been of great value to me. To the National Heart Institute of the National Institutes of Health of the United States Public Health Service is extended my appreciation for the funds which allowed the completion of this work. I wish to thank my colleagues for the help and stimulation I have received from them during numerous discussions. My special thanks go to Dr. Robert S. Levy for his close cooperation in certain phases of this work. I also wish to thank my sister, Fumi, who so generously gave her time in the typing of this dissertation. TABLE OF CONTENTS CHAPTER PAGE I. HISTORICAL REVIEW ................ . 1 v Isolation and Analysis of Lipoproteins from Serum ...................... 1 Metabolic Interrelationships ........... ' 13 II. MATERIALS AND METHODS..........' ................23 Dextran Sulfate ..... .................. 23 r Rice Starch Sulfate . . . . . . . . . . . . 23 Bovine Plasma Albumin . .................... 23 Serum............................ 23 Ultracentrifugal Isolation of Lipoproteins 25 Zone Electrophoresis on Starch ...... 25 Cholesterol Determination 27 ! 1 Lipid Phosphorus Determination 28 j j Paper Electrophoresis . 28 j , i Moving Boundary Electrophoresis ........... 29 ] Analytical Centrifugation . ............... 31 Total Weight of the Lipoprotein Fractions by Dry Weight Determinations............. 32 Delipidation of the Lipoprotein Fractions . 33 Total Weight of the Lipid Extracts by Dry Weight Determinations . ..................3^ Protein Content.......... 3^ Purification of the Lipid Extracts .... 3^ V CHAPTER PAGE Hydrolysis of the Purified Lipid Extracts . 35 Extraction of Sphingosine ................... 35 Concentration of the Water Soluble Nitrogenous Bases after Hydrolysis . . . 36 Choline Determination ............... ...37 Nitrogen Determination ..................... 37 Density Determinations . ............. . 37 III. EXPERIMENTAL RESULTS.................... 39 Electrophoresis of Serum Lipoproteins on Starch....................................39 Separation of |3-Lipoprotein by Flotation . 39 Separation of Lipoproteins/with Dextran Sulfate ....... 44 Bulk Isolation" of Lipoprotein Species from Serum . . .......................... 47 Concentration of Lipoproteins with a Hydrated Density Less than I.O63 gm. per ml. ....... .................... 50 Recovery of Low Density Lipoproteins . . . 50 Recovery of High Density Lipoproteins . . . 51 Density of the Gradient Layers ............ 53 Analytical Centrifugation ................... 54 Moving Boundary Electrophoresis ............ 57 Composition of the Lipoproteins ............ 57 vi CHAPTER PAGE Estimation of the Nitrogenous Constituents of Lipoprotein Phospholipids ...... 60 IV. DISCUSSION................................. 63 V. SUMMARY . ......................................76 BIBLIOGRAPHY..................................... .. . 77 LIST OF TABLES TABLE PAGE I. Per Cent Composition of Various Plasma Lipoproteins ........................ . 2 II. Lipoproteins of Human Serum .... 4 III. The Lipid Composition of IsolatedtFractions in Three Normal Humans . . . ............... 9 IV. Estimation of the Composition of Human Serum Lipoproteins ................................. 10 V. Composition of Serum Lipoprotein Fractions . . 12 VI. Properties of Sulfated Polysaccharides Used for Lipoprotein Fractionation ....... 24 VII. Reagents Used in the Ninhydrin Method of Moore and S t e i n ......................................30 VIII. Density of the Gradient Layers Containing the Lipoproteins.............. .................. 55 IX. Sf Values of Lipoproteins Prepared by Rice Starch Sulfate Precipitation . . ........... 58 X. Per Cent Composition of Serum Lipoprotein Fractions ...... .................. 59 XI. Nitrogenous Constituents of Human Serum Lipoprotein Lipids ................... 61 LIST OF FIGURES FIGURE PAGE 1 . Starch block apparatus with sample inserter . 26 2A. Starch block electrophoresis of serum at an ionic strength of 0 . 1 0 .................... 39 2B. Starch block electrophoresis of serum at an ionic strength of 0 . 0 5 ............. 39 3A. Starch block electrophoresis of ultracen- trifugally isolated lipoproteins before dialysis..................... 41 3B. Starch block electrophoresis of ultracen- trifugally isolated lipoproteins (24 hour flotation period) after dialysis ......... 41' 4 . Paper electrophoretic analyses of serum and lipoproteins — isolated with a 24 hour ultracentrifugal period by flotation (1.063 gm./ml. salt density) ............. 42 5A. Starch block electrophoresis of ultracen- trifugally isolated lipoproteins (48 hour flotation period) after 24 hours ......... 43 5B. Starch block electrophoresis of ultracen- trifugally isolated lipoproteins (48 hour flotation period) after .5^ h o u r s ......... 43 6 . Paper electrophoretic analysis of serum after dextran sulfate precipitation .... 46 ix FIGURE PAGE 7 . Starch block electrophoresis of dextran sulfate precipitated lipoproteins ........... 48 8 . Displacement apparatus ...................... . 52 9 . Flotation patterns,of serum lipoproteins isolated by the precipitation-flotation method.............................. 56 CHAPTER I i HISTORICAL REVIEW I Isolation and Analysis of Lipoproteins from Serum j - i The concept that lipids in plasma do not exist in a 'free state but are combined with each other and with pro- j iteins as definite entities is of relatively recent origin. I | The first indication of this came in 1929-when i jMacheboeuf isolated a lipoprotein fraction of constant i ^ |composition from horse serum by the use of ammonium sulfate j i iprecipitation (l). Eleven years later Blix, Tiselius, and !Svensson (2) reported the presence of an appreciable amount |of lipid associated with the oC-and ^-globulins isolated i ; I by electrophoresis from normal human plasma. By ammonium j |sulfate precipitation Adair and Adair (3) in 19^3 isolated a lipoprotein from human serum which layered on the surface ; during centrifugation due to its low density (thought to ! j j ;be about 1.1 gm. per ml.). In 19^5., Pedersen reported (A) j .the ”X-protein" of plasma which he isolated by high-speed j i j jcentrifugation of human plasma diluted with saturated mag- J ! jnesium sulfate solution. He reported its density and i !molecular weight as 1.03 gm. per ml. and 10^* respectively, j The composition of the materials isolated by the above !investigators is presented in Table I. TABLE I PER CENT COMPOSITION OF VARIOUS PLASMA LIPOPROTEINS Cholesterol Cholesterol^/ Lipoproteins Total Free Ester Phospholipid Protein Phospholipid Macheboeuf (Horse) 10.4 0 17-9 22.7 59.3 0.46 Blix. Tiselius, and Svensson /S -Globulin 8.6 10.0 0.86 ^-Globulin 4.4 - - 7.2 - 0.61 Adair and Adair 16.4 - - 8.5 40 1.9 Pedersen X-protein 23.4 1/ By weight 3 The modern history of the lipoproteins can be con- j sidered to have originated with the development of two i i radically different procedures for the isolation of lipo- :proteins. The first contribution was made by the Harvard i Sgroup from studies utilizing the cold alcohol fractionation • procedures developed by them during the years 19^6 - 1950 | (5-8). They found that the serum lipids were precipitated ; into two separate fractions combined with two quite dis- i jtinct types of lipoproteins. Electrophoretically, one i [group moved with the characteristics of an ot~ -globulin; j i :the other of the /S -globulins. The second procedure, as i 1 j suggested by Pedersen (4) and refined by Llndgren, Elliott, | and Gofman (9), takes advantage of the relatively low den- | sity of the lipoproteins to separate them from the other I j serum proteins by ultracentrifugal flotation. Differences in density among the various lipoproteins permit the sepa- j I ration of the lipoproteins into two general classes, low : 1 | density lipoproteins and high density lipoproteins. The | i ' i i earlier studies conducted by these investigators (10) led ! I f * 1 |the way for Jones et_ al. (11) in 1951 to report on the I existence of a considerable number of lipoprotein species I in human serum. In the so-called low density group (lipo protein species with a density less than 1.063 gm. per ml.) at least 7 discrete species were visualized by analytical centrifugation (Table II). At least 2 species were observed for the high density group (lipoproteins with a density less TABLE II LIPOPROTEINS OF HUMAN SERUM Species Density1/ (gm. per ml.) Low Density Group Sf 2 1.050 Sf 4 1.040 Sf 6 1.035 Sf 8 1.029 Sf 10 1.023 Sf 13 1.015 Sf 17 0.99 Sf 17-40,000 0.99 High Density Group HDL-1 1.075 HDL-2 1.145 1/ Density of the hydrated molecule 5 than 1.20 gm. per ml. but greater than 1.063 gm. per ml.) (Table II). Kunkel and Slater (12) in 1952 reporting on the analysis of serum by starch electrophoresis indicated the j presence of two main lipoprotein peaks at an ionic strength of 0.1. The mobilities of the two peaks corresponded to i that of the -globulins and of -globulin, respectively. | At a lower ionic strength the peak corresponding to the ; |S -globulins was observed to split into several peaks. A study was made by Herbst et. al. (13) in 1955 to determine the relationship between lipoproteins isolated j by cold alcohol fractionation and ultracentrifugal flota- I j tion. Their results indicated that the low density 1 j lipoproteins migrated on paper similarly to the lipo proteins obtained from Fraction I +- II +- III (^-lipopro- ; teins) of Cohn's Method 10 (14). The high density ; I lipoproteins were shown to migrate in essentially the same > t way as the lipoproteins from Fraction IV + V (oC^-lipo- ! proteins). j i Various investigators have studied the lipoproteins 1 ; Isolated by cold alcohol fractionation. Oncley, Gurd, and Melin (7) in 1950 found the composition of the ^3-lipo- . ! protein to be 8$ free cholesterol, 39$ cholesterol ester, 29$ phospholipid, and 23$ protein. The total cholesterol to phospholipid ratio was reported as I.0 5. The molecular weight was estimated to be 1,300,000 (15)* Cohn, Strong, 6 and Blanchard (l6) reported the composition of the oC- lipoprotein to be 3$ free cholesterol, 15$ cholesterol ester, 21$ phospholipid, and 57$ protein. The total cho lesterol to phospholipid ratio was reported as O.5 7. The free to esterified cholesterol ratio was reported to be 0.27 for both lipoprotein species (16). The amounts of the o(_“ and ^-lipoprotein present in normal human plasma pools used for fractionation were reported to be about 3$ and 5$ of the total plasma protein, respectively" (7* 8). i / x j Later studies by Russ, Eder, and Barr (17)* and Lever et_ 1 J aT. (18) upon the distribution of these two lipoproteins ■ indicated somewhat higher concentrations, of the order of ! 