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The hydrolysis and fatty acid exchange of cholesterol esters administered in lipoproteins to Cebus monkeys
The Hydrolysis
and
Administered
Fatty Acid
Exchange
in Lipoproteins
of Cholesterol
Esters
to Cebus Monkeys’
‘Tile dist ril)rlt ion of rwdioactivity was mc:tsured in tlifl’f~lenl, ~l~c~lcsicr~~l ester cow p~llcnts of plasrn:~ of Cebus monkeys at, varying int~erwls after injection of nirv:~lon:tt e-L-(:‘l or tlte intrxvcnous :ldministr:itioli of tllr rliol~~st.trol-l-C~ll esters of cllcir, prlnritic, or linoleic acid which Irad I)een soluhilized I,\- tllr procedure of Avigm [,I. Nio!. C’hcw. 234, 787 (1959)]. The pl:tsnm of mollkcys wllicll Ilad IXPI~ t reatcd with Illevalo!l:tt,c-2-C1” had dist,rihutions of r:~dio:tctivity ill t Ite different c~holest ~rol ester fr:wtions, :ts selxuxted hy thin l:~yrr chrom:rtcl~~:~l~l~~, wllich WPW ren~:wk:hly sinrilar to the distrilmtion of f:tt,ty acids in tile cllolestcrol rstcr fr:trtiotl wg:trdlrss of tile tinlr interm after t reattnent. b’llell individwtl I:heled ~trolesterol esters were adn~inist~erctl, t IIP rPdistrit)ution of C”-cl~olrst,crol within the other cllolrsierol mters proceedrtl until the specific :ictivit its of all of tllenl wew nearly eqn:tl I)rtwrrn tile first, ;u~tl tllird day :tft,rr administration of that test material. It was wlmlated tll:it t Ile pool of J~lasm:~ c*lrolcsterol rstclrs was Irydrolyzpd al~~ut one tinle lwr tl:ry.
rat plasma cholwtcrol esters and 65 ‘G of that fracCon from chimpanzees. The distrihut~ion of different fatty acids It appears that thwe spccics-wlatcd in the plasma cholest’erol ester fraction of a patterns may best he explained as rcsult,itg single species retains a relatively constant from a specificity of the plasma or livcl pattern except during fairly marked dietary variations in the quantit,y and qualit~y of cliolrstcrol cst,er synthesis enz.yme or cnfat. On t,hc other hand, individual sprcies zymes rat8tw than from diffrrenccs ill the of animals havr rat’her specific patt,erns of composit,ion of the fat’ty acid suhstrat,c types of cholesterol esters (CR., I). 1~01~ from which the cholesterol clster fat,ty acid is drrivrd. example, on a given synthetic diet containAIorr information al)out ttw rates of ing corn oil as the sole source of fat,, cliolesfat,ty acid excllangc of individual clwlcsteryl arachidonate made up mow than 45 ‘,;t of t,lir cholesterol esters in the plasma of b.01 csttw is nwdcd, hut littlt is klwwll about tjhc ratIt‘s of synthesis and hydrolysis rat,s aiid less than i3.0 ‘,‘Gof the cholcxterol of t,hc different cholcstwol &Cars in plasma est,er fraction of the chimpanzee, while cholrstcryl linoleat,c comprised 30 “; of t,tlc of the iutact, animal. lilei~l atld Rlart,ill (2) Ilax:c studied ttw scclucnce of appraralw of differrnt, nrwly formed cholcstcrol cxst,cw in rat’ liver alld have dernonstratcd a hctwog(w~us patJtcrii of laheliiig in t,he chotc~two1 moiety at 30 min. after administrat,ioii of radioawtattl but ilot, at 1 hr. aftfq trcat8mcnt witti the cholcste~~~l prcc~u’sor. l’inter (~1nl. (3) have dcmonst,ratrd Irc%cw,gciicitJy of specific acti\+icls of dil’friwlt
cholesterol esters in t’lie liver and blood of rats after the administration of cholesterolNY4 in monoglyccridc rmulsiolw through a gastric cannula. 111this sbudy the rates of incorporation of cholesterol derived from mrvalonate%(‘I4 illto different plasma cholest8crol cstcrs and the rates of disappearance of radioactivity from thcsc fractions were studied. The rates of disappearance and alteration of individual cholrsterok-
Ferrule (Jcbrrs nlcjnkeys weiglling htwren 1200 anti 1800 g. were used in tl1cse experinlents. They were fed 3 synthetic diet, tl1r composition of wlricl1 11:~sbeen previously descritxd (5). Corn oil was supplied in the diet :tt 8”;, 1)~ wright or 15’;;) on the IAs of calories. Monkeys were given single meals supplying 300 calories per kg. of Iwdy weight each d:ty, and were fed after all blood samples had lwn drawn for that day.
