significantly with respect to the rate of oxidation Despite the differences in the rate of incorporation

Transcription

1 THE COMPOSITION AND BIOSYNTHESIS OF LIPIDS IN HUMAN ADIPOSE TISSUES * By ALFRED GELLHORN AND PAUL A. MARKS (From the Deprtmnent of Medicine, Columlbi University College of Physicins nd Surgeons, nd the Medicl Service, Frncis Delfield Hospitl, New York, N. Y.) (Submitted for publiction Jnury 9, 1961; ccepted Februry 3, 1961) The cpcity of dipose tissue in rts nd mice to synthesize ftty cids nd triglycerides from cette, glucose nd long chin ftty cids nd to oxidize these substrtes to CO. hs been well estblished (1-5). In this investigtion, study hs been mde of lipid biosynthesis nd lipid composition of humn dipose tissues. Humn subjects provide unique opportunity to compre the metbolism of norml subcutneous dipose tissue with benign neoplsm of ft tissue, the lipom. Comprtive studies of most tumors with their norml tissue counterprt hve been difficult becuse of cellulr nonhomogeneity of both norml tissues nd tumors nd the inbility to obtin tumor nd norml control tissue from the sme subject. The lipom, on the other hnd, rises most frequently in the subcutneous dipose tissue nd morphologiclly is indistinguishble from norml dipose tissue. This mkes it possible to exmine the synthetic nd ctbolic ctivities nd composition of the tumor nd control tissue in the sme subject under identicl environmentl conditions. In the present studies, it hs been found, in vitro, tht lipoms hd significntly higher rte of cette incorportion into the mixed lipids thn the norml subcutneous dipose tissue of the sme subject. Although the rte of synthesis of individul ftty cids ws greter in the lipom thn in the norml ft, the reltive distribution of rdioctivity mong the ftty cids ws the sme in the two tissues. These tissues did not differ significntly with respect to the rte of oxidtion of cette to CO., or the rte of trnsfer of newly synthesized lipids into the medium. * This work ws supported in prt by Grnt CY-2332 of the Ntionl Cncer Institute, U. S. Public Helth Service, The Alm Toorock Memoril Fund nd the Evelyn R. Hill Fund. A portion of these dt hs been presented in the Proceedings of the Interntionl Symposium on Drugs Affecting Lipid Metbolism, Miln, 196. Despite the differences in the rte of incorportion of cette into the mixed lipids, the ftty cid composition of the norml subcutneous ft nd the lipom did not differ qulittively or quntittively. In ddition, there ws impressive constncy of the ftty cid composition of the norml ft nd lipom mong the subjects studied despite wide vrition in the rtes of lipid synthesis by these tissues from one person to the next. MATERIALS AND METHODS Twenty ptients with subcutneous lipoms provided the mteril for this study. The tumors were removed under block locl nesthesi together with smple of d- 925 jcent norml dipose tissue. All ptients hd eten 3 to 5 hours prior to the opertion. On removl, the tissues were kept t bout 5' C until they were prepred for incubtion. The fibrous tissue cpsules of the lipoms were dissected wy nd tissue slices of the tumor nd the norml subcutneous dipose tissue were prepred with Stdie tissue slicer (6). Usully 1 hour elpsed between the opertive removl of tissue nd the beginning of incubtion. Incubtion ws crried out in 5 ml Wrburg-type flsks employing rubber serum bottle stoppers to sel the flsks. One to two lic of cette-l-c",.1 ml 1 M glucose nd vrying mounts of nonrdioctive sodium cette were plced in the side-rm. Into the min comprtment were dded 15 to 3 mg of tissue nd n mount of Krebs-Ringer bicrbonte solution (7), contining 5 to 1 per cent of the ptient's own serum, sufficient to mke finl volume of 5 ml. In most experiments flsk ws set up, identicl with those just described except tht boiled rther thn fresh tissue ws employed. The flsks were gssed with 95 per cent 2, 5 per cent CO. for 5 minutes. After 2 minutes of temperture equilibrtion t 37' C in Dubnoff metbolic shker-bth, the rection ws begun by tipping into the min comprtment the contents of the side-rm. The durtion of incubtion ws usully 2 nd 4 hours. In preliminry experiments it ws estblished tht, under the present experimentl conditions, the rte of incorportion of cette-1-c'4 into the mixed lipids or into C"42 by norml dipose tissue nd lipom slices ws liner over 6 hour period. At the completion of incubtion, the flsks were chilled in chipped ice. A 3 per cent solution of KOH ws in-