4$ and 6$ for oL - and B -lipoprotein, respectively. The 1 1 j composition of the lipoproteins given above was determined i • in the course of studies upon large plasma pools, whose I , X j composition was reported (19) to be remarkably constant. Later studies on individual plasma samples (17 » 18) indi cated that considerable variations in the lipid composition of the cA- and ^-lipoproteins existed, which prompted Dr. ! J. L. Oncley and Dr. F. R. N. Gurd to write: "The composi tion of these lipoproteins, while falling within certain limits, do exhibit considerable variation or these oL- and ^ -lipoprotein fractions define families of closely related lipoproteins of accurately defined composition, just as the electrophoretic plasma components (albumin, oL-, and /'-globulins) represent families of molecules of similar 7 surface charge distribution." Various investigations have also been performed on | material isolated by ultracentrifugal flotation. For l | practical reasons of yield and technique, the chemical composition data reported by Lindgren, Nichols, and Freeman I (20) were for relatively broad flotation bands of ultra- ; J centrifugally isolated lipoproteins. The following bands j were studied by these workers: | (1) S^3 20-400. All lipoproteins in this i | group are of a density lower than 1.007 gm. per | | ml. ! (2.) S ° 0-20. All lipoproteins of density ! between 1.007 gm. per ml. and 1.04 gm. per ml. ! are in this group. A part of the 1.05 gm. per | ■ | ml. lipoprotein is present here also, although j i the usual level of 1.05 lipoprotein is much too j low relative to the other Sf° 0-20 lipoproteins o ' i to influence appreciably an estimate of i | . | ! 0-20 lipoprotein composition. j • j (3) The high density lipoproteins. Lipo- | proteins of density 1.07 gm. per ml. and 1.12 j ! gm. per ml. are in this fraction. Part of the ■ i j 1.05 lipoprotein, generally reported to be pre- ; sent in low concentration relative to the other r ! two lipoproteins, is also present in this group. i i i | The lipid composition of each of the three fractions, i as reported by these workers from infrared measurements | ! (20), is presented in Table III. If the average value of ; the lipid-protein ratios estimated by Gofman et al. (21) J for the three fractions is combined with the mean value of : the lipids presented in Table III, the per cent composition i ‘ of the lipoprotein in each of the three classes can be estimated and is shown in Table IV. A detailed study of the cholesterol to phospholipid ratio of ultracentrifugally ) separated lipoproteins was made by Havel, Eder, and Bragdon , (22) in 1955* By their method of isolation (repeated i 1 ultracentrifugations after progressively raising the sol vent density), they were able not only to subfractionate < i : the low density lipoproteins into two groups but also to j i obtain some information relative to the composition of each i of the two high density lipoproteins. The total choles terol to phospholipid ratio for the lipoproteins with a ! 1 density less than 1.019 gm. per ml. and with a density be tween I.OI9-I.O63 gm. per ml. was reported as 0.84 and 1.41, respectively. The ratio for the combined high den- I sity lipoproteins (lipoproteins with a density greater than1 ■ I.O63 gm. per ml.) was reported as O.3 8. Further fraction- 1 j ation of the high density lipoproteins indicated only a s small difference between the lipoproteins with a density ! between 1.063-1.125 gm. per ml. and those between 1.125- ; 1.21 gm. per ml., the cholesterol to phospholipid ratio 9 TABLE III THE LIPID COMPOSITION OF ISOLATED FRACTIONS IN THREE NORMAL HUMANS Subject Choi.1/ Choi. Ester P'Lipid^/ Glyceride Free, FA 2/ o 0 1 o CVJ o CO Class 1 9.5 13.4 17.6 58.4 1.1 2 5.4 12.9 20.9 6 0 .6 0.3 3 5-5 9.3 20.4 64.2 0.2 Mean Value 6.8 11.9 19.6 6 1 .1 0.5 Sfo 0-20 Class 1 9.7 51.3 24.5 13.9 0 .6 2 10.8 45-0 26.5 16.7 1 .0 3 11.6 47.0 24.4 15.9 1 .2 Mean Value 10.7 47.8 25.1 15.5 r 0 .9 High Density Lipoproteins 1 5.4 28 .6 46.5 14.6 4 .9 2 6.3 15.4 51.5 19 .8 6. 9 3 6.3 19-7 38.4 30.4 5.2 Mean Value 6.1 . 21.2 45.5 21.6 5.7 j 1/ Cholesterol | 2/ Phospholipid i i 3/ Fatty Acid TABLE IV ESTIMATION OF THE COMPOSITION OF HUMAN SERUM LIPOPROTEINS —^ Class Free Cholesterol Cholesterol Ester Phospholipid Glyceride Free Fatty Acid Protein Sf° 20-400 6.0 10.4 17.2 53.5 0.4 12.5 Sf° 0- 20 8.3 37.0 19.5 12.0 0.7 22.5 High Density Lipoproteins 2.7 9.5 20.5 9.7 2.6 55.0 1/ Values shown are percentages I 11 I i being reported as 0.50 and 0.47 respectively. A greater jprotein content was reported for the 1.125-1 .2 1 lipoprotein | fraction as compared with the 1.063-1 .1 25 high density | fraction. Bragdon, Havel, and Boyle (23) in 1956 reported 'a somewhat more complete study of the chemical composition J 1 | of lipoproteins isolated by the method (22) developed in ! 1955. In this study they segregated the low density lipo proteins into three classes: Chylomicrons, lipoproteins ; with a density between 1.005-1.019 gm. per ml., and lipo- i 1 proteins with a density greater than 1.019 but less than | I.O63 gm. per ml. The high density lipoproteins were ana- : lyzed as a group. The results of their study are presented I I ' in Table V. Fraction 2 in Table V also includes some chy- 1 , lomicrons, as no attempt was made by these investigators toj | completely remove the chylomicrons from serum. The chemi- j i cal composition of the chylomicrons reported in Table V compares favorably with the chylomicron analysis of Laurell, (24). Laurell found free cholesterol 1.3$* cholesterol ‘ ester 6.7$* phospholipid 7*3$* triglyceride 84.7$* and j j protein 1.7$. ' A study was made by Kunkel and Trautman (25) in 1956 t 1 ! to determine the electrophoretic mobility of the light low ' density lipoproteins (lipoproteins with a density less than 1.019). Earlier work by numerous investigators using fil ter paper electrophoresis had indicated another fraction aside from the two main "alpha" and "beta" lipoprotein TABLE V COMPOSITION OP SERUM LIPOPROTEIN FRACTIONS Fraction Density No. of Pools No. Of w Ind.±7 Free 9/ Chol.^ Choi. Esters P'Lipid^/ Glyceride Protein FC^/ TC TC—^ PL 1 Chylomicrons 2 3.2 6.4 7.0 81.2 2.1 0.46 1.0 2 3.0 5.7 7.1 81.4 2.8 0.47 0.90 2 < 1.019 11 6 .0 16.2 17.9 31.8 7.1 0.37 0.90 3 1.019-1*063 16 7.5 39.4 23.1 9.3 20.7 0.24 1.3 4 8.5 42.3 23.6 5.1 20.5 0.25 1.4 4 1.063-1.21 11 2.0 17.4 26.1 8.1 46.4 0.16 0.48 5 2.3 18.5 26.9 4.6 47.7 0.17 0.50 1/ Individuals 2/ Cholesterol ! | 3/ Phospholipid 4/ Free cholesterol/Total cholesterol 5/ Total cholesterol/Phospholipid H ro 13 components. This fraction observed at the site of origin was considered to represent adsorbed lipoproteins (26, 27), particularly the chylomicrons (28). The results of Kunkel and Trautman using zone electrophoresis on starch indicated that the lipoproteins with a density less than 1.0 19 gm* ! J per ml. migrate on starch as a distinct fraction in the j ! 0^2_gl°frulin region rather than as part of the yS - j lipoproteins. j Metabolic Interrelationships It has been suggested by Jones et_ al. (11) that the | lipoprotein distribution found in plasma represents the ! ! sequence of molecules in the metabolic chain which accom- j : plishes lipid transport and that lipoproteins in the high | ' | Sf range are progressively transformed into those of lower j ! Sf classes (and possibly into the high density classes) ! 1 with the concomitant release of glyceryl esters and/or i fatty acids. In this sense the cholesterol, phospholipids i i i and protein may be considered to be part of a prosthetic I | fragment. ! To support this contention Jones et_ al. (11) cite r | evidence of a transient elevation of molecular classes i above Sf 60 followed by a stepwise conversion of such spe cies to those of progressively lower Sf classes upon the administration of high fat meals to normal humanb. They have followed the lipemlc load down to Sf 30, with the 14 lowest species in this range increasing at the same time that the higher Sf species are decreasing in concentration. They observed no detectable change in the levels of the |serum lipoproteins from Sf 30 to Sf 4. They postulate that jeither these latter groups must be equilibrated with a very much larger pool of lipids than is the case for the higher Sf molecules or the utilization rate of molecules below Sf 30 may increase sufficiently in response to small incre ments in load so that the normal steady state level is I | maintained. | j Similar findings have been reported by Pierce (29)* i jwho injected into normal rabbits lipoproteins of the Sf ! s -15> 15-20, 20-100, 100-400, and 400+ classes isolated ! Ifrom the serum of cholesterol-fed and alloxanized rabbits. i He noted a serial conversion of all lipoprotein classes ! above the Sf 5-15 class to the Sf 5-15 class itself, the |clearance from the serum of all classes above Sf 15 occur- j 1 jring in a matter of hours. As each class disappeared the ! ! !concentration of the next class increased and then dis- jappeared. This continued until only the Sf 5-15 class was !above normal. In several days this class decreased to a [normal concentration. This conversion always occurred in a i direction from high Sf rate to low Sf rate. Pierce suggestsj a different metabolic fate for the Sf 5-15 class as compared with the higher S^ elasses and bases this contention on the continuous presence of the Sf 5-15 class in normal animals | as well as on the different clearing rate of this class. ! j His findings corroborate and extend observations made ear- I lier by him in the conversion of lipoproteins found under i i other experimental conditions. Cortisone (30) and, in | some cases, radiation (31) were observed to produce large i ! quantities of high Sf lipoproteins in rabbit serum. Upon i j recovery these lipoproteins were serially converted to : lipoproteins of lower Sf rate. Somewhat similar observations have been reported by j Bragdon, Boyle, and Havel (32), who injected various serum I I lipoprotein fractions from humans into rats. Chylomicrons i i j when injected into rats resulted in the appearance of lipo- I J proteins with Sf values between 30 and 3 0 0. There was no | i increase in the Sf 0-10 class or in the high density lipo- ! ! ji ' proteins. Rats receiving lipoproteins with a density less ! j ; ' than 1.019 (Sf 10-400) showed an increase in the Sf 0-10 I j class. The lipoprotein pattern of rats injected with the < J Sf 0-10 class showed an increase in that class and a slight; i . i elevation of the Sf 40 to 400 species. Rats injected with j | the high density lipoproteins (lipoproteins with a density » I i between 1.063-1.21 gm. per ml.) gave unexpected results. Not only was a large increase in the high density lipo- j ! proteins noted (which would be expected) but also a large | [ increase (tenfold) in the Sf 50-300 class and a two - s ; threefold elevation in the Sf 0-10 class were observed. j I One rat was also injected with high density lipoproteins j 16 isolated from a large pool of rat serum. A sixfold in- i | crease in the Sf 40-400 class was observed but no increase ! { in the Sf 0-10 class. Bragdon et_ al♦ (32) point out that t ; although a conversion of high density to low density lipo- ! proteins may have occurred, the large amount of high density lipoproteins injected may have resulted in a block- I I ing of the normal mechanism necessary for the removal of i low density lipoproteins, thus causing an accumulation of ! the low density lipoproteins. I j Heparin administration to humans and rabbits was l I ■ shown by Graham et_ al. (33) to cause a reorientation in I I the distribution of the low density lipoproteins, charac- i i terized by a shift of high Sf lipoproteins to those