ESTl!XS cst,rrs were prepared ty The cl~olesterol-l~(:” rr:trt,ing small qunnt,itirs of Inbeled cholest.erol (0.9 1ng. containing 100 PC. of Cl’) wit,11 tenfold excesses of the appropriate :wyl chlorides for 10 n1in. at 70” under :I nitrogen at~nrosphere (6). Tlw reaction mixture was dissolved in a few lrlt. of ligroine and this fraction was washed 3 times wit11 0.01 ,T KC)H and 1 times wit11 water. The crude fr:tct,ion was then c~t1ror11:~togr:tpl1ed on silicic acid wtriclr had been xt~ivatrtl t)y heating in an {wen at 120” overnight. IsZution elf tl1c rt1olesterol est.er wit11 :l mixture of ligroinc and ctlloroforrn (50:50, v:v) resulted in :I fraction wtricl1 contained OO(;, of t,l1e added r:tdiouctivi~y and was free of that port,ion of the acyl c*l1loridr wl1icl1 was not relnoved in tllr washing procedure and of free cl1olesteroI. Tl1in layer clrror11:rtogr:1~11~ of t hr purified p1 otlrrcts wit,l1 standard rnist.ures of cl1olesterol casters indicated that not less tlr:~n 95(‘;, of t,lre radioactivity was in t,lre :hppropriato chrc,n1:tt.~)gr:1l)tli(* I,:rnd. Higher limits of purity could not b(, est,:rl)lishc~d wit.h tllc> thin I:ryPr systerlr used.
In t tic studies of tire pattern of labeling of difl’erent ctlotesterol esters from endogenously derived c~trolestcrol, mevalonic acid-2-C’-’ as tl1r tli:~rrinioniunr salt wus inje:cted int,raperitonrnlly in O.VS, saline. In t.he st,udies involving the injection of ct1olestcrol-1~C’” cstrrs of individual f:lt,ty :wids,t he following procedurr was used [tl1r metl1otl is fwcnti:tlly t,l1:tt of Avigan (1)]: The 111onkcg.s were blctl from an :wn vein, :md the hlootl was tr:rnsfcrred 1.0 iced tubes conhining a smnll :trnc,unt of t1rpwin. 1’lasnr;t was obtained after centrifuging the t)l~,c,tl at 800~/ and 3” for 30 111in. Four 1111.of glnsrna were then transferred to st,rril(l 25-ml. :rn~poulcs which contained 500 mg. of :rcitlwxstietl 0lite 515 whictr 11~1 been watrd wit t1 about 1.1 ~nioles (50 pc.) of cllolesteryl-&(l’l linohtr, oleut,c, or p:rlmit:~te. The ampoules wer’c‘ sealed under nitrogen and were placed wit11 their long xws at. :I 10” angle to tl1r horizontal in :1 Illrt:ibc~lic sl1:lker at 37” and incubated in this \~a>for 5 t1r. At, t trc end of I tw incubation period I Ire :mlporiles wew opened and the Cclite-pl:ts1rl:\. n1ist)ures filtewd t t1ri)ugh il Swinney filter into :1 st,erilt! culture tulw wl1ich was then stored in ice until it was injected intravenously (less :I small aliquot for analysis) hck into the donor monkey c,n the following day. l<:ven wittl tt1rse very small clmtntities of c~l1~~lrsterol esters in t lie solubiliz:ition syst,ru1, not n1orc tl1:m 8’;;, of the added cholesterol cste1 was solut~ilizctl. This is :L mucl1 less efficient solut~itization than tt1at chscrved using i1 sirrrilal cluuntity of tlrc more po1:rr unesterified chc)lest.erol. As was t,lre case for rmestrrified clil1lesterol (1), JI1ost, of t,llc sc~lul)iliacd ~l1olest.erol cstrrs t)rt1:tved in t l1e prep:Lr:tt.ive ultfwentrifuge as conlponrnts of twta and alpha lipoproteins. l,ess tt1nn5’ ;, of tI1e dioactivity was oht:Gnect in the c~hylornic~rori I’r:wtion. There was invariably 3--5’ 1 nonc~nzywitir 11ydrol,vsis of the strlut)ilized cholesterol rst,ers (a similar percentage of t.l1e labeled cholesterol ester was l1ydrolyzed during incukxttion witl1 phr11a that, had been previously l1rntcd to GO” for X0 min. :d was free of cl~olest~erol ester synt,trrtic xtivity), This factor, togettrer with :I 5’ ; loss of ch~1lesterol ester f’r~~rrr the supposed :1uttientic tantl due to impurities or failure of the :In:rlytic*:cl procedure, resulted in :L zero t,irnfx valrle of :~l)orlt !)O’ (, for tlie perwntnge of r:ldioactivity in tl1r prc,per cholest,erol ester hntl.
