Proten-Lpd Relatonshps n Normal Dog Plasma pplcaton of Cohn Method 0 By ELL M. EUSS ND JULIE RYMUNT lthough Cohn's merofmetonaton method number 0 was specfcally devsed to meet the condtons of proten concentraton and dstrbuton n human plasma, t s shown n ths contrbuton that t may be appled to studes of canne plasma. Essentally all of the lpds of plasma are recovered n Fractons IV + V + VI and I + III. Only neglgble amounts appear n Fracton II. Electrophoretc studes proved that the lpoprotens of Fracton IV + V + VI have the moblty of alpha globulns and those n Fracton I -f III have the moblty of beta globulns. WITH the use of Cohn's rncrofractonaton method number 0, almost all of the lpds of normal human plasma may be quanttatvely recovered from two dstnct fractons. In one, they are bound to the alpha globulns as alpha lpoprotens, and n the other to beta globulns as beta lpoprotens. The method has been found applcable to the separaton of lpoprotens n a wde varety of normal and pathologc plasmas. - 7 That the greatest relatve amount of lpd n dog plasma, n contrast to human plasma, s bound to alpha globulns whle only a small part s bound to beta globulns was frst demonstrated by Zelds, lng, McCoord, and Kulka. 8 By electrophoretc analyses of dog plasma before and after an elaborate alcoholether extracton, they showed marked changes n the confguraton and number of alpha globuln peaks, wth lttle or no change n the remanng electrophoretc components. The present communcaton s a report of the effectveness of the Cohn method n separatng the lpd-bearng globulns of dog plasma. It ncludes some prelmnary observatons on ther lpd composton. From the Department of Medcne, The New York Hosptal-Cornell Medcal Center, New York. These studes have been aded by a grant from the U. S. Publc Health Servce, by support from the Commonwealth Fund and the lbert and Mary Lasker Foundaton, and by gfts from Mrs. Katherne Llly Conroy. Receved for publcaton, December, 954. PROCEDURES Studes were made on plasma from sx apparently normal dogs three to four years of age. Blood was drawn from the jugular ven nto CD soluton,* and the plasma was fractonated by the technque dentcal to that employed n prevously reported studes of human plasma. Three proten fractons were obtaned under the same precse condtons necessary for the solaton of Fractons TV + V + VI, II, and I + III from human plasma. Snce, however, dog plasma s known to contan electrophoretc components never found n human plasma, the Cohn nomenclature for proten fractons was avoded and the desgnaton of Fractons, B and C + D was substtuted. Proten analyses were made by the standard Kjeldahl and buret procedures and, whenever necessary, by the mcro methods of Jacox' or Lowry and coworkers. 0 Cholesterol and phospholpd determnatons were performed as descrbed elsewhere wth an adaptaton of the phospholpd method to a mcro scale whenever t was essental. Movng boundary electrophoretc analyses of dog plasma and ts fractons were carred out wth the ad of an mnco-stern apparatus usng veronalctrate buffer ph S.6, onc strength 0. at C. The samples were prepared for electrophoress n the same manner as descrbed for human plasma. For rapd dentfcaton of lpoprotens or other proten components, paper electrophoress was employed. The fractons were prepared as for movng boundary analyses, but dalyss was not essental. Ths onophoretc system was a modfcaton and combnaton of several methods. M " To a strp of Whatman MM flter paper held tautly across two plastc rods, a potental of 00 V was appled for sx * Exactly 0.6 ml. of acd ctrate dextrose soluton contanng.0 gm. traodum ctrate (.0 HO), 8.0 gm. ctrc acd monohydrate, and.0 gm. dextrose per lter was used for each.0 ml. of blood. 94 Crculaton Rttarck, Volume III, Mara IBM