of l jsuccessively lower Sf rates. With the decrease in concen- t tration of molecules of the high Sf classes, a concomitant increase in concentration in those of the lower Sf classes \ was observed, which suggested to these investigators a i i progressive transformation of the higher Sf lipoproteins ; ! I into those of lower Sf classes. i » 1 Utilizing Anderson's (34) findings that rapid clear- i ■ing of alimentary lipemic plasma could be brought about in i I vitro by heparin under certain conditions, Anfinsen, Boyle, land Brown (35) isolated an ’ 'active clearing factor" which upon incubation with serum produced an accumulation of low density lipoproteins at the expense of higher Sf components. When incubated with this factor, one serum from a patient with a proven myocardial infarct showed a decrease in the concentration of the Sf 30-150 and Sf 2 0 -3 0 classes with an increase in the Sf 10-20 class. No change in distribu tion or concentration was observed for the Sf 3-10 jlipoproteins. j j In a later study, Boyle, Bragdon, and Brown (36) !demonstrated by ultracentrifugal and chemical means a i jquantitative increase in the high density lipoproteins, and ■a concomitant decrease in the Sf 1 0 -8 0 class upon incuba- ;tion of plasma obtained from normal young male adults given i ;a high fat breakfast followed by heparin injection. These i !changes suggested to the investigators an enzymatic conver- |sion not only of the high Sf classes to those of lower Sf classes, but also a conversion to the high density lipo proteins. Lipid analyses indicated that triglyceride and fatty acids, phospholipids, and to a lesser extent choles- I I i terol esters were liberated from the low density j lipoproteins during the in. vitro conversion. j A similar viewpoint was taken by Nikkila and J jGrasbeck (37), who studied the effects of heparin adminis- j |tration to nephrotic individuals by analyses of the serum pattern obtained by paper electrophoresis. Their results suggested a conversion of the {3 -lipoprotein to the oi1^- lipoprotein as indicated by a large increase in the lipid staining area corresponding to the ot-^-globulin. However, this theory of an jun vivo transformation of -lipoprotein | ^ 18 j into oCf-lipoprotein by heparin has been challenged by jHerbst et al. (13)* They postulate that the increase in lipid stain in the oLf-globulin region is due to a libera- | tion of fatty acids from the high Sf lipoproteins which I i when bound to the lower Sf lipoproteins causes them to i , : migrate with the mobility of an (X.-,-lipoprotein. This i ! concept is strengthened by the studies of (l) Shore, I ^ I ■Nichols, and Freeman (38), who observed that in vivo ! j heparinized plasma caused a release of fatty acids from jhigh Sf lipoproteins and (2) of Gordon (39)* who produced |an increase in the mobility of low Sf lipoproteins by the iaddition of oleate. I t \ The report of Shore and Shore (40) on the amino acid ;composition of the protein moieties of low and high density ! ! |lipoproteins would support the possibility of an in vivo j I conversion of low density to high density lipoproteins. I ( i By the paper chromatographic method of Levy and Chung (4) j t l the authors found no quantitative differences in the amino 1 f { ‘acid composition of the different lipoprotein fractions. j ; • I |If interconversion occurs directly through stripping down j !of lower density material by delipidation, they reason that j |the protein moieties of source and product should be iden- i | I ;tical. J A similar study has been reported by Brown et al. !(42). They found a similarity in amino acid composition I ■for the 0 .9 1 5 and 1.048 gm. per ml. lipoprotein species ! 19 i | but, contrary to the findings of Shore and Shore (40), a ! | difference in the amino acid composition of the low and i high density lipoproteins. • i i ! The recent finding of Levy (43) disagrees with both | I the studies of Shore and Shore (42) and Brown et_ al. (40). By the ion exchange method of Moore and Stein (44) he found i i | quantitative differences in amino acid composition not only ; between the low and high density lipoproteins but between j j the 1.000-1.019 and 1.019 to 1.063 gm. per ml. low density 5 ; lipoprotein classes. j j N-terminal residue analyses of delipidated and | native low density (1.019-1.063 gm. per ml. fraction) and \ high density (1.063-1-21 gm. per ml. fraction) lipoproteins | by Avigan, Redfield and Steinberg (45) indicate structural j i ' j j differences in the protein moieties of these two classes. 1 ’ Their finding that glutamic acid appears to be N-terminal j j i in the low density lipoprotein and aspartic acid N-terminalj i in the high density lipoprotein tends to rule out direct | ; interconversion, at least for these two materials. They i i also report that tracer experiments show no significant in 1 j I i vivo interchange of labeled protein moieties between the j ' i l high density and low density lipoproteins in rabbits. j With respect to tracer experiments, Gitlan et_ al♦ j | (46) observed a similar situation upon intravenous injec- i . | tion of low and high density lipoproteins, labeled in the i j peptide moiety with radioiodine, to children with the J nephrotic syndrome and to normal individuals. They detec- ! ted no significant radioactivity in the chyiomicra or in ! | lipoproteins of lower density upon injection of the high J density lipoprotein. Similarly, radioactivity originally j injected in association with the peptide moiety of the j labeled low density lipoproteins (Sf 3-9) was not found ! subsequently in either the high density lipoproteins or in i t j the chyiomicra. In contrast, they observed that the | labeled peptide moiety of low density Sf 10-100 class lipo- ! proteins rapidly appeared in the low density Sf 3-9 i j lipoprotein class. They suggest on the basis of their ! findings that the metabolism of the peptide moiety of the i I ! high density lipoprotein is independent of the metabolism ! of the peptide moiety of the low density lipoproteins and ! ' 1 that there is no direct conversion of high or low density lipoproteins in the formation of chyiomicra. They further j ! ‘ indicate that there normally appears to be a system oper- j t ating in vivo for the unidirectional conversion of low : ! density lipoproteins (Sf 10-100) to lipoproteins of higher t ; density (Sf 3-9)* This concept of lipoprotein metabolism ! is in accord with the immunologic studies performed by ; Aladjem, Lieberman and Gofman (47) on several low and high i (density lipoprotein species. Their results indicate that the 1.145 gm. per ml. high density lipoproteins are anti- genically distinct from all other lipoproteins, while the 1.075 gm. per ml. high density lipoproteins contain several 21 antigenic components, at least one peculiar to itself and j others in common with low density lipoproteins. The low | density lipoproteins (Sf 6 and Sf 13) contain at least one i set of antigens different from the high density lipopro teins. These workers' findings as regards quantitative j precipitation with specific antisera indicate that the low | density Sf 6. and .Sf 13 lipoproteins are immunochemically ! ! very similar. They postulate that in lipoproteins of still I j lower density additional antigenic components may be pre- - I sent but that some of those associated with Sf 6 or Sf 13 i | might be absent, this type of distribution continuing up I j to the chylomicron range. I j . It is apparent upon consideration of the findings s ! reported above that the metabolic interrelationship of the i j various lipoproteins is not well understood. The initial i i j suggestion of Jones et_ al. of the possibility of a pros- j thetic fragment consisting of cholesterol, phospholipids j and protein cannot be easily reconciled with the available j ! i ! information as to the chemical composition of the ! ! ' i I lipoproteins. I ! i i In the experimental work reported in this disserta- | tion, lipoproteins representing two low density classes apd ! one high density class have been obtained in bulk by the development of a precipitation-flotation method for the isolation and purification of lipoproteins. The three | classes have been analyzed by physical and chemical means. 22 The purpose of this investigation has been to gain further i information relative to the chemical composition of the ! j lipoproteins. j CHAPTER II i MATERIALS AND METHODS Dextran Sulfate Two preparations of dextran sulfate were kindly pro- ;vided by Dr* A. Keltz. The properties of the preparations i are shown in Table VI. i !Rice Starch Sulfate I ................. n n i " ' " I " " ' ' ' ........... I I j A commercial preparation of rice starch (Morning- | star, Nicol, Inc.) served as the starting material. It was |sulfated by treatment with chlorosulfonic acid in pyridine for 5 hours at 70° (48) and was isolated as the potassium salt (Table VI). |Bovine Plasma Albumin I I A crystalline, salt-free product from Armour and j % i j ' i :Company was used. | j . 1 Serum j _ — — . 1 The serum was obtained from whole blood collected from normal, white, male donors under 40 years of age, who were presumably in the fasting state. TABLE V I PROPERTIES OP SULFATED POLYSACCHARIDES USED FOR LIPOPROTEIN FRACTIONATION Polyanion Specific Viscosity Per Cent S Appearance of Powder Dextran Sulfatei/ Preparation 1 0.67 17*6 White Preparation 2 O .6 5 1 6 .0 Slightly Yellow Rice Starch Sulfate^ - - -- White | 1/ Provided through the courtesy of Dr. A. Keltz. 2/ Prepared in this study. i Ultracentrifugal Isolation of Lipoproteins Lipoproteins with hydrated densities less than il.063 gm. per ml. were isolated essentially as described by De Lalla and Gofman (49) using NaCl and NaBr to increase jthe density for flotation. However, a Spinco Wo. 40 head |was used in the Spinco Model L preparative ultracentrifuge at a speed of 35*GOO RPM instead of the Wo. 40.3 rotor used by the above investigators. j I Zone Electrophoresis on Starch Starch electrophoresis was carried out essentially : j ias described by Kunkel and Slater (12). Potato starch was j ; 1 used as the supporting medium in all cases, and in most ; instances diethylbarbiturate, pH 8.6, ionic strength 0.05 | jwas employed as the buffer. All runs were made at 0°, i t usually in a lucite block designed by Dr. J. W. Mehl (Figure jl). The starch was thoroughly washed with at least three [volumes of buffer prior to pouring of the starch block. ! This has been found to be important, particularly with the ! 1 jO.05 ionic strength buffer, to prevent pH changes during the course of the experiments due to the ionic exchange at the surface of the granules (27). The entire system was encased jin polyethylene sheeting. The sample (serum or lipo- j protein) was inserted into the block by the use of a sample inserter (Figure 1) after mixing with dry starch to a j -INSERTER SAMPLE CONTAINER CHANNEL nq I ______l oooo QOOOO oooo 00000 DIVIDER FIG. 1 STARCH BLOCK APPARATUS WITH SAMPLE INSERTER ro ON paste-like consistency. Separation was generally carried ;out employing a field strength of approximately 3*5 volts jper cm. for a period of 24 to 48 hours after a preliminary i ; equilibration period of 12 hours. Following the separa- i } | tion, the protein-containing areas were located by placing j a strip of filter paper edgedown into the starch along the i |long axis. The strips were dried at 100° and stained for | protein with bromphenol blue. The bromphenol blue stain j I was prepared by dissolving 200 mg. bromphenol blue in 50 | ml. 95% ethanol, adding 100 gm. ZnSO^.THgO and diluting to ! 