l-‘lnsnra samples were I reated with :I ~lllorof~~r111~t~~etl1nnol 111ixturc (2: 1) and the cst r:wl s wcrc srparatetl in dlll)lic:1te Intel cholesterol cstrr
salt, and blood samples were drawn at various intervals after the injections for the determination of the radioactivity in free cholesterol and the different cholest,erol esters. In accord with the usual pattern after the administration of a cholesterol precursor (!1), the labeled sterol in plasma appeared first in the fret form, but within l-3 days, the specific activity of the cholesterol in the ester fraction was approximately that, of the free form. I’igure 1 illustrates t,he extraordinary constancy in the percelltagc dist,ribution of radioactivity in the different cholesterol ester fractions---from 2 hr. to :30 days aft’er injection of mevalonatr for a composite of two of the six monkeys treated with labeled mevalonate. The stew1 ester fraction did not have sufficielltl activity for accurate determinations prior t,o 2 hr. after inject,ion of the mevalonatc. The distribut’ion of radioactivit’y in the different cholesterol ester fractions as separated by thill layer chromatography was ill good agreement wth the composition of the total cliolest~rrol ester fatty acids determined by gas liqilid chromatography. DISTRII~CTI~X OF I~AI)IoACTI~ITY IN I)IFFERENT CHOLESTEROL ESTERS OF PL.MM.~ AFTER THE AD~~I~TKATION OF CHOLESTEROI,-4-C'4 I':STERS OF ()LEIC, PALMITIC, AND LINOLlMC akI1)
Figures 2-4 illustrate t,lie percentage distribution of radioactivity in the different plasma cholesterol esters after the intravenous injection of the t,reated plasma cholcsteryl-l-Cl4 oleate, chocontaining lestcryl-4-C’4 palmitatc, or cholestrryl-4-P linoleate. A single animal was used for each labeled cholesterol ester. The radioactive material found in the different, cholesterol ester fractions was changed from a preponderance of the administered labeled ester at the bleedings performed at 20 min., 1 hr., and 5 hr. aft’er injection to a pattern which resembled that of the existing composition of the total cholesterol ester fraction by 3 days after injection. The only difference of possible significance between the radioactivity distribution and the cholcstcrol ester fattv acid distribution at 3 days and longer at& administration
of the labeled cholesterol ester was in the cholesteryl arachidonate. The proportion of radioactivity in this latter fraction was consistently somewhat greater t,han the arachidonate content of the cholesterol ester fatty acids analyzed by gas liquid chromatography.
IkwIvmIiA~
CHOLESTEROL
I':sTERH
Figure L, illustrates t,he pattern of change in t,lie total plasma cholesterol ester and in t’hc plasma cholesteryl oleate specific activit#ies after administration of cholest,wyl+
chol. linoleate -----oleate .................... palmitate arachidonate -,-.-.-.radioactivity i ‘\‘. \ \ \ \ \ ‘---z./’
/ /&.W&a?* 1
... . . “.-.. ‘“-~-“““.y--_.
‘. .----------T..*.m. .....,...,.,...,... .. . .. . .. . . . . .. . . . .. ,.......... ,-,. - ,_._._*,_._. - ._.-.-.-.-.
2
3
4
-.-.-.-.-..-
5
6
.-._
74
::z
+
DAYS Fra. 2. The percentage distribution of rndioactivitr or fatty acids (ylc) in tlx diffcreut plas~na cholesterol esters of a C’ebm moukeq- which had been injected intravenously wit11 cl~olesteryl-~-C1l-ole:~te st,al)ilixed in lipoprotein form using the procedure of Avigun (4).
chol. linoleate ___ e-w-0. oleate . .............,.... palmitate arachidonate-.-.-.-radioactivity
FIG. 3. Tile percentage distribution of rr?dioartivitJor fatty acids iu the different, phsur:r cholesterol esters of :i P&US monkey which had been iujected with ~llolrst,ct~~l--L-(‘” palmitat~e.
chol. linoleate oleate palmitate arachidonate
~ .----... .. .. . . .... -.-.-.-.-
rad ioxsay
II
.(
..* / ,) .,,~ .._.......... ./ . . ...’ ~:~.:.~.-..-‘-.-‘-‘-‘-’ P
*..-----
c/
4)
______ ---------,-_
_. _. -. -....... . . . . . . . . . . . . . . . . . . . . . . . . . .._..............~~~ _,*._.-.-.
2
1
5
4
3
a..
DAYS FIG. 4. The percentage esters of a Cebrcsmonkey
of radioactivity or fatty which had been injected
. ..... ...cholesterol esters \
.,---cholesteryl
oleate
i
\
‘\ ;
‘\
\
*......... ‘\ “. ....._‘\ \ ‘-. .._, \\ “b... ‘=C:,,,_-;;
I)
I
2
3
.................~_..._..~.~ ----_ --._.
4
5
6
1
DAYS
Frc;. 5, The specific activities of cholest,rryl oleate and the t,ot,al chr)lesterol ester fraction of plasma of a C’eb~s monkey injected mitlr cholestrrvl-4-P’ oleute.
acids in the different with cholesteryl-4-C’”
plasma cholest,erol linoleate.