ELL M. RUSS ND JULIE RV'MUXT 95 horn's. The experments were carred out n a constant vapor chamber at 5 C. usng veronal-ctrate buffer ph 8.6, onc strength 0.05. The lpoprotens were specfcally dentfed wth Sudan black B, whle all proten components were staned wth bromphenol blue. 4 ' " Starch electrophoress was carred out at 5 C. usng veronal-etrate buffer ph 8.6, onc strength 0., employng the system descrbed by Kunkel and Slater." potental of 00 V was appled to a starch block 0 cm. by 45 cm. and cm. thck for approxmately twenty hours, or untl complete resoluton had taken place. Pror to electrophoress, Fracton of dog plasma was concentrated wth znc reagent and redssolved wth ctrate-sodum chlorde reagent to a concentraton of 4.0 gn. per cent as descrbed prevously for human Fracton IV + V + VI. Of ths soluton, 4.0 ml. was appled to the starch block. Followng complete electrophoretc separaton of the proten components, the block was cut nto.0 cm. segments. The proten was eluted wth 0.S5 per cent sodum chlorde. Cholesterol, phospholpfl, and proten analyses were made on each segment employng, when necessary, the mcro procedures descrbed elsewhere n ths communcaton. RESULTS The type of separaton characterstcally obtaned from the Cohn fractonaton of dog plasma may be seen from the movng boundary electrophoretc patterns shown n fgure. LI *LJ I ^m *l t, - - ' m % >- DOG PLSM The percentage area dstrbuton of the electrophoretc components n plasma and Fractons, B, and C+D from three dog plasmas are lsted n table. lbumn, whch on an average represents approxmately 5 to 40 per cent of the total proten, srecoveredalmost entrely n Fracton ; only very small amounts are found n Fracton C+D. Two extra peaks together representng approxmately 0 per cent of the plasma total area are desgnated as alpha- and alpha^t on the bass of ther mobltes and postons relatve to the alpha- and beta- peaks. In ths scheme of desgnaton, the beta- component mgrates somewhat more slowly than does the beta- of human plasma. The alpha- globulns appear to be concentrated entrely n Fracton whle the alpha-, alpha-, and alpha-4 components are scattered n varous amounts between Fractons and C+D. Resoluton of the alpha-], beta-, and beta- components n Fracton vares consderably n the dogs. In one, there s a mergng of the alpha-4 and beta- peaks, and, n another, of the beta- and beta- peaks, resultng n one electrophoretc component B r 4 L <LI C+D «LI «SCENDING DESCENDING -^ LI FIG.. Movng boundary electrophoreto patterns of dog plasma and three fractons.
96 PROTEIX-LIPID RELTIONSHIPS IX DOG PLSM TBLE. Percentage rea Dstrbuton of Proten Components from Movng Boundary Electrophoretc nalm* of Dog Plasma and Three Fracton* Specmen DOR Xo. lbumn m at a* P ft Plasma Fracton Fracton B 5. 4. 40.5 69. 70.S 6S.7.7.4.4 8.5 9.5 7. S.6 6.S 8. 7.7 7.S 6.0 9. 8. 7. 5.0. 7. 4.8 5.5 8.6 5.9 6. 6.9 9. 7.8.5 4.5 5..5.. 6.7..6 4..0 4.5 4..7.9.6. 6.9 9.S 5.0 5.. 8.7 5. 4.9 50.4 47.7 44.S.5 9.0.6.7 9.S. 48.0 49. 5.6 0.S IS.6 0.S whch mgrates between the alpha- and beta- areas. Beta- globulns, asde from the small amount found n Fracton, are localzed chefly n Fracton C+D where they represent approxmately 7 per cent of the fracton. The large concentratons of beta- globulns and fbrnogenrangngfrom 9 to 5 per cent of the total protens n unfractonated plasma are recovered n both Fractons B and C+D. pproxmately 60 per cent of the plasma gamma globulns are recovered n Fracton B; the remander s found n Fracton C+D. Sudan black B lpd stans of the paper electrophoretc patterns of dog plasma and ts fractons ndcate that most of the lpds of dog plasma mgrate wth protens from the mddle of the albumn to the alpha- area, whle very small amounts appear to be bound to the beta globulns. The patterns also ndcate that the alpha lpoprotens are concentrated entrely n Fracton and appear to be effectvely separated from the beta lpoprotens whch are localzed entrely n Fracton C+D. Ths lpoproten dstrbuton n dog plasma s a marked contrast to that n the human, where approxmately three-fourths of the lpds are bound to beta globulns. In table, the mean concentraton and dstrbuton of cholesterol, phospholpds, and proten, ncludng the cholesterol-phospholpd ratos from the plasma