2 liters with 5$ acetic acid. Starch segments approximate- \ i j ly 5 x 1.5 x 1 cm. in size were then stirred with 3 ml. of I I 1% NaCl. After thorough mixing, the starch was allowed to i | j settle, and aliquots removed for quantitative protein ana- j : lyses by the method of Lowry, Rosebrough, Farr, and Randall! (50). The remainder was allowed to air-dry and was then ' I extracted three times with acetone-ethanol (1:1), heating i to boiling and filtering. The combined extracts were ! brought to 25 ml., and aliquots were taken for lipid ana- i i lyses. 1 Cholesterol Determination I ' " T ......— ; j ! Several methods of cholesterol assay were utilized during the course of this study. Initially, cholesterol was determined by the FeCl^ method proposed by Zlatkis, I Zak, and Boyle (51)• This method, due to its greater ! 28 jsensitivity was especially adapted to the analysis of 1 samples from starch electrophoresis. Later, starch seg- ! i Iments were also analyzed for cholesterol by the Pearson, i |Stern, and McGavack (52) modification of the Liebermann- |Burchard reaction. i ! For the analysis of lipoprotein fractions isolated i |by the precipitation-flotation method, the modification of |the Sperry-Schoenheimer method as reported by Nieft and I i Deuel (53) was used for the determination of* free and esterified cholesterol. j Lipid Phosphorus Determination i | Lipid phosphorus was determined by the method of I iLowry, Roberts, Leiner, Wu, and Farr (5*0* Phospholipid | :was estimated by multiplying, the lipid phosphorus value by j I ! |the factor 2 5.0. i : Paper Electrophoresis j \ : j j Electrophoretic examination on filter paper was per- I I formed on serum and lipoprotein fractions isolated either i t !by ultracentrifugal flotation alone or by precipitation procedures followed by flotation. The Spinco-Durrum appa- I ratus was used, the buffer generally being 0.1 M sodium barbital, pH 8.6. Electrophoresis was in most instances carried out at room temperature at 100 - 150 volts for 18 - j20 hours. After electrophoresis, the paper strips were 29 dried for 30 minutes at 95 - 100° and then stained with Oil jRed 0 for lipid as described by Jencks, Jetton, and Durrum j (55) and with bromphenol blue for protein. 1 ; Paper electrophoresis was also utilized for the i jseparation of the ninhydrin positive, nitrogenous consti- I j tuents of serum and lipoprotein phospholipids following | jlipid extraction, purification and hydrolysis. Electro- » [phoresis was performed in the Spinco-Durrum apparatus at 1 _ jroom temperature using veronal buffer, pH 8.6, ionic [strength 0.1. The length, of the run was generally 3 hours at 150 volts. After electrophoresis, the paper strips con taining standard serine and ethanolamine were dried at 95 - 100° for 30 minutes and stained with ninhydrin solu tion. Development of the color was performed by placing the paper- strips for 5-10 minutes in a 95 - 100° air oveni 1 The "standard" strips were then placed alongside the sample] \ • ! strips and areas corresponding to the serine and ethanola- i 1 mine cut for elution and subsequent ninhydrin analyses by 1 .the modified ninhydrin reagent of Moore and Stein (5 6). \ 1 I j The reagents used in this procedure are outlined in Table I VII. Moving Boundary Electrophoresis All analyses were carried out in a Spinco Model H electrophoresis-diffusion instrument. The standard cell 1 j (11 ml.) was utilized for the 1.019-1-063 and 1.063-1.200 30 TABLE VII REAGENTS USED IN THE NINHYDRIN METHOD OP MOORE AND STEIN (56) Ninhydrin, Ninhydrin sufficiently pure so that recrystal lization was unnecessary was purchased from Dougherty | Chemicals, Richmond Hill, New York. I Hydrindantin. Crystalline hydrindantin prepared by the i reduction of ninhydrin with ascorbic acid was pur- ! chased from Dougherty Chemicals. ! A N_ Sodium Acetate Buffer (pH 5»5). Five hundred forty- four gm. of sodium acetate trihydrate was added to 400 ml. of water and stirred on a hot water bath un til solution was complete. The solution was filtered | while still hot into a 1 liter volumetric flask, j After it had cooled to room temperature, 100 ml. of | glacial acetic acid was added and the solution brought to 1 liter with water. The solution had a pH ! of 5-5. The buffer was stored at 0°. i I Methyl Cellosolve. Dowanol J (ethylene glycol monomethyl ether) was purchased from the Braun Corporation, Los j i Angeles. After distillation in glass it gave a nega-i J tive peroxide test with 10 per cent aqueous KI. j Ninhydrin Solution. Two gm. of ninhydrin and 0.3.gm. of j hydrindantin were dissolved in 75 ml. of methyl j cellosolve with stirring. Twenty-five ml. of the pH j 5 .5 sodium acetate buffer was then added to the solu-: 1 tion. The reddish reagent solution was used I immediately. ; ■ Diluent Solution. A mixture of equal volumes of 95 per J j cent ethanol and water was used for dilution of the I ; ninhydrin solution and eluted fractions. 31 | gm. per ml. lipoprotein fractions while the 1.000-1 .0 1 9 gm. ; per ml. fraction was analyzed in the micro cell (2 ml.). ; Suitable aliquots of the lipoprotein preparations were j ! dialyzed against 0.05 M NaCl containing 0.01 M versene, j followed by another dialysis against veronal buffer, pH | 8.6, ionic strength 0.05 containing 0.001 M versene. ! Chelation of trace divalent metallic cations has been shown to increase the stability of lipoproteins by Ray et_ al. (57). Rutin was also added to the dialyzing solutions to I { \ a final concentration of 10 microgram per ml. in order to | \ ; protect the lipoproteins from oxidation (57). In most in- j ; - ; stances, bovine albumin was added to the lipoprotein i , i ; I j fractions to bind fatty acids which would otherwise in- j ; crease lipoprotein mobilities (39)* Where necessary, the i 1 dialyzed preparations were diluted with veronal dialysate I to a final volume of 13 ml. for the standard cell and 2.5 ml. for the micro cell. The initial protein-buffer bound- j ary was displaced 9 to 11 mm. into the limb of the cell, and a current of 0 .2 5 ma. for the micro cell and 4.0 ma. ! for the standard cell was applied for approximately 1000 i land 400 minutes, respectively. The moving boundaries were | ! recorded photographically by means of a schlieren optical i I i I system. j Analytical Centrifugation I j j Analyses were carried out in a 1.2 cm. cell of a ; Spinco Model E analytical ultracentrifuge essentially according to the methods outlined by De Lalla and Gofman (49). Before analysis* the 1.000-1.019 and 1.019-1.063 gm per ml. lipoprotein fractions were adjusted to a density of 1.063 gm. per ml. Centrifugation was carried out for j30 minutes at 26° and 52*640 RPM. Records of the schlieren | 'patterns were made on raetallographic plates when the rotor • had accelerated to the desired speed (up-to-speed time) and j :at 6* 12, 22 and 30 minutes thereafter. The 1.063-1.200 1 1 jgm. per ml. lipoprotein fraction was adjusted to a density | |of 1 .2 0 gm. per ml. and centrifuged for 64 minutes at 26° I |and 52,640 RPM. Film records of the schlieren pattern jwere obtained at up-to-speed, 16, 3 2* 48 and 64 minutes. j ' • | Total Weight of the Lipoprotein Fractions by Dry Weight 1 - 1 j Determinations i " Lipoprotein fractions were dialyzed with stirring for 24 hours at 0° against at least a 100 fold excess of ,0.05 M NaCl. Aliquots of lipoprotein solution and dialy- !sate were dried over concentrated HgSO^ under reduced 1 pressure at room temperature and then dried in vacuo in an |Abderhalden pistol over ^2^5 5 6.5° to a constant weight. ;A salt correction was made by subtracting the dry weight, of the dialysate from the dry weight of the lipoprotein sample. 33 ; Delipidation of the Lipoprotein Fractions j Delipidation of the lipoproteins was carried out by i jthe method of Delsal (58) as modified by Fillerup and Mead I (59)* Before lipid extraction* the lipoproteins were dialyzed against 0.05 M NaCl at 0° for 24 hours. Although dialysis may have some adverse effects on the state of the lipoproteins* this procedure was adopted in order to remove i [the high salt concentration which would interfere with the i ’ ■ ■ delipidation procedure. Previously* dialysis against dis- i 1 tilled water was attempted. However* this was found to i ihasten the denaturation of the lipoproteins. Following i i - jdialysis* the lipoprotein solutions were added dropwise with I [stirring at room temperature to a 4:1 solution of methylal-'! i [methanol. The ratio of solvent to lipoprotein solution was i i [maintained at 15*1 on a volume basis. Under.these condi- | Jtions* the delipidated protein settled out of solution as I I a relatively uniform flocculent precipitate. The mixture ! i was centrifuged at 2500 RPM (International Centrifuge) at j 'room temperature for 15 minutes. The supernatant contain- j |ing the total lipids was carefully decanted into a fine isintered glass filter and filtered with suction. To insure jremoval of adsorbed lipids* the protein precipitate was washed three times with methylal-methanol, and the washings filtered and combined with the main lipid extract. The iprotein precipitate was further treated with distilled water 34 until the water wash gave a negative test for Cl” with j AgNO^. The water wash was filtered and added to the lipid t |extract. Total Weight of the Lipid Extracts by Dry Weight Determinations i The lipid extracts of the lipoprotein fractions were ;taken to dryness under a stream of nitrogen in a 40 - 50° | |water bath. The samples were then dried in a vacuum oven I at 60° to a constant weight. A salt correction was made by i !taking the difference in weight between equal volumes of i jlipid extract and dialysate. It is assumed that the salt I ■ j concentration of the dialysis residue is equal to that of | jthe dialysate. i i i Protein Content j f » ^ i j The protein content of the lipoprotein fractions was j j obtained by subtracting the dry weight of the total lipid j I jfrom the dry weight of the lipoprotein. In a few instan- | joes* the protein content was directly determined by lcollecting the protein residue following delipidation and i drying to a constant weight at 56.5° under vacuum in an Abderhalden pistol over ?2®5’ i Purification of the Lipid Extracts I j The removal of non-lipid contaminants from the lipid ; 35 I j extracts of the lipoprotein fractions was carried out l according to the method of Folch, Ascoli, Lees, Meath, and Le Baron (60). Before submitting the lipid extracts to hydrolysis, their purity was verified on 0.5 to 2 mg. of I j lipids by descending paper chromatography. Whatman No. 1 1 I filter paper was used with chloroform-ethanol (chloroform i | saturated with H^O 80, ethanol 20) as the solvent. The i ] runs were performed at room temperature for 24 hours. The | chromatographic strips were stained with ninhydrin and | bromphenol blue to confirm the absence of amino acids, pep- |tides, and proteins. j j i i j Hydrolysis of the Purified Lipid Extracts i I The purified lipid extracts were hydrolyzed accord- I jing to Levine and Chargaff (61) for 40 hours at 110° with ! |6N HC1 (redistilled HC1) in sealed tubes under vacuum. The quantities submitted to hydrolysis were calculated on the basis of the total lipid necessary to contain between 40 and 50 mg. of phospholipid. 