rates of hydrolysis of individual cholesterol esters. The apparent turnover rate of the cholcsteryl oleatc due only to elimination of oleat’e but without removal of the labeled cholesterol from the plasma, the rate of loss of the original fatty acid, was calculated by subtracting the turnover rate of the total cholesterol ester fraction from the turnover rate of the cholesteryl-oleate fract’ion. The rate of loss of the original fatt,y acid was then corrected for re-esterification of the cholest’erol with oleate itself in order t#oobtain a rate of hydrolysis of plasma cholesteryl oleate. The correct,ion for reesterification with oleate was made by assuming that, there was homogeneous labeling of newly formed cholest8erol esters (as appeared to be justified from the experiment with mevalollate-2-C’“). Thus, the hydrolysis rate is rate of loss of olcat’e 1.OO- fraction of ester cholest’erol est’rrified with oleate. Table I presents the turnover rates (based on the specific activities at t’he 5th and 24th hours after injection of bhe ester)
removed from the blood strram by the livrr. Ttir cliolrstrrol then appears, first Isrgel) as frer cholestrrol (13) in lipoproteins. This cholestrrol is progressively estcrifird until the specific activitirs of t,hr cst,er cholestrrol are rqual to or greater that t t,tiat) of the fra: ctiolest’rrot. l‘trr disapprarance of this tipoprotciii c~tiolrstrrol from t’hr blood is wlativety stow with turnovrr times of many days duratioil. It teas bcrn very difficult, t)o obtain iufolmat,ion about tlir rates of hydrolysis of .klministered the diffrrrntJ fattv acids which are estrrifird with cholrsterol in the blood. In fact,, thrrc is considrrablc unccrt~ainty about t,hr rrlatiw importance of the diffrrellt sitrs of 2.2 0.X rstIrrificatioil of cholcstcrot (II). c’liolrs~ 2.0 0.8 trro1 cstw synthetic activit)y has brr11 demonstrat’ed iu livrr and plasma, a11d 1.1 0.5 ctiolestrrol cstrr hydrolyt8ic acti\.ity has beru fomld itr thr liver. A\ drfiuition of’ t,llr rates of fatt*y acid hydrolysis in the rtrotrsterol rst#rr moleculr i/l r?rv~ brays 011 thr qriestiotl of the relative rotitribut,iotl that could be provided from thr livrr alit1 plasma ctiolrstcrol estw syiit,het~ic act iritirs n-hirh have bre11 demollstratrd 2’~ c?tw. ‘I’hr trrel oi’ rhotrstri~ol rstrr synttiet,ic activity which has 1wrt1 dcnionst,ratctl in blood by (:lomsrt, (I.;), as n-r11 as ottw for thr individual stcrol rstrr, for the tot,al illvrstSigat,ors, lvoutd probably lw sufficirtlt cholrsterol es& fraction (this is also Ohe to off& t,he chotrst~erol rstrt tIydrotytic turnover rate of thr individusl cholrstcrol activity demollstrnted in Go it1 thr prrsrnt, estrr relat’ed to disappcaraucc of t’hc choles- st,udy. Although t,hr rvidrtlrr from ot,tlrr t,rrol from the plasma), for the individual studies is uot, co~lclusiw, it appears likcty cholrsterol estrr due to toss of thr original that the pertinrilt cholcstct~ol estw h~tltw fat,ty acid moiety, and for hydrolysis of thr lytic act’ivity is found lat,grty in the ti\.rt, and individual cholesterol rster when choles- is essentially absent, from the periphctxl tcty-1-C’” 01&e, cholrstrryt-4-C’” linoblood in most qxxirs. lratr, and cllolrstclyl-l-C“ palmit#ate v,wc Onr approach to a drterminat,ion of the injrcted in lipoprot,riu form. It caln bp sren in Go rate of chotrst,erol rster hydrolysis that. for all three chotest8erol esters three was and rr-est,erificat,iotl is the admitlistrat,ion of a rate of hydrolysis equivalent to about one labrlrd ct~olrsterot rstrrificd with a specific pool prr day. Hecausr of thr limitrd nurnbel fatty acid atld drtermillation of the rates of of studies carried out (one monkry pel hydrolysis from calculations of that ratr of cholesterol ester), the differrncrs or lack of disappcsrancr of the itldividual chotrst~rl~ol diffrrences bet~ww~ the ratrs of hydrolysis rster bryotld that, rrlat’rd t,o disappraralxt: of the diffcrellt cholrsterol rsters ww Ilot, of cholesterol from the pool. It’ had not brcll rstnhlished. possible for us (r.g., 16) to obt#ain pat,trrtls of disappraraticr from the plasma of iiijrrted ctiolrst,r~ol which wrr comparable to t t~osc C’hylomici~on cholesterol is targcly ii1 the seen wit,h rt~dogrnous labeled ctiolrst.rt~ot c&r form (12), but it is usually rapidly unt,it tile procrd~tre of ,I\-igall (4) was ilsrd.
342
PORTMAN
ANI)
The values for the hydrolysis rates of the individual cholesterol esters iu the Cebus monkey are, at least, qualitatively compatible with observations previously made on the rates of repletion and depletion of polyunsaturated fatty acids in the same species (7). REFERENCES L., FIEI.D, H., JR., AND TREAD\VELL, C. R., Proc. Sot. Esptl. Biol. Med. 104, 325 (1960). lir.~n, P. I)., AND MARTIN, K. A., J. Biol.
1. SWELL,
2.
C’hem. 234, 1685 (1959). 3. PINTER,K.G.,MILLER,O.X.,
J. G., Federation
AND HAMILTON,
Proc. 22,376 (1963).
4. AVIG~N, J., J. Rio/. Chem. 234, 787 (1959). 5. M.4NN, G. V.. ASDRuS, 8. B., MCNAI>LY, A., END STARE, F. J., J. E’rptl. Med. 98, 195
(1953). 6. PAGE, 1. H., AND RUI)B, H., Biochem. 220, 30-2 (1930).
SUGANO
F. E., J. Niol. C’hem. 195, 357 (1952). 9. Gour,u, 11. G., LEROY, 0. V., OKUA, G. T., KAB~RA, J. J., KEEGAN, I'., AXD BERGEFJ~etl. 46, 3i’-1 S'L'AI,, I). M., J. Lab. Clin. (1955). Il., AND BERMAN,M., 10. AVIGAN, J., STEINBERG, J. Iipid 12es. 3, 21G (1962). of Radiobiochen11. ARBNOYF, s., “Techniques istry," p. 80. Iowa State Kniv. Press, Smes, Iowa, 1956. 12. CHAIK~FF, I. I,., BLOOM, B., SIIJERSTEIN, 31. l)., KIYASU, J. Y., REINHBRDT, W. O., KENDALL,
I)ANBEN, W. c;., AND EASTHAM, Hiol. (‘hem. 194, 407 (1952).