and fractons of sx dogs, may be compared to those obtaned from human plasma. lthough the concentraton of cholesterol n the dog plasmas vares greatly, t s n general lower than n humans. The concentraton of the phospholpds n the two speces s essentally the same. Thus the cholesterol-phospholpd rato s consderably lower n canne plasma, n whch the fgure of 0.5 may be compared wth the human value of 0.84. In Fracton of dogs, the concentratons of both the cholesterol and phospholpds are approxmately twce as great as n the correspondng human Fracton IV+V+VI, wth no essental dfference n the cholesterol-phospholpd ratos. The concentraton of cholesterol n Fracton C+D s less than one-tenth, and the phospholpd s less than one-ffth of that n the human Fracton I+III. The cholesterolphospholpd rato whch averages only 0.6 n Fracton C+D n comparson to.5 for humans would ndcate the possblty of a speces varaton n the consttuton of the lpoprotens or perhaps unusual mxtures of lpoprotens. The decreased albumn concentraton characterstc of dog plasma (table ) accounts n part for the dmnshed total plasma proten concentraton. The greatest speces dfference appeare n Fracton, where the proten concentraton s markedly reduced. Ths may be attrbuted to the dmnuton of albumn n the unfractonated plasma, as 96 per cent of the total albumn s recovered n Fracton. Fractons B and C+D show no essental dfference. Values for the relatve dstrbuton of lpds
ELL M. RUSS XD JULIE RYMUXT 97 TBI.K. The verage Concentraton and Comparalve Dstrbuton of Cholesterol, Phospholpd, and Proten n the Plasma of Dogs and Man, Includng the Cholesterol:Phospholpd Ratos Concentraton n Dstrbuton "n Dog Plasma Human Plaama Dog Human Mean Range Mean ± S.D. Cholesterol Unfractonated plasma Fracton Fracton B... Phospholpd Unfractonated plasma Cholesterol:Phospholpd Rato Unf ractonated plasma Fracton. ll- % 4.0 4.0 l.s 0. 57.0 4 0.7 0.6 0.50 0.6 lt- % S-79 64-55 0- - 5S-0-94 9-8 0.46-0.5S 0.44-0.5S 0.46-0.80 Ht- % S9 ± 5 6 ± ± ± 5 ± 8 7 ± 0 9S ± 0.S4 ± 0. 0.5 ± 0 06.5 ± 0.6 % of Total S5.4.6. 90 9.6 % of Total..6 65. 64 5 45. Proten Unfractonated plasma (Kjeldahl)... Fracton (Buret)... Fracton B Gm.% 5.7.4 0.S.6S Gn. % 5.09-5.99.0-.60 0.66-.08.59-.94 Cm. % 6.9S ± 0.5 5.04 ± 0. 0.S7 ± 0.5.7 ± 0.9 56.9 4. 8.6 69..9 9.0 n Fractons and C+D emphasze the strkng dfferences between the speces; 85.4 per cent of the canne plasma cholesterol n recovered n Fracton as compared to. per cent for humans. The speces dfference n phospholpd dstrbuton s almost as great. Recoveres for cholesterol and phospholpds were 94 per cent and 00 per cent, respectvely; the proten recovery averaged 0 per cent. The analytcal results of an electrophoretc proten separaton of Fracton employng a starch medum are shown n fgure. Ths analyss was made on a pool of Fracton from three dogs. The concentratons of proten, cholesterol, and phospholpds are plotted for each.0 cm. segment of the block accordng to the legend descrbed on the graph. The proten curve ndcates the same type of electrophoretc dstrbuton of the components n Fracton as was shown by the movng boundary patterns n fgure and also by the paper electrophoretc patterns. The curves of the cholesterol and phospholpd concentratons ndcate that these lpds are concentrated chefly between the alpha- and alpha- areas, wth a spread from the start of the albumn through the alpha- area. In ths experment, the proten recovery was 96 per cent, whle the cholesterol and phospholpds each showed an 80 per cent recovery. In three smlar experments employng starch electrophoress of dog Fracton the proten recoveres ranged from 9 to 0 per cent, whle that for the cholesterol was 65 to 80 per cent, and the phospholpds 65-75 per cent. The rather poor recovery for the lpds may be MWTO* COttCCNTIUTIOM CHOLXtTCROL COMCINTRTIOII PHOLJPD COHCCNTRTIOW FIG.. Quanttatve separaton of proten components n pooled dog Fracton by the starch electrophoretc method; dstrbuton and concentratons of cholesterol, phospholpd, and proten.