2 926 ALFRED GELLHORN AND PAUL A. MARKS troduced into the center well, 1 ml.2 N H2SO4 ws dded to the medium in the min comprtment nd the flsks were shken in ice for nother 3 minutes. The KOH nd H2SO4 dditions were mde by needle puncture through the rubber stopper. In nine experiments, the rdioctivity of the lipids in the tissue nd in the medium ws nlyzed seprtely. In these studies, t the completion of the incubtion period, the flsk contents were poured through fine-mesh, stinless steel screen. The tissue ws repetedly wshed with either distilled wter or dilute mmonicl ethnol. Lipids were then extrcted from the medium nd tissue s described below. C142 ssy. The.2 ml of 3 per cent KOH solution in the center well ws trnsferred quntittively to centrifuge tubes nd sturted BCl2 ws dded to precipitte the crbonte. The BCO, ws wshed repetedly with wter until the smples obtined from flsks with boiled tissue (blnks) showed negligible rdioctive contmintion (less thn.6 per cent of the dded counts). The BCO, ws suspended in mixture of diphenyloxzole,.3 per cent, nd thixin, 2.5 per cent, in toluene nd _1. Lipoo z z w jt7. ;67.4 * Normol 19. I~~~Q AGE IN YEARS 13. FIG. 1. THE RATE OF INCORPORATION OF ACETATE INTO NORMAL ADIPOSE TISSUE AND LIPOMA CORRELATED WITH THE AGE OF EACH OF 2 SUBJECTS. The clcultion of cette incorported into the totl mixed lipids is bsed on the initil specific ctivities of the cette in the incubtion medium nd the totl rdioctivity incorported into the lipids. TABLE I Nitrogen nd lipid content of norml dipose tissue nd lipom No. of subjects* Nitrogen SEMt Lipid SEMt mg/g mg/g wet wt wet wt Norml ±9 Lipom ± * At lest 4 liquots of tissue were nlyzed from ech subject. t SEM is the stndrd error of the men. counted in liquid scintilltion spectrometer (). It ws demonstrted in preliminry experiments tht quenching of rdioctivity did not occur when s much s 2.5 g BCO3 ws dded. Rdioctivity ws mesured in this system with n bsolute efficiency of 75 per cent nd bckground of 3 cpm. Extrction of lipids. The mixed lipids of the tissues or medium were extrcted nd prepred for nlysis by modifiction of the methods described by Folch, Lees nd Slone Stnley (9). The smple to be extrcted ws homogenized in multimixer employing to 1 ml CHCl3-CH3OH (2: 1). In order to free the lipid extrcts of nonlipid rdioctive compounds, they were plced in viscose tubing nd dilyzed ginst running tp wter in rottory dilyzer for 12 to 16 hours. It ws demonstrted tht this method completely removed rdioctive mterils from boiled* tissue smples which were crried through ll steps of the experimentl procedure. It ws lso found tht there ws no detectble loss of C14-lbeled triplmitin dded to blnk smple fter 24 hours of continuous dilysis. After dilysis, the mixed lipid extrct ws tken to dryness under reduced pressure nd mde up to known volume with petroleum ether (bp 3 to 6 C). An liquot ws tken for nlysis of rdioctivity nd lipid content. Lipid frctiontion. Free ftty cid, triglyceride nd triglyceride ftty cids were seprted by modifiction of the method of Borgstr6m (1). An liquot of the mixed lipid extrct in petroleum ether ws tken to dryness nd redissolved in CCl4. To obtin the free ftty cids, the CCl4 ws repetedly wshed with.5 per cent NH4OH,.2 M KCl in 4 per cent CH3OH. Crrier, nonrdioctive plmitic cid ws dded to the CCl4 to fcilitte the free ftty cid extrction. The mmonicl methnol solution of ftty cids ws cidified with H2SO4 nd the free ftty cids extrcted with petroleum ether (bp 3 to 6 C). The petroleum ether extrct of the ftty cids ws wshed with wter until the wsh ws neutrl nd then dried over nhydrous N2SO4. The CCI4 solution contining the triglycerides from which the free ftty cids hd been extrcted ws divided into two prts, one for the determintion of the rdioctivity of the triglycerides nd the other for hydrolysis (by refluxing in 5 per cent H2SO, in CH3OH for 2 hours) to recover the triglyceride ftty cids. The