7. PoRTYAN,O.W.,PINTER,K., AND H~uaa~~u.4, T., Ant. J. C'lin. ,$'utr. 7, G3 (1959). 8. ABELL, 1,. L., I,EVY, B. B., BRODIE, B. B., ASD
J.
13. HELLMAN,
I,., ROSENFELD, It. S., EIDINOFF, &I. L., FCKl!SHIMA, 11. K., GALLAGHER, T. P'., WAXG, C. I., AND ADLERSBERG, I)., J. Clin. kwest.
14. BROT, Ztschr.
J. F.,
N.,
34, 48 (1955). W. J., AKD CHAIKOFF, Res. 3, 113 (1962).
1.
Ada
65,
T,ossow,
I,., J. I.ipid 15. GLOMSE'L., J. A., Biorhem. Riophys. 128 (1962). Ifi. PoRTMA.U, (1. W., AND 8INISTERR.4, E’rptl. 31ed. 106, 727 (1957).
L.,
.f.
and
Administered
Fatty Acid
Exchange
in Lipoproteins
of Cholesterol
Esters
to Cebus Monkeys’
‘Tile dist ril)rlt ion of rwdioactivity was mc:tsured in tlifl’f~lenl, ~l~c~lcsicr~~l ester cow p~llcnts of plasrn:~ of Cebus monkeys at, varying int~erwls after injection of nirv:~lon:tt e-L-(:‘l or tlte intrxvcnous :ldministr:itioli of tllr rliol~~st.trol-l-C~ll esters of cllcir, prlnritic, or linoleic acid which Irad I)een soluhilized I,\- tllr procedure of Avigm [,I. Nio!. C’hcw. 234, 787 (1959)]. The pl:tsnm of mollkcys wllicll Ilad IXPI~ t reatcd with Illevalo!l:tt,c-2-C1” had dist,rihutions of r:~dio:tctivity ill t Ite different c~holest ~rol ester fr:wtions, :ts selxuxted hy thin l:~yrr chrom:rtcl~~:~l~l~~, wllich WPW ren~:wk:hly sinrilar to the distrilmtion of f:tt,ty acids in tile cllolestcrol rstcr fr:trtiotl wg:trdlrss of tile tinlr interm after t reattnent. b’llell individwtl I:heled ~trolesterol esters were adn~inist~erctl, t IIP rPdistrit)ution of C”-cl~olrst,crol within the other cllolrsierol mters proceedrtl until the specific :ictivit its of all of tllenl wew nearly eqn:tl I)rtwrrn tile first, ;u~tl tllird day :tft,rr administration of that test material. It was wlmlated tll:it t Ile pool of J~lasm:~ c*lrolcsterol rstclrs was Irydrolyzpd al~~ut one tinle lwr tl:ry.
rat plasma cholwtcrol esters and 65 ‘G of that fracCon from chimpanzees. The distrihut~ion of different fatty acids It appears that thwe spccics-wlatcd in the plasma cholest’erol ester fraction of a patterns may best he explained as rcsult,itg single species retains a relatively constant from a specificity of the plasma or livcl pattern except during fairly marked dietary variations in the quantit,y and qualit~y of cliolrstcrol cst,er synthesis enz.yme or cnfat. On t,hc other hand, individual sprcies zymes rat8tw than from diffrrenccs ill the of animals havr rat’her specific patt,erns of composit,ion of the fat’ty acid suhstrat,c types of cholesterol esters (CR., I). 1~01~ from which the cholesterol clster fat,ty acid is drrivrd. example, on a given synthetic diet containAIorr information al)out ttw rates of ing corn oil as the sole source of fat,, cliolesfat,ty acid excllangc of individual clwlcsteryl arachidonate made up mow than 45 ‘,;t of t,lir cholesterol esters in the plasma of b.01 csttw is nwdcd, hut littlt is klwwll about tjhc ratIt‘s of synthesis and hydrolysis rat,s aiid less than i3.0 ‘,‘Gof the cholcxterol of t,hc different cholcstwol &Cars in plasma est,er fraction of the chimpanzee, while cholrstcryl linoleat,c comprised 30 “; of t,tlc of the iutact, animal. lilei~l atld Rlart,ill (2) Ilax:c studied ttw scclucnce of appraralw of differrnt, nrwly formed cholcstcrol cxst,cw in rat’ liver alld have dernonstratcd a hctwog(w~us patJtcrii of laheliiig in t,he chotc~two1 moiety at 30 min. after administrat,ioii of radioawtattl but ilot, at 1 hr. aftfq trcat8mcnt witti the cholcste~~~l prcc~u’sor. l’inter (~1nl. (3) have dcmonst,ratrd Irc%cw,gciicitJy of specific acti\+icls of dil’friwlt
cholesterol esters in t’lie liver and blood of rats after the administration of cholesterolNY4 in monoglyccridc rmulsiolw through a gastric cannula. 111this sbudy the rates of incorporation of cholesterol derived from mrvalonate%(‘I4 illto different plasma cholest8crol cstcrs and the rates of disappearance of radioactivity from thcsc fractions were studied. The rates of disappearance and alteration of individual cholrsterok-
Ferrule (Jcbrrs nlcjnkeys weiglling htwren 1200 anti 1800 g. were used in tl1cse experinlents. They were fed 3 synthetic diet, tl1r composition of wlricl1 11:~sbeen previously descritxd (5). Corn oil was supplied in the diet :tt 8”;, 1)~ wright or 15’;;) on the IAs of calories. Monkeys were given single meals supplying 300 calories per kg. of Iwdy weight each d:ty, and were fed after all blood samples had lwn drawn for that day.