6n HC1 was added in an amount 200 times the weight of total lipid. Extraction of Sphingosine The extraction of sphingosine was carried out essen tially by the method of McKibbin and Taylor (62). At the end of the hydrolysis period the contents of the tube were transferred to a 125 ml. separatory funnel with about 10 ml ! 36 i !of CHCI3 . The tube was dried overnight at 55° and then I ! | rinsed first with CHCI3, then with warm water. The rins- |ings were added to the separatory funnel. The volumes of ]the aqueous and CHClo phases were about 75 and 25 ml., i j respectively. After vigorous shaking, the chloroform layer | I iwas transferred into a 40 ml. centrifuge tube; 10 to 15 ml. j jmore CHClg was added to the separatory funnel, and the ex traction repeated. The combined CHCI3 extracts were I centrifuged and then decanted into a 50 ml. volumetric 1 1 i flask. Sphingosine was then determined from the nitrogen i | content of the chloroform extract. I I Concentration of the Water Soluble Nitrogenous Bases i ^ 1 1 < after Hydrolysis J ! ! I ; ; i In order to concentrate the water soluble nitro genous bases (choline, serine and ethanolamine) after t • j ,hydrolysis, the aqueous phase following sphingosine extrac- ! tion was filtered with suction through a fine sintered j : j glass filter (to remove charred material) and then taken toj dryness in a 300 ml. round bottom flask by the use of a i ! Rinco evaporator. To remove trace amounts of HC1 water was j added and removed by evaporation twice. The flask was thenj I placed in a desiccator under vacuum over KOH. The contents of the flask were analyzed for choline, phosphorus, and the ninhydrin positive phospholipid bases, - serine and f j ! ethanolamine. j ' Choline Determination l ~ ~ ' .... I Choline was determined as the enneaiodide by the I (method of Appleton, La Du Jr., Levy, Steele, and Brodie | (63). ! I [ Nitrogen Determination | j | Total lipid nitrogen was determined by the micro- s [Kjeldahl method as modified by Meacham (64) using the | (digestion mixture recommended by the Association of Official i [Agricultural Chemists ( 6 5) and with selenium as catalyst. i 1 (Sphingosine nitrogen was determined according to the method i ' ! jof Koch and McMeekin (66) with superoxol addition as recom- j mended by Miller and Miller (6 7)• \ 1 j j ( Density Determinations, i j ! The density of the various salt solutions was- deter- i mined with a 25 ml. Weld pycnometer. Five and 10 ml. Weld [ pycnometers were used to determine the density of the vari- j 1 ; i ous lipoprotein preparations. All measurements were made at! 126°. ! CHAPTER I I I I EXPERIMENTAL RESULTS i i i I Electrophoresis of Serum Lipoproteins on Starch i ; • » I In order to confirm the observations of Kunkel and i iSlater (27) serum samples were analyzed by starch electro- ! I phoresis. The results obtained were like those previously idescribed (25, 27). At an ionic strength of 0.1, pH 8.6, | i there did not appear to be clear evidence of more than two !lipoprotein peaks - the and f£ . However, at an ionic !strength of 0.05, pH 8.6, the peak clearly appeared to !be heterogeneous. In general, one peak was observed a ilittle behind the yS^-globulin and the other a little ahead,j |but not migrating as rapidly as the peak of the o^g-protein* j I j The oL-i peak was also observed to split into several com- j j X ! v - I j ponents with some serum samples at this, lower ionic !strength. The results for cholesterol and phospholipid at , i ^ ;an ionic strength of 0.1 are shown in Figure 2A. Figure 2B !shows the results at an ionic strength of 0.05. ; : . ! !Separation of |3-Lipoprotein by Flotation j j t i \ i I Initially, the preparation of lipoproteins with hy- j i i !drated densities less than 1.063 gm. per ml. was carried j ! , i I out using a centrifugal period of 24 hours (No. 40 head, i ;35,000 RPM). Analysis of the preparation by starch and 4 6 8 10 12 14 16 18 2 0 | MIGRATION IN CENTIMETERS ! ' > ' ;Plg. 2A Starch block electrophoresis of serum at an ionic ; strength of 0.10. | Cholesterol -------- | Phospholipid - - - - i 24 hours 3-5 volts/centimeter | Temperature of run 0° i • j 4 6 8 10 12 14 16 18 0 2 MIGRATION IN CENTIMETERS I . ■ ;Fig. 2B Starch.block electrophoresis of serum at an ionic strength of 0.0 5. i ! ! 40 i i (paper electrophoresis indicated that the lipoprotein ob- jtained by flotation was quite homogeneous, that the !mobility differed from the mobility observed in serum, and |that dialysis resulted in the appearance of a lipid-free protein peak. Cholesterol and phospholipid values of a j typical preparation analyzed on starch are shown in Figure i ' ■ > !3A• The results of cholesterol, phospholipid, and protein i .analysis after dialysis are shown in Figure 3B. However, ■ ,paper electrophoretic examination of preparations before :dialysis, after dialysis, and retreated with NaCl to a salt f jcontent equivalent to that of the original preparation in- t i jdicated that the effects noted above were artifacts due to i the effects of the high salt concentration on the electro phoretic results. In the 0.05 ionic strength buffer, the ' high salt concentration remaining from the ultracentrifugal i i jflotation had the effect of shorting out the region in j which the sample was applied. The lipid-free protein peak j ( which appeared on dialysis was albumin contaminating the i i l , samples. These results are shown in Figure 4. In order | i > |to remove the contaminating albumin, the period of centri- ;fugation was increased from 24 to 48 hours. Samples prepared in this manner contained no detectable albumin as 'determined by paper and starch electrophoresis. The re sults of cholesterol and phospholipid analysis of a dialyzed preparation after starch electrophoresis for 24 jhours are shown in Figure 5A. Although the preparation is s r\ h (’ i*i i I i i I i i I > 4 i _L I 1 ------------ 1 ------ 1 — ----------! — MIGRATION IN CENTIMETERS Eig. 3A Starch block electrophoresis of ultracentrifugally I isolated lipoproteins before dialysis. i I ! Cholesterol ---------- | Phospholipid - - - - - i Protein __ I 24 hours 3-5 volts/centimeter I Temperature of run 0° 4 6 8 10 12 14 16 0 2 MIGRATION IN CENTIMETERS Fig. 3B Starch block electrophoresis of ultracentrifugally ’ isolated lipoproteins (24 hour flotation period) i after dialysis. A SERUM B LIPOPROTEIN BEFORE DIALYSIS B A LIPOPROTEIN AFTER DIALYSIS B A DIALYZED LIPO PROTEIN RE TREATED WITH NaCl B • • • • Fig. 4 Paper electrophoretic analyses of serum and lipo- j proteins -- isolated with a 24 hour ultracentrifugal period by flotation (I.O63 gm./ml. salt density). j i A -- Bromphenol blue protein stain. B -- Oil Red 0 lipid stain. I 0 - 2 4 6 8 10 12 14 16 18 20 22 | MIGRATION IN CENTIMETERS i |Plg. 5-4 Starch block electrophoresis of ultracentrifugally ; isolated lipoproteins (48 hour flotation period) | after 24 hours. i | Cholesterol ---------- i Phospholipid - - - - - 3.5 volts/centimeter Temperature of run 0° MIGRATION IN CENTIMETERS Pig. 5B Starch block electrophoresis of ultracentrifugally i isolated lipoproteins (48 hour flotation period) i after 54 hours. not homogeneous,as indicated by the asymmetry of the peak, j there is less evidence of heterogeneity than is normally | found for ^3-lipoprotein under similar conditions in serum. I | However, another preparation migrated on starch for 54 I i i hours clearly indicates the heterogeneity of the prepara- i j tion (Figure 5B) which resembles the pattern seen in J ! serum. i i I . Separation of Lipoproteins with Dextran Sulfate Information regarding dextran sulfate as a complex- ! ing agent for -lipoprotein was obtained through the 1 | courtesy of Drs. A. Keltz and J. L. Oncley. As mentioned | previously, two preparations of dextran sulfate were pro- ! vided by the above investigators. Their information i ! I ' regarding the conditions for precipitation showed that ! ; j ! they had included a small amount of alcohol as they were i i _ i interested in the removal of p> -lipoprotein after precipi- j ! tation of Fraction II (cold alcohol, fractionation method). ' To determine whether the precipitation procedure could be i applied directly to serum, samples of serum were treated ! with increasing amounts of dextran sulfate. The amounts j corresponded to 10, 25, 50, and 100 mg. dextran sulfate per 100 ml. serum, and were added as a solution containing 10 mg. dextran sulfate per ml. in 0.15 M NaCl at 0°. The resulting mixture was allowed to stand for 1 hour at 0°. The mixture was then centrifuged and the supernatant i 45 isubjected to paper electrophoresis. The paper strips were jstained with Oil Red 0 and then analyzed by scanning in the Spinco Analytrol densitometer. The results of the pattern obtained with dextran sulfate preparation No. 1, are shown in Figure 6. It is evident that essentially all the fi- |lipoprotein is removed with 50 mg. of dextran sulfate per i |100 ml. of serum. Also, there appears to be no indication !of reaction between -lipoprotein and dextran sulfate, 1 1 |assuming that such a reaction would at least alter the mo- Jbility of this component at the electrophoretic pH of 8.6. Complete precipitation was not achieved at the same concen tration for dextran sulfate preparation No. 2. A total of 2 mg. dextran sulfate per ml. serum was found necessary. i iThis indicates that the optimum ratio of polyanion to serum I i !may vary between preparations and emphasizes the importance I jof determining experimentally the amount of polyanion 1 necessary for complete precipitation for any given polyan- ; i ion preparation. i -Lipoprotein was prepared by adding 5 ml. of j t idextran sulfate (10 mg. per ml. in 0.15 M NaCl) to 100 ml. i t :of serum at 0°. The resulting mixture was allowed to stand i jfor 2 hours and then centrifuged at 35*000 RPM for 20 minutes. The precipitate was washed with 0.1 M calcium acetate, and then dissolved in 2 M NaCl. The solution was centrifuged for 20 hours at 35*000 RPM (No. 4-0 rotor) and the pigmented material removed from the upper layer. This c I i i i i i i DEXTRAN SULFATE MG./100 ML. SERUM ALPHA - v LIPOPROTEIN I i I i i ! BETA- LIPO PROTEIN 10 I t ! I I 25 i j ! 