98 PROTEIX-LIPID RELTIONSHIPS IN DOG PLSM partally explaned by the nevtable adherence of some of the proten to the waxed paper whch s used to envelop the starch block durng elcctrophoress. lso, the large amounts of lpd-bearng protens characterstcally found n dog Fracton may be extremely dffcult to elute completely from the starch granules. nalyss of Fracton pror to electrophoress showed a cholesterol-phospholpd rato of 0.57. Snce the recoveres for both cholesterol and phospholpds were of the same order, cholesterol-phospholpd ratos were calculated for each segment. These showed a range of 0.40 to 0.65 except for the two segments 6 and 7. Ths dscrepancy was not apparent n observatons on Fracton of other dogs. It s beleved that n ths experment t s attrbutable to faulty analyss of phospholpds n these two segments. SUMMRY Cohn method number 0, although orgnally developed for human plasma, may be appled to canne plasma for an effectve separaton of many proten components. In dog as n man the plasma lpds are combned chefly wth alpha and beta globulns. In the dog and unlke man, however, the lpds are bound almost entrely to alpha globulns. s judged from the cholesterol-phospholpd ratos, the alpha lpoprotens of dogs are smlar to those of man. The beta lpoprotens have, however, a much lower cholesterol-phospholpd rato than those n human bengs. Ths may ndcate that n the dog there are qualtatve dfferences or unusual mxtures of the beta lpoprotens. REFERENCES COHN, E. J., CURD, F. R. N., SURGENOR, D. M., BRNES, B.., BROWN, R. K., DEROUUX, G., GILLESPIE, J. M., KHNT, F. W., LEVER, \V. F., LIU, C. H., MITTLEMN, D., MOUTON, R. F., SCHMID, K. ND UROM, E.: system for the separaton of the components of human blood: Quanttatve procedures for the separaton of the proten components of human plasma. J. m. Chem. Soc. 7: 465, 950. 5 LEVER, W. E., CUED, F. R. N., UROM, E., BROWN, R. K., BRNE.S, B.., SCHIID, K. ND SCHULTZ, E. L.: Chemcal, clncal, and mmunologcal studes on the products of human plasma fractonaton. XL. Quanttatve separaton and determnaton of the proten components n small amounts of normal human plasma. J. Cln. Invest. 0: 99, 95. Russ, E. M., EDER, H.. ND BRR, D. P.: Proten-lpd relatonshps n human plasma. I. In normal ndvduals. m. J. Med. : 46S, 95. * BRB, D. P., Russ, E. M. ND EDER, H..: Proten lpd relatonshps n human plasma. II. In atheroscleross and related condtons. m. J. Med. : 480, 95 'BRR, D. P., Russ, E. M. ND EDER, H..: Influence of estrogens on lpoprotens n atheroscleross. Trans.. m. Physcans 66: 0, 95. LEVER, W. F., HURLEY, N.. ND BLNEY,. E.: The protens n pemphgus vulgars. IV. Determnaton of plasma protens by electrophoress and chemcal fractonaton n patents wth pemphgus under treatment wth CTH or cortsone. J. Invest. Dermat. 9: 55, 95. 7 LEVER, W. F., SMITH, P.. J. ND HURLEY, N..: Idopathc hyperlpema and prmary hypercholesterolemc xanthomatoss. II. nalyss of the plasma protens and lpds by means of electrophoress and fractonaton of the plasma protens; effect of hgh speed centrfugaton and of attracton wth ether on the plasma protens and lpds. J. Invest. Dermat. : 5, 954. 8 ZELDIS, L. J., LLJNG, E. L., MCCOORD,. B. ND KULK, J. P.: Plasma proten metabolsm electrophoretc studes; the nfluence of plasma lpds on electrophoretc patterns of human and dog plasma. J. Exper. Med. 8: 4, 945. 9 JCOX, R. F.: Quanttatve fractonaton of component protens of human serum wth catonc detergents. J. Cln. Invest. 7: 66, 95. 0 LOWHY, 0. H., ROSEBROUQH, N. R., FRR,. L. ND RNDLL, R. J.: Proten measurement wth the Foln phenol reagent. J. Bol. Chem. 9: 65, 95. CREMER, H. ND TISELIUS,.: Electophorese von Ewess n Fltrer Paper. Bochem. Ztschr. 0: 7, 950. U FLYNN, F. V. ND DE MYO, P.: Mcro-electrophoress of proten on flter paper. Lancet : 5, 95. MCDONLD, H. J.: Ionography: new fronter n electrophoress. J. Chem. Educat. 9: 4S, 95. 4 SWHN, B.: Studes n blood lpds. II. mcro method for determnaton. Scand..). Cln. and Lab. Invest. 5: 44, 95, Suppl. 9. 5 GO, J.: smplfed method for the separaton and quanttatve determnaton of serum protens by paper electrophoress. Scand. J. Cln. and Lab. Invest. : 6, 95. "KUNKEL, H. G. ND SLTER, R. J.: Zone electrophoress n a starch-supportng medum. Proc. Soc. Exper. Bol. & Med. 80: 4, 95.