ESTl!XS cst,rrs were prepared ty The cl~olesterol-l~(:” rr:trt,ing small qunnt,itirs of Inbeled cholest.erol (0.9 1ng. containing 100 PC. of Cl’) wit,11 tenfold excesses of the appropriate :wyl chlorides for 10 n1in. at 70” under :I nitrogen at~nrosphere (6). Tlw reaction mixture was dissolved in a few lrlt. of ligroine and this fraction was washed 3 times wit11 0.01 ,T KC)H and 1 times wit11 water. The crude fr:tct,ion was then c~t1ror11:~togr:tpl1ed on silicic acid wtriclr had been xt~ivatrtl t)y heating in an {wen at 120” overnight. IsZution elf tl1c rt1olesterol est.er wit11 :l mixture of ligroinc and ctlloroforrn (50:50, v:v) resulted in :I fraction wtricl1 contained OO(;, of t,l1e added r:tdiouctivi~y and was free of that port,ion of the acyl c*l1loridr wl1icl1 was not relnoved in tllr washing procedure and of free cl1olesteroI. Tl1in layer clrror11:rtogr:1~11~ of t hr purified p1 otlrrcts wit,l1 standard rnist.ures of cl1olesterol casters indicated that not less tlr:~n 95(‘;, of t,lre radioactivity was in t,lre :hppropriato chrc,n1:tt.~)gr:1l)tli(* I,:rnd. Higher limits of purity could not b(, est,:rl)lishc~d wit.h tllc> thin I:ryPr systerlr used.
In t tic studies of tire pattern of labeling of difl’erent ctlotesterol esters from endogenously derived c~trolestcrol, mevalonic acid-2-C’-’ as tl1r tli:~rrinioniunr salt wus inje:cted int,raperitonrnlly in O.VS, saline. In t.he st,udies involving the injection of ct1olestcrol-1~C’” cstrrs of individual f:lt,ty :wids,t he following procedurr was used [tl1r metl1otl is fwcnti:tlly t,l1:tt of Avigan (1)]: The 111onkcg.s were blctl from an :wn vein, :md the hlootl was tr:rnsfcrred 1.0 iced tubes conhining a smnll :trnc,unt of t1rpwin. 1’lasnr;t was obtained after centrifuging the t)l~,c,tl at 800~/ and 3” for 30 111in. Four 1111.of glnsrna were then transferred to st,rril(l 25-ml. :rn~poulcs which contained 500 mg. of :rcitlwxstietl 0lite 515 whictr 11~1 been watrd wit t1 about 1.1 ~nioles (50 pc.) of cllolesteryl-&(l’l linohtr, oleut,c, or p:rlmit:~te. The ampoules wer’c‘ sealed under nitrogen and were placed wit11 their long xws at. :I 10” angle to tl1r horizontal in :1 Illrt:ibc~lic sl1:lker at 37” and incubated in this \~a>for 5 t1r. At, t trc end of I tw incubation period I Ire :mlporiles wew opened and the Cclite-pl:ts1rl:\. n1ist)ures filtewd t t1ri)ugh il Swinney filter into :1 st,erilt! culture tulw wl1ich was then stored in ice until it was injected intravenously (less :I small aliquot for analysis) hck into the donor monkey c,n the following day. l<:ven wittl tt1rse very small clmtntities of c~l1~~lrsterol esters in t lie solubiliz:ition syst,ru1, not n1orc tl1:m 8’;;, of the added cholesterol cste1 was solut~ilizctl. This is :L mucl1 less efficient solut~itization than tt1at chscrved using i1 sirrrilal cluuntity of tlrc more po1:rr unesterified chc)lest.erol. As was t,lre case for rmestrrified clil1lesterol (1), JI1ost, of t,llc sc~lul)iliacd ~l1olest.erol cstrrs t)rt1:tved in t l1e prep:Lr:tt.ive ultfwentrifuge as conlponrnts of twta and alpha lipoproteins. l,ess tt1nn5’ ;, of tI1e dioactivity was oht:Gnect in the c~hylornic~rori I’r:wtion. There was invariably 3--5’ 1 nonc~nzywitir 11ydrol,vsis of the strlut)ilized cholesterol rst,ers (a similar percentage of t.l1e labeled cholesterol ester was l1ydrolyzed during incukxttion witl1 phr11a that, had been previously l1rntcd to GO” for X0 min. :d was free of cl~olest~erol ester synt,trrtic xtivity), This factor, togettrer with :I 5’ ; loss of ch~1lesterol ester f’r~~rrr the supposed :1uttientic tantl due to impurities or failure of the :In:rlytic*:cl procedure, resulted in :L zero t,irnfx valrle of :~l)orlt !)O’ (, for tlie perwntnge of r:ldioactivity in tl1r prc,per cholest,erol ester hntl.