50 100 i Fig. 6 Paper electrophoretic analysis of serum after i dextran sulfate precipitation. ! ’ ■ 47 Jsolution was dialyzed and subjected to starch electropho ne sis at pH 8.6, ionic strength 0.05 for 24 hours. Another i preparation was migrated on starch for 48 hours. As indi cated in Figure 7A, heterogeneity is not entirely apparent after 24 hours of electrophoresis. However, two well re- j 'solved peaks were obtained when the electrophoretic period \ i iwas extended to 48 hours (Figure 7B). An additional step of ultracentrifugal flotation in ,a salt density of 1.019 gm. per ml. was performed on an aliquot of the preparation used for the 48 hour starch Jelectrophoresis. When the top layer (lipoproteins with hy- jdrated densities less than 1.019 gm. per ml.) was removed I land the remaining material (lipoproteins with hydrated den- jsities between 1.019-1.063 gm. per ml.) subjected to starch j electrophoresis for 48 hours, the leading peak was elimina- ■ i ted as shown in Figure 7C. Although the mobility appears ; 1 i [to be somewhat greater than it had been before removal of , !the material of low density, this may be due to some degra- | Nation and the presence of free fatty acids (39)- i j j Bulk Isolation of Lipoprotein Species from Serum i With the finding that lipoproteins prepared by precipitation resembled very closely those prepared by ultracentrifugal flotation, the way seemed clear for the development of a method which would be sufficiently rapid !and involve sufficiently little manipulation to allow 0 2 4 6 8 10 12 14 16 18 20 22 Cholesterol ------- Phospholipid - - - - 3.5 volts/centimeter Temperature of run O' 18 20 22 | MIGRATION IN-CENTIMETERS Pig. 7 Starch block electrophoresis of dextran sulfate precipitated lipoproteins. A -- 20 hours B -- 48 hours C -- 48 hours after removal of lipoproteins with densities less than 1.019 gm./ml. irecovery of the lipoproteins in a natural state and yet in isufficient yield to allow detailed chemical and physical |examination. Concentration of the low density lipoprotein iwould be achieved by treating serum with a polyanion capa- I ble of complexing with the low density lipoproteins and causing them to precipitate out of solution. The high den- i jsity lipoproteins would be separated from the remainder of i ;the serum by flotation methods. The complex would be |solubilized by the addition of NaCl, the 1.000-1.019 gm. ;per ml. hydrated density fraction being separated from the |1.019-1.063 gm. per ml. fraction by ultracentrifugation in j a. salt density gradient. | Rice starch sulfate was utilized as the complexing |polyanion in the preparative scheme given below. This 1 1 change was necessitated by the unavailability of commercial dextran with the necessary properties as recommended by j j Drs. A. Keltz and J. L. Oncley. As indicated by Dr. P. | i t Bernfeld in a personal communication, commercial rice | i i ■starch when sulfated acts as a complexing agent similar to ' I other sulfated polysaccharides. The optimum ratio of rice j I starch sulfate to serum was found to be 1 mg. rice starch j I 1 j sulfate to 1 ml. of serum. This compares quite favorably j with the results of Bernfeld, Donahue, and Berkowitz (68) with sulfated amylopectin. 50 Concentration of Lipoproteins with a_ Hydrated Density Less than 1.063 gm. per ml. ; At the Los Angeles County Hospital Blood Bank* five i pints of whole blood were collected from normal, white, / male donors under 40 years of age, who were presumably in jthe fasting state. The blood was allowed to clot for one |hour at room temperature and then centrifuged at 5 - 7° fov f I an additional hour. The serum, transferred from above the ! clot by suction, was again centrifuged at 5° and pooled. j I A predetermined amount of rice starch sulfate in 0.15 M I | NaCl was added dropwise with stirring to serum which had i I been previously cooled to 0°. The lipoprotein-rice starch I - |sulfate precipitate was resuspended by shaking and the I resulting mixture was centrifuged at 20,000 RPM for 30 minutes. The supernatant was decanted and retained for !recovery of the high density lipoprotein species (1.063- 1.200 gm. per ml.). |Recovery of Low Density Lipoproteins j The soft, orange-yellow paste-like precipitate was ! . I washed once with 0.1 M calcium acetate (pH 7*0) to remove I !adsorbed proteins. The precipitate was then dissolved in i j 3.2 M NaCl containing 5$ BaCl2 and 2 x 10~^V rutin (57)- The BaCl2 was added in order to precipitate the sulfated starch. ! 51 j ! Five ml. of the solubilized precipitate was care- j fully delivered with a calibrated syringe into the bottom |of a capped 13*5 ml. lusteroid centrifuge tube containing j j7 ml. of 0.15 M NaCl, 2 x 10”^ in rutin. By careful I manipulation two distinct phases were obtained. Ultracen- j trifugation was then carried out in a No. 40 rotor of a |Spinco Model L ultracentrifuge at 35*000 RPM (80,730 x g) ;for 30 hours with refrigeration. After centrifugation a ‘milky zone was observed at the top of the tube. Immedi- j 1 _ 1 j ately beneath this was a colorless region followed by a jheavily pigmented layer slightly below the midpoint of the j j jlusteroid tube. This layer was located immediately above 1 1 another colorless region which extended to the bottom of | the tube. By use of a displacement apparatus (Figure 8), i |the top milky zone (lipoprotein fraction 1.000-1.019 gm. ! I ; Jper ml.) and the pigmented middle layer (lipoprotein frac- j tion 1.019-1.063 gm. per ml.) were obtained as separate , 1 |fractions. 1 i i 1 I 1 Recovery of the High Density Lipoproteins i ; The supernatant obtained after the removal of the i |rice starch sulfate precipitate’was adjusted to a density I of 1.200 gm. per ml. by the addition of solid NaBr. The i jsolution was placed in a No. 30 rotor of the Spinco ultra- =centrifuge and centrifuged for 64 hours at 30,000 RPM I j (78,410 x g) with refrigeration. After centrifugation a SYRINGE THREE WAY STOPCOCK STAINLESS STEEL TUBING PLASTIC INSERT RESERVOIR FOR ! HIGH DENSITY SALT SOLUTION ; RUBBER GASKET CENTRIFUGE CAP CENTRIFUGE TUBE Displacement apparatus Fig 53 pigmented layer of lipoprotein was concentrated at the top ! of the tube. This layer was followed by a clear, color less region occupying approximately 1 /8 of the length of i | the tube. Below this region the characteristic color of i serum was observed. By the use of a bulb pipette, the top | layer of pigmented lipoproteins was carefully removed from the tube. i In order to remove any lipoproteins with a density | j less than 1.063 gm. per ml. (in the event that complete i precipitation had not been achieved), 7 ml. of the "top | cut" was layered beneath 5 ml. of a 1.058 gm. per ml. | solution of NaCl in a 13*5 ml. lusteroid tube.. After ! j ultracentrifugation in a No. 40 rotor for 38 hours at ' 37*500 RPM (92,660 x g), a pigmented top fraction was } observed followed by a narrow, clear region and then a . slightly pigmented relatively wider zone. The area below ' this zone extending to the bottom of the tube was color- J less. Again by displacement (Figure 8) the two pigmented : layers were obtained as separate fractions, the "top cut" | ! t J containing the low density lipoproteins, the bottom frac- | tion the I.O6 3-1 .2 0 0 gm. per ml. hydrated density species. Two preparations were obtained by this method and j ' were designated preparation No. 1 and No. 2. Density of the Gradient Layers The density of the various gradient layers from : 54 I [which the lipoproteins were isolated are shown in Table IVIII. Since the boundaries of a lipoprotein class within i [the gradient tube are not sharply defined, the density of !the gradient layer is near but not necessarily equivalent I jto the average density of the lipoprotein class. j i I Analytical Centrifugation i | The three lipoprotein fractions isolated were sub- j jjected to analytical centrifugation. The analytical j I patterns for preparation No. 1 are shown in Figure 9. It |is evident that the 1.000-1.019 gm. per ml. fraction con sists of at least two components — a slower moving principal component and a small amount of a second, faster [moving component. Since no sedimenting components were idetected, it is concluded that neither high density lipo- * ! ’ ■ ' I !proteins nor albumin is present. The 1.019-1*063 fraction j i 1 shows only one component. This fraction apparently is free! i of the other two low density components, as well as of the j > I 1 ihigh density lipoproteins and albumin. The film record ; i " J ifor the I.O6 3-1*200 fraction also indicates only one com- ! iponent. The presence of low density lipoproteins would be I indicated by faster moving peaks. No sedimenting material i !is observed, which eliminates the presence of albumin. I Essentially the same results were observed for preparation No. 2 -- the only notable difference being a !reversal in the quantities of the slower and faster moving j 55 TABLE VIII DENSITY OF THE GRADIENT LAYERS CONTAINING THE LIPOPROTEINS Lipoprotein Fraction Gm./ml. Preparation 1 Gm./ml. Preparation 2 Gm./ml. < 1.019 1.020 1.022 1.019-1.063 1.041 1.043 1.063-1 .2 0 1.132 1.128 56 Pig. O C j SALT 1.063 TIME MIN. OC 16 22 30 48 60 i SALT 1.200 ? Flotation patterns of serum lipoproteins isolated by the precipitation-flotation method. oC2 ~ Less than 1.019 gm./ml. density fraction f3 -- 1.019-1.063 gm./ml. density fraction 0CX — 1 .063-1.200 gm./ml. density fraction 57 |components in the 1.000-1.019 fraction in preparation No. j2. The S^, values for the two preparations are shown in iTable IX. i i ! I Moving Boundary Electrophoresis j j Electrophoretic analyses were performed on the lipo- i i I protein fractions from preparation No. 1 and No. 2. i iExamination of the film patterns indicated that comparable j :lipoprotein fractions of preparation No. 1 and No. 2 were j | iessentially identical. Only one symmetrical peak was ob- i i |served for both the 1.019-1.063 and 1.063-1-200 gm. per ml. I ! jfractions. Comparison with the albumin peak showed the j [ 1.019-1.063 fraction migrating slightly behind the midpointj t i • ! [between the albumin peak and the origin and the 1.063-1-200| |fraction migrating as a tail on the albumin peak. A very j I ] (diffuse peak which migrated slightly ahead of the midpoint | 1 • between the albumin and the origin was observed for the j 1.000-1.019 gm. per ml. fraction. > A { i ! i Composition of the Lipoproteins ; i •' ! | The per cent composition of the three fractions of (lipoproteins are presented in Table X along with their free cholesterol to total cholesterol, and total cholesterol to phospholipid ratios. The most obvious difference among the (fractions is the relative increase in protein content with (increasing density. The triglyceride is the most variable TABLE IX Sf VALUES OF LIPOPROTEINS PREPARED BY RICE STARCH SULFATE PRECIPITATION Preparation < 1.019 gm. Faster Moving , Component per ml. Slower Moving Component I.OI9-I.O63 gm. per ml. No. 1 * 5 3 [ Mj ? e a k j s*.11 tel Sf 7 No, 2 sf 15 tei Sf 3 TABLE X PER CENT COMPOSITION OF SERUM LIPOPROTEIN FRACTIONS Fraction Density Gm./ml* Prep. Free Cholesterol Cholesterol Ester Phospho lipid Glyceride Protein FCi/ TCi/ TC PL 1 6.4 6.3 16.6 16.2 18.3 18.4 46.1 46.4 12.6 0.39 0.88 1 < 1.019 2 4.9 5*1 11*5 10.9 17*1 17*1 58.2 59*1 7*9 0.44 0.66 1 9*1 8.5 37*6 39*5 21.0 21.3 12.9 11.3 19*4 0.28 1.49 2 1.019-1.063 2 6.7 6.4 43.2 44.3 20.7 20.4 8.3 7*9 21.1 0.20 1.57 1 2.5 2.5 17*4 17*4 24.1 24.5 9.0 8.7 47*0 0.20 0.52 3 1.063-1.200 2 2.5 2.5 20.7 20.7 26.1 25*9 8.8 8.9 42.0 0.17 0.56 1/ Free cholesterol/Total cholesterol 2/ Total cholesterol/Phospholipid ^ 60 lipid component, varying from 46 - 60 per cent of Fraction i 1 to 9 per cent of Fraction 3. There is a significant