l-‘lnsnra samples were I reated with :I ~lllorof~~r111~t~~etl1nnol 111ixturc (2: 1) and the cst r:wl s wcrc srparatetl in dlll)lic:1te Intel cholesterol cstrr
salt, and blood samples were drawn at various intervals after the injections for the determination of the radioactivity in free cholesterol and the different cholest,erol esters. In accord with the usual pattern after the administration of a cholesterol precursor (!1), the labeled sterol in plasma appeared first in the fret form, but within l-3 days, the specific activity of the cholesterol in the ester fraction was approximately that, of the free form. I’igure 1 illustrates t,he extraordinary constancy in the percelltagc dist,ribution of radioactivity in the different cholesterol ester fractions---from 2 hr. to :30 days aft’er injection of mevalonatr for a composite of two of the six monkeys treated with labeled mevalonate. The stew1 ester fraction did not have sufficielltl activity for accurate determinations prior t,o 2 hr. after inject,ion of the mevalonatc. The distribut’ion of radioactivit’y in the different cholesterol ester fractions as separated by thill layer chromatography was ill good agreement wth the composition of the total cliolest~rrol ester fatty acids determined by gas liqilid chromatography. DISTRII~CTI~X OF I~AI)IoACTI~ITY IN I)IFFERENT CHOLESTEROL ESTERS OF PL.MM.~ AFTER THE AD~~I~TKATION OF CHOLESTEROI,-4-C'4 I':STERS OF ()LEIC, PALMITIC, AND LINOLlMC akI1)
Figures 2-4 illustrate t,lie percentage distribution of radioactivity in the different plasma cholesterol esters after the intravenous injection of the t,reated plasma cholcsteryl-l-Cl4 oleate, chocontaining lestcryl-4-C’4 palmitatc, or cholestrryl-4-P linoleate. A single animal was used for each labeled cholesterol ester. The radioactive material found in the different, cholesterol ester fractions was changed from a preponderance of the administered labeled ester at the bleedings performed at 20 min., 1 hr., and 5 hr. aft’er injection to a pattern which resembled that of the existing composition of the total cholesterol ester fraction by 3 days after injection. The only difference of possible significance between the radioactivity distribution and the cholcstcrol ester fattv acid distribution at 3 days and longer at& administration
of the labeled cholesterol ester was in the cholesteryl arachidonate. The proportion of radioactivity in this latter fraction was consistently somewhat greater t,han the arachidonate content of the cholesterol ester fatty acids analyzed by gas liquid chromatography.
IkwIvmIiA~
CHOLESTEROL
I':sTERH
Figure L, illustrates t,he pattern of change in t,lie total plasma cholesterol ester and in t’hc plasma cholesteryl oleate specific activit#ies after administration of cholest,wyl+
chol. linoleate -----oleate .................... palmitate arachidonate -,-.-.-.radioactivity i ‘\‘. \ \ \ \ \ ‘---z./’
/ /&.W&a?* 1
... . . “.-.. ‘“-~-“““.y--_.
‘. .----------T..*.m. .....,...,.,...,... .. . .. . .. . . . . .. . . . .. ,.......... ,-,. - ,_._._*,_._. - ._.-.-.-.-.
2
3
4
-.-.-.-.-..-
5
6
.-._
74
::z
+
DAYS Fra. 2. The percentage distribution of rndioactivitr or fatty acids (ylc) in tlx diffcreut plas~na cholesterol esters of a C’ebm moukeq- which had been injected intravenously wit11 cl~olesteryl-~-C1l-ole:~te st,al)ilixed in lipoprotein form using the procedure of Avigun (4).
chol. linoleate ___ e-w-0. oleate . .............,.... palmitate arachidonate-.-.-.-radioactivity
FIG. 3. Tile percentage distribution of rr?dioartivitJor fatty acids iu the different, phsur:r cholesterol esters of :i P&US monkey which had been iujected with ~llolrst,ct~~l--L-(‘” palmitat~e.
chol. linoleate oleate palmitate arachidonate
~ .----... .. .. . . .... -.-.-.-.-
rad ioxsay
II
.(
..* / ,) .,,~ .._.......... ./ . . ...’ ~:~.:.~.-..-‘-.-‘-‘-‘-’ P
*..-----
c/
4)
______ ---------,-_
_. _. -. -....... . . . . . . . . . . . . . . . . . . . . . . . . . .._..............~~~ _,*._.-.-.
2
1
5
4
3
a..
DAYS FIG. 4. The percentage esters of a Cebrcsmonkey
of radioactivity or fatty which had been injected
. ..... ...cholesterol esters \
.,---cholesteryl
oleate
i
\
‘\ ;
‘\
\
*......... ‘\ “. ....._‘\ \ ‘-. .._, \\ “b... ‘=C:,,,_-;;
I)
I
2
3
.................~_..._..~.~ ----_ --._.
4
5
6
1
DAYS
Frc;. 5, The specific activities of cholest,rryl oleate and the t,ot,al chr)lesterol ester fraction of plasma of a C’eb~s monkey injected mitlr cholestrrvl-4-P’ oleute.
acids in the different with cholesteryl-4-C’”
plasma cholest,erol linoleate.
rates of hydrolysis of individual cholesterol esters. The apparent turnover rate of the cholcsteryl oleatc due only to elimination of oleat’e but without removal of the labeled cholesterol from the plasma, the rate of loss of the original fatty acid, was calculated by subtracting the turnover rate of the total cholesterol ester fraction from the turnover rate of the cholesteryl-oleate fract’ion. The rate of loss of the original fatt,y acid was then corrected for re-esterification of the cholest’erol with oleate itself in order t#oobtain a rate of hydrolysis of plasma cholesteryl oleate. The correct,ion for reesterification with oleate was made by assuming that, there was homogeneous labeling of newly formed cholest8erol esters (as appeared to be justified from the experiment with mevalollate-2-C’“). Thus, the hydrolysis rate is rate of loss of olcat’e 1.OO- fraction of ester cholest’erol est’rrified with oleate. Table I presents the turnover rates (based on the specific activities at t’he 5th and 24th hours after injection of bhe ester)
removed from the blood strram by the livrr. Ttir cliolrstrrol then appears, first Isrgel) as frer cholestrrol (13) in lipoproteins. This cholestrrol is progressively estcrifird until the specific activitirs of t,hr cst,er cholestrrol are rqual to or greater that t t,tiat) of the fra: ctiolest’rrot. l‘trr disapprarance of this tipoprotciii c~tiolrstrrol from t’hr blood is wlativety stow with turnovrr times of many days duratioil. It teas bcrn very difficult, t)o obtain iufolmat,ion about tlir rates of hydrolysis of .klministered the diffrrrntJ fattv acids which are estrrifird with cholrsterol in the blood. In fact,, thrrc is considrrablc unccrt~ainty about t,hr rrlatiw importance of the diffrrellt sitrs of 2.2 0.X rstIrrificatioil of cholcstcrot (II). c’liolrs~ 2.0 0.8 trro1 cstw synthetic activit)y has brr11 demonstrat’ed iu livrr and plasma, a11d 1.1 0.5 ctiolestrrol cstrr hydrolyt8ic acti\.ity has beru fomld itr thr liver. A\ drfiuition of’ t,llr rates of fatt*y acid hydrolysis in the rtrotrsterol rst#rr moleculr i/l r?rv~ brays 011 thr qriestiotl of the relative rotitribut,iotl that could be provided from thr livrr alit1 plasma ctiolrstcrol estw syiit,het~ic act iritirs n-hirh have bre11 demollstratrd 2’~ c?tw. ‘I’hr trrel oi’ rhotrstri~ol rstrr synttiet,ic activity which has 1wrt1 dcnionst,ratctl in blood by (:lomsrt, (I.;), as n-r11 as ottw for thr individual stcrol rstrr, for the tot,al illvrstSigat,ors, lvoutd probably lw sufficirtlt cholrsterol es& fraction (this is also Ohe to off& t,he chotrst~erol rstrt tIydrotytic turnover rate of thr individusl cholrstcrol activity demollstrnted in Go it1 thr prrsrnt, estrr relat’ed to disappcaraucc of t’hc choles- st,udy. Although t,hr rvidrtlrr from ot,tlrr t,rrol from the plasma), for the individual studies is uot, co~lclusiw, it appears likcty cholrsterol estrr due to toss of thr original that the pertinrilt cholcstct~ol estw h~tltw fat,ty acid moiety, and for hydrolysis of thr lytic act’ivity is found lat,grty in the ti\.rt, and individual cholesterol rster when choles- is essentially absent, from the periphctxl tcty-1-C’” 01&e, cholrstrryt-4-C’” linoblood in most qxxirs. lratr, and cllolrstclyl-l-C“ palmit#ate v,wc Onr approach to a drterminat,ion of the injrcted in lipoprot,riu form. It caln bp sren in Go rate of chotrst,erol rster hydrolysis that. for all three chotest8erol esters three was and rr-est,erificat,iotl is the admitlistrat,ion of a rate of hydrolysis equivalent to about one labrlrd ct~olrsterot rstrrificd with a specific pool prr day. Hecausr of thr limitrd nurnbel fatty acid atld drtermillation of the rates of of studies carried out (one monkry pel hydrolysis from calculations of that ratr of cholesterol ester), the differrncrs or lack of disappcsrancr of the itldividual chotrst~rl~ol diffrrences bet~ww~ the ratrs of hydrolysis rster bryotld that, rrlat’rd t,o disappraralxt: of the diffcrellt cholrsterol rsters ww Ilot, of cholesterol from the pool. It’ had not brcll rstnhlished. possible for us (r.g., 16) to obt#ain pat,trrtls of disappraraticr from the plasma of iiijrrted ctiolrst,r~ol which wrr comparable to t t~osc C’hylomici~on cholesterol is targcly ii1 the seen wit,h rt~dogrnous labeled ctiolrst.rt~ot c&r form (12), but it is usually rapidly unt,it tile procrd~tre of ,I\-igall (4) was ilsrd.
342
PORTMAN
ANI)
The values for the hydrolysis rates of the individual cholesterol esters iu the Cebus monkey are, at least, qualitatively compatible with observations previously made on the rates of repletion and depletion of polyunsaturated fatty acids in the same species (7). REFERENCES L., FIEI.D, H., JR., AND TREAD\VELL, C. R., Proc. Sot. Esptl. Biol. Med. 104, 325 (1960). lir.~n, P. I)., AND MARTIN, K. A., J. Biol.
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SUGANO
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13. HELLMAN,
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34, 48 (1955). W. J., AKD CHAIKOFF, Res. 3, 113 (1962).
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T,ossow,
I,., J. I.ipid 15. GLOMSE'L., J. A., Biorhem. Riophys. 128 (1962). Ifi. PoRTMA.U, (1. W., AND 8INISTERR.4, E’rptl. 31ed. 106, 727 (1957).
L.,
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