in crease in phospholipid content with increasing density. I The ratio of free to total cholesterol diminishes progres sively with increasing density. Since the triglyceride value was obtained from the total lipid weight minus the ' I | cholesterol and phospholipid values, any unesterified fatty !acids that might be bound to lipoproteins, either in_vivo ; or as an artifact would be included in the value for i !triglyceride. I i i j Estimation of the Nitrogenous Constituents of | Lipoprotein Phospholipids ! ! The purified lipid extract from each lipoprotein I fraction of preparation No. 1 was hydrolyzed and analyzed j for choline, sphingosine, serine, and ethanolamine. The I I results for choline, sphingosine and ninhydrin positive j J material (after sphingosine extraction) are presented in :Table XI. Quantitation of serine and ethanolamine was not j j j |achieved due primarily to the low concentration of these ^substances in each of the hydrolyzed lipid fractions. However, their presence was verified, visually, by examination of ninhydrin stained paper strips following electrophoresis. The ninhydrin positive values as given in Table XI are calculated on the assumption that serine and ethanolamine are the only contributors and are present TABLE XI NITROGENOUS CONSTITUENTS Of HUMAN SERUM LIPOPROTEIN LIPIDS Density Fraction v . Gm./ml. Mi/ P Hydrolysis Sample Choline % Total Lipid Nitrogen Ninhydrin Sphingosine Positive^/ Undetermined A 68.2 13.7 4.2 13.9 < 1.019 ' 1.44 B 73.3 15.9 3.8 7.0 Average 70.8 14.8 4.0 10.4 A 72.0 15.8 3-3 8 .9 1.019-1.063 1.42 B 71.7 14.8 3.6 9.9 • Average 71.9 15.3 3.5 9.3 A 78.0 10.8 6.0 5.2 1.063-1.20 1.24 B 79.3 10.0 4.8 5.9 Average 78.7 10.4 5.4 5.5 1/ Total lipid nitrogen/total lipid phosphorus — mole ratio 2/ Assuming ninhydrin color is due to the presence of equimolar serine and ^ ethanolamine h 62 in equimolar amounts. It should be emphasized that the |values for'the individual bases presented in Table XI may I ' * I include unrecognized substances which determine in the j i same manner (6l). I i j [ CHAPTER IV | j DISCUSSION i I i J The present investigation of the preparative I methods for serum lipoproteins was undertaken with the i | objective of finding a relatively rapid means of isolating I these materials in relatively large quantities. A purely I ultracentrifugal procedure, such as that employed^by i v ; Havel e_t al. (22) requires a total period of 88 hours in • the ultracentrifuge in order to obtain the two low density j ■ j fractions (1.019-1.063 gm. per ml. and < 1.019 gm. per ml.) I j from 100-120 ml. of serum. Making use of the initial ! i i precipitation with dextran sulfate or sulfated rice starch j i ■ 1 j reduces the time in the ultracentrifuge to 30 hours for ! I * ! batches of about 1000 ml* of serum. I i ! i The utility of the method of precipitation with sul-[ ! fated macromolecular polysaccharides was first reported by | Oncley and Mannick ( 6 9)* who originated their study from j \ ' I ' the observations of Walton (70) on the formation of an 1 i ! j insoluble complex of fibrinogen with dextran sulfate. j Information regarding the conditions for precipitation of j I j ! low density lipoproteins with dextran sulfate was obtained j through the courtesy of Dr. A. Keltz. Since the completion of this work, a detailed des cription of the procedure employing dextran sulfate has ; 64 I been reported by Oncley, Walton, and Cornwell (71). This I jappears to offer no advantage over the use of sulfated rice 1 (starch, which is more readily obtained than the dextran j sulfate. The mechanism of interaction of the macromolecular i f ipolysulfated compounds and low density lipoproteins is not i junderstood at present, although the formation of complexes Ibetween many sulfated polysaccharides and low density lipo proteins which change both solubility and electrophoretic I ^mobility have been reported (7 2, 73)♦ It is known that the I size of the polyanion appears to be one determining factor in the solubility of the complex, low molecular weight dex tran sulfate forming soluble complexes with fibrinogen and j I 'low density lipoproteins while high molecular weight dex- I i j tran sulfate results in precipitation of these substances 1 (71). The recent report by Bernfeld, Donahue, and ! Berkowitz (68) on the interaction of low density lipopro- J i teins with various polyanions suggests that the structure iof the polysaccharide is closely related to the properties | ;of the complex formed. The present study as well as the ! iwork of Bernfeld et_ al_. (68) and Oncley et_ al_. (71) indi cate that the high density lipoproteins are not iprecipitated under the conditions employed for the precipi tation of the low density lipoproteins. A few experiments !at lower pH values indicate that the failure to obtain pre- I » Icipitation of high density lipoproteins is not just due to 65 a higher net negative charge on the high density lipopro- i ; teins. I Other preparative methods developed and utilized in | | the past several years all present certain limitations. I j The Cohn cold alcohol fractionation procedure permits de- | termination of the lipid components of the alpha and beta | lipoproteins but further subfractionation of these groups ; is not possible with this technique. It also fails to i I : separate lipoproteins from other serum proteins. Neverthe- ; less, one of the earliest isolations of |3-lipoprotein i | involved the use of this technique in combination with the I technique of ultracentrifugal flotation. The method of | zone electrophoresis with quantitation either by staining I (28) or by chemical analysis of eluates from the support- j i ■ i j • ; ing medium (26, 2 7).has been widely used in the past. I i i j However, electrophoretic techniques also fail to separate lipoproteins from other serum proteins, thus making i impossible the study of the protein moiety. i In this study, zone electrophoresis has proven use- ! ; . i j ful as an index of comparison between lipoproteins prepared ‘ by ultracentrifugal flotation and by precipitation- i i flotation. It should be pointed out, however, that several ! . | problems are inherent in the application of starch electro phoresis in this manner. If the extent of migration (mobility) is used as a means of comparison, differences ; may be suspected between preparations which may in jactuality only reflect differences in the mechanics invol ved in starch block preparation. The wetness of the starch block, for example, is difficult to control exactly. Con versely, similarity in mobility cannot be considered to be | * jproof of similarity in composition. This is indicated by ithe cholesterol and phospholipid values shown for the i various starch block electrophoretic results (Figures 2A, i I2B, and 7)* Lipoprotein peaks with similar mobilities show varying cholesterol to phospholipid ratios. It is impossi ble to decide, from the present study, whether these ■differences result from analytical difficulties or are i ilargely a reflection of the known variations among the i 'cholesterol to phospholipid ratios of serum samples from different individuals, and of the subfractions from such serum samples. For example, Barr, Russ, and Eder (74) ■found the ratio of cholesterol to phospholipid to vary from about O.78 to 1.14 in a series of individual plasmas from normal males. Cholesterol:phospholipid ratios between 0 . 32 - 0.62 were reported for the high density lipoproteins i and between 1.10-1.4-9 for the low density lipoproteins. 1 The variation in cholesterol:phospholipid ratio observed in , 1 the present study may also be a reflection of the known j difficulties involved in the extraction of lipids from : i starch. A wide literature is available on the affinity of certain lipids for the starch granule particularly in the 1 presence of polar solvents (75)• 67 If the lipoprotein preparations are compared on the i I basis of their homogeneity under similar conditions (time i j of electrophoresis, ionic strength, pH, etc.) a striking 1 | similarity in pattern is observed for the ultracentrifu- | gaily isolated and polyanion precipitated lipoproteins.^ | Both techniques of preparation yield lipoproteins which i appear relatively homogeneous after a 24 hour migration i | period whereas heterogeneity is quite apparent for both i j preparations after a 48 hour period - the faster moving i peak migrating with almost the mobility of an “globulin* Additionally, the leading peak of the polyanion precipita- i ted lipoproteins can be eliminated by centrifugation in a salt density of 1.019 gm. per ml. This has also been | observed for the lipoprotein prepared by ultracentrifugal flotation (25). Therefore, it appears that both techniques yield preparations which contain at least two components j i with differing mobilities in starch, the faster moving i component being capable of removal by flotation at a salt ' \ density of 1.019 gm, per ml. | The apparent homogeneity of the lipoprotein prepara- i i i tions isolated either by ultracentrifugal flotation or by precipitation-flotation after 24 hours of migration in starch (ionic strength 0.0 5) is not consistent with the heterogeneity of these components when observed under simi lar conditions in whole serum. Since a study to clarify this observation was not attempted, any explanation must be 6 8 considered theoretical. It is possible that dialysis may ihave been incomplete, resulting in a preparation containing a higher content of salt than is normally present in serum. I !This might produce a lipoprotein-lipoprotein complex which is not readily separable electrophoretically until suffici ent time has elapsed for the excess salt to be lowered in concentration by diffusing into the low ionic strength !buffer. This may be analogous to the difference in results ! j - obtained with serum at ionic strengths of 0.1 and 0.05, j 1 respectively. One of the difficulties in evaluating a preparation of lipoproteins is the establishment of suitable criteria of purity. By the methods of analytical centrifugation and I ! (electrophoresis, the low density fraction of I.OI9-I.O63 j Igm. per ml. and the high density fraction of 1.063-1*200 ! t Igm. per ml. show only single peaks. However, according to j i De Lalla and Gofman's analysis of analytical centrifugation• patterns (^9)> each peak, while sharp, represents a whole ! ' spectrum of lipoprotein molecules of very similar hydrated { ! 1 !densities. This has also been pointed out by Oncley, Walton, and Cornwell (71) by the use of a distribution j function, developed by Williams and Saunders ( 7 6) for heterogeneous high polymer systems. Since the 1.000-1.019 gm. per ml. lipoprotein fraction presented two peaks upon analytical centrifugation, the presence of molecules of j even less similarity in their hydrated densities is 69 indicated for this fraction. A discrete peak could not be ! demonstrated by moving boundary electrophoresis although a I I single peak was observed by zone electrophoresis in starch. I j Apparently, each of these lipoprotein classes that is so i ] heterogeneous in respect to Sf values appears relatively homogeneous., at least, in starch block electrophoresis. | The absence of a second peak in the centrifugal i ! ! pattern of the 1.063-1*200 gm. per ml. high density class appears to rule out the presence of one of the two high density lipoproteins normally found in serum. Since the j 1*075 gm. per ml. component occurs in small amounts in | serum (ll) and, since the high density class isolated in j this study was recovered from a gradient layer with an i | average density of 1.130 gm. per ml., it appears likely ' that it consists solely of the 1.145 gm. per ml. fraction. The chemical composition of the three fraction as presented in Table X does not include all the substances which are known to be present in the various lipoproteins. For example, carotene and tocopherol have been shown to accompany serum lipoproteins (7* 71, 78)• Other fat- soluble vitamins may be present. These substances would be present only in negligible quantities. Unesterified fatty acids would make up a more significant portion of the total weight, the available information indicating a range of values from less than 1 f o to 2% of the total weight (20, 2 3). 70 The composition of the various lipoprotein classes reported here can be compared with the results of the study i jof Bragdon, Havel* and Boyle (23)*who analyzed fractions of !similar density (Table V). The similarity in values for the < 1 .0 1 9 gm. per ml. and 1.019-1.063 gm. per ml. frac- jtions offers further evidence for the postulate that the jmethod of precipitation-flotation yields material similar 1 !to that obtained by ultracentrifugal flotation. Since the 5 1 'high density lipoprotein value reported by Bragdon et_ al. ; includes both high density species (1 .0 7 5 and 1.145 gm. per ml.) and since as indicated above the high density fraction obtained in this study appears to consist only of the 1.145 gm. per ml. species, some indication as to the composition of the 1.075 gm. per ml. lipoproteins can be deduced by a I comparison of the two high density values. The favorable j I comparison suggests that the 1 .0 7 5 gm. per ml. fraction is j I I very similar in composition to the 1.145 gm. per ml. frac- 1 1 tion or it is present in such low amounts relative to the 1.145 gm. per ml. fraction that its value does not appre- I jciably affect the over-all composition of a mixture. Comparison with the composition as reported by I jLindgren, Nichols, and Freeman (20) indicates a general similarity in values although the cholesterol ester value of these workers for the high density lipoprotein is con siderably lower and the protein value somewhat higher (Table IV) than the present findings. 71 The 1.019-1.063 gm. per ml. low density fraction which contains the major -lipoprotein species of serum has been characterized by numerous investigators. The chemical composition and total cholesterol to phospholipid values are in adequate agreement with.similar values ob- 1 | tained by other workers (16, 17, 20, 22, 27, 79)• Investigators utilizing the cold-alcohol fractionation procedure, however, report the absence of triglyceride. ; The present study confirms the previous findings t 1 j (22) that the free cholesterol to total cholesterol ratio for the <1.019 gm* per ml. fraction is significantly dif- j ferent from the 1.019-1.063 gm. per ml. fraction and high | density lipoproteins. Some differences in partition of 1 the various types of phospholipids between the different ; lipoprotein fractions are indicated by the present study. ! 1 This latter finding is somewhat surprising in view of the i : i work of Kunkel and Bearn (80), who indicate that the phos- j pholipids on the high density lipoproteins are exchangeable with those of low density and vice versa. Upon P^2 admin- ! I 1 j istration, very little difference in specific activity of the different serum lipoprotein fractions was observed by j these workers. However, they report that in some sera slightly higher specific activities were encountered for the o^-lipoprotein, the /S - and oC2-lipoproteins having similar but lower specific activities. The relative levels of the nitrogenous phospholipid bases of the different 72 lipoprotein fractions observed in this study can be recon ciled with the findings of Kunkel and Bearn if It can be assumed that various phospholipids are differentially labeled. Evidence of differential labeling has been noted in certain tissues (81). j It is apparent that the work of the last decade i . j along with the present study has done little but scratch |the surface in the study of the serum lipoproteins. Con- I sideration of what is known, however, indicates that the serum lipoproteins can be segregated into broad groups from jthe standpoint of ultracentrifugal analysis. The lipopro teins may also be distinguished as three major groups by !electrophoresis. There are definite differences between ! {the groups with respect to the per cent composition, free I y ! cholesterol to total cholesterol ratio and total choles terol to phospholipid ratio. There appear to be some differences in the amino acid composition and N-terminal residue of the protein moiety between the groups. Some I differences in partition of the phospholipid bases between l j the group's are indicated. Immunochemical differences have jalso been reported by several workers. Differences are also found within the lipoprotein groups. There are clear ultracentrifugal differences with in the o £ - and -lipoprotein groups. The recent work of Oncley et_ al. (71) indicates small but definite differences within the ^-lipoprotein group. The ether extraction | 73 | studies of Avigan (8 2) suggest that the ^-lipoprotein i group and possibly the c<2-lipoprotein group could each ! represent one protein-phospholipid complex with varying ! amounts of free cholesterol, esterified cholesterol, i | triglyceride and free fatty acid. j It is of interest to speculate on the possible j metabolic role of the serum lipoproteins in the light of | | the present physical and chemical information. It is not I j difficult to visualize the serum lipoproteins as providing ! j a mechanism for the transport of large amounts of lipid. i j | The chemical specificity of these serum lipoproteins would ! ! seem to argue against their formation by simple colloidal i i principles. These lipoproteins seem rather to represent | | ! specialized carrier molecules synthesized by metabolic re- i ! i • I ! actions in more or less the molecular form observed for the' i ; | purified components. It is difficult, however, to estab- j j lish whether the lipoprotein distribution found in serum < ! represents a dynamic equilibrium comprising a sequence of ; , metabolic reactions whereby one lipoprotein group is con- ! j j ! verted to another group. By virtue of isotope, ! i | ! immunochemical, N-terminal residue and protein composition J ! ■ ' i | studies, it seems unlikely that the low density lipopro- i ! I I teins are converted to the high density lipoproteins. Thexej | would appear to be less evidence in opposition to a mecha- J nism whereby the 0 6 0-lipoproteins are converted to the j | j /3-lipoproteins. Iodinated c>£2~lipoprotelns aPPear to be j 74 transformed in a unidirectional manner to jB -lipoproteins; i iimmunochemicalj N-terminal residue, and protein composition i studies show little or no differences in the protein imoiety between these groups; and structurally, the o^2- and !^3-lipoproteins appear to be similar on the basis of ether I (extraction and polyanion complexing studies. I ! The conversion of o£2"liP0Pro‘ t : 'e:3- ns t lipoproteins would not involve a simple release of gly- ,ceryl esters and/or fatty acids as suggested by Jones et_ I I Ial. (ll). The present information on the chemical compo- jsition of these groups would argue against a prosthetic fragment consisting of protein, cholesterol and phospho lipids. The differences in the phospholipid and i {cholesterol content of the two low density fractions must j be explained by any valid theory. These changes could be considered to take place secondarily as a function of the increase or decrease in neutral fat. The apparent ease I with which unesterified plasma cholesterol can exchange ! i (With that of the red blood cell (8 3) is indicative of the { , 1 1 ;labile nature of the lipid-protein complex in these lipo- iproteins. Similar interchange, although of a smaller imagnitude, has been observed for the phospholipids (84). i . As a working hypothesis, the <x.2-lipoproteins may be assumed to consist of one protein-phospholipid complex |with varying amounts of free cholesterol, esterified |cholesterol, triglyceride and free fatty acid. The core jof a representative species in this group, would consist i mainly of phospholipids, the surface consisting of areas of lipids and peptide, possibly in a configuration similar to j jthe postulated mosaic structure in protoplasmic membranes | (16). The different species observed within this group i i I could represent the conversion products of the lowest den- i 1 jsity species in this fraction. This conversion could be j (assumed to take place in a stepwise manner by a simple re- \ i jtnoval of the appropriate quantity of lipid material. ! ’ Conversion of the o^2“liP0Pro' t;eins ^-lipoproteins ~ 1 I [would probably Involve the removal of not only the appro- i jpriate quantity of lipid material but perhaps even a (peptide fragment. Small but definite conversions could 1 joccur within the ^ -lipoprotein group in a similar manner j las described for the o^2“1:i-P0Pr0te:5- n group, thus accounting ! [for the spectrum of lipoprotein species observed in this ; group. i The conversion scheme postulated above would, in ' i 1 1 ' i jgeneral, be compatible with the available physical and j |Chemical knowledge''of the serum lipoproteins. CHAPTER V SUMMARY A method of bulk isolation of lipoproteins from human serum has been developed. The method involves the use of rice starch sulfate as a complexing agent for the low density lipoproteins. Three lipoprotein fractions have been separated by this method from pooled normal human serum. The chemical and physical properties of these fractions have been studied in detail. It has been shown that chemical and physical differences exist between all three fractions. The findings neither prove nor disprove interconversion of the lipoproteins. The process, t if considered to occur, must involve a mechanism more | complex than removal of glyceryl esters and/or fatty acids.; BIBLIOGRAPHY BIBLIOGRAPHY Macheboeuf, M., Bull. Soc. Chim. biol., 11, 268, 483 (1929). ~~ Blix, G., Tlselius, A., and Svensson, H., J. Biol. Ghem., 137* 485 (1941). ^ ^ Adair, G. S., and Adair, M. E., J. Physiol., 102, 17P (1943). ~ 1 Pedersen, K. 0., Ultracentrifugal Studies on Serum and Serum Fractions (Almquist and Wiksells, Uppsala, 19^5). Cohn, E. J., Strong, L. E., Hughes, W. L*, Jr., Mulford, D. J., Ashworth, J. N., Melin, M. and Taylor, H. L., J. Ain. Chem. Soc ♦, 68, 459 (1946). Oncley, J. 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Creator Ushiyama, Takao (author)

Core Title The lipid composition of human serum lipoproteins.

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Degree Doctor of Philosophy

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