3 LIPID SYNThESIS IN HUMAN ADIPOSE TISSUES 927 TABLE The men rtios of cette incorportion into mixed lipids nd cette oxidtion to CO2 of lipom to norml Lipom/norml No.* Acette incorported into mixed lipid (mamoles/hr) Acette oxidized to C2 (mimoles/hr) Per g wet wt Per mg N Per g lipid-free wt Per g wet wt Per mg N Per g lipid-free wt i 1.29t i ± ± i.4 * No. refers to the number of seprte experiments on which the men is bsed. t Stndrd error of the men. rdioctivity of the vrious lipid frctions ws ssyed in liquid scintilltion spectrometer employing toluene contining.3 per cent 2,5-diphenyloxzole nd.3 per cent p-bis [2-(phenyloxzolyl)] benzene s solventphosphor system. Ftty cid nlysis. An liquot of the mixed lipid extrct ws hydrolyzed nd methylted by refluxing for 2 to 3 hours with 5 per cent H2S4 in dry methnol t 7 C. The methyl esters of the ftty cids thus formed were extrcted with petroleum ether nd the extrct ws tken to dryness in nitrogen tmosphere. The ftty cid methyl esters were nlyzed by gs-liquid chromtogrphy t 15 C in the Brber-Colemn pprtus with rdium source ioniztion detector. The mobile phse ws rgon gs nd the sttionry phse ws either ethylene glycol dipte or ethylene glycol succinte, 12 to 17 per cent, on to 1 mesh Chromosorb W (11-13). The determintion of the rdioctivity present in the individul ftty cids nd their specific ctivities ws chieved by splitting the effluent gs strem coming from the column. One prt of the effluent gs pssed through the detector cell, the other to n nthrcene-filled glss crtridge outside of the pprtus which effectively trpped the ftty cid (14). The crtridges were chnged mnully to collect the desired frctions s indicted by the trcing which ws being recorded simultneously. The rdioctivity on the nthrcene ws determined in the scintilltion spectrometer by inserting the glss crtridge into the well of Lucite sphere which hd the sme dimensions s the conventionl liquid counting bottle. RESULTS Lipid biosynthesis nd cette oxidtion in norml dipose tissue nd lipom. In 1 of 2 sub- II jects, the rte of cette incorportion into mixed, lipids of lipom ws higher thn tht of the djcent norml dipose tissue (Figure 1). In some persons the norml dipose tissue synthesized ft more rpidly thn did the lipom of other ptients. Among the subjects studied, there ws gret vrition in the rte of cette incorportion into mixed lipids by norml subcutneous dipose tissue. In the rt, it hs been found tht there is decrese in the in vitro rte of cette incorportion into mixed lipids by dipose tissues with ging.1 As cn be seen in Figure 1, the vrition in lipid synthesis mong the subjects studied bers no systemtic reltionship to their ges. The rtes of cette incorportion into mixed lipids mentioned bove hve been relted to the unit of wet weight of tissue. Norml dipose tissue ws found to hve reltively higher nitrogen nd lower lipid content thn lipom (Tble I). When the rtes of cette incorportion into the mixed lipids re referred to these units, the difference in the rtes of lipid synthesis between the two tissues re mgnified (Tble II). The rtio of the rte of oxidtion of cette to CO, by lipoms to tht of the norml dipose tissue of the sme subject did not differ significntly from 1. whether relted to units of wet weight, nitrogen, or lipid- TABLE III Ftty cids in norml dipose tissue nd lipom * 1 Benjmin, W., Kundel, H., nd Gellhorn, A. Unpublished observtions. Ftty cidst Norml Lipom Ftty cidst Norml Lipom C12.35 ± :4.17 C1 6. ± C ± :1:.53 C ± i.4 C i C ± ±.29 C ± ±.14 * These dt re weighted men vlues, the stndrd error of the men for 4 to 14 seprte smples of norml dipose tissue nd lipom from ech of 9 subjects. The figures re given s percentges ± the stndrd error of the men. t Designtion of ftty cids s in Figure 2.

4 92 ALFRED GELLHORN AND PAUL A. MARKS _._ LIPOMA C -' I Sttionry phose-ethylone glycol #dipolr Argon flow rtes115cc/,ein Temperoture ' /5' Sensitivit.3z/ Voltge z 5 C14 S CIO-1 CleS2 Cis *v Co q6 c ' '.?3 tx i> NORMAL TISSUE 6 Cle-' A I I Stetionry Phese =Ethylnee/yce/ e~~~~d/ipete Argon flow re e=15 Temperture /5 SensitIvIty c3j IO Voltge 5 /s. Cis *1 ' fir FIG. 2. See legend t bottom of pge 929.

5 LIPID SYNTHESIS IN HUMAN ADIPOSE TISSUES 929 TABLE IV Acette-i-C'4 incorportion into lipid components of norml dipose tissue nd medium * Mixed lipid Mixed Mixed lipid free ftty Triglyceride lipidt Sp. ct.t triglyceridet Sp. ct. cidt Sp. ct. ftty cidt Sp. ct. Tissue 33. ( ).47 (.3-.6) 35. ( ).4 (.3-.59).35 ( ).7 ( ) 2.4 ( ).33 (.2-.5) Medium.35 ( ).6 (.4-.7).5 ( ).1 (.9-.15).3 ( ).26 ( ) * These re dt for single experiment. The men vlue for rdioctivity in the totl lipid nd the other lipid components ws clculted by multipliction of the specific ctivity of the component by the totl milligrms present per grm wet weight of dipose tissue. The vlues in prentheses indicte the rnge of determintions on 4 seprte smples. Vlues shown re jsc X 12/g wet weight. t Sp. ct. =specific ctivity in pc XlO-2/mg. free weights of the tissues (Tble II). Thus, the incresed rte of ft biosynthesis by lipoms ws 5 to 13 times greter thn the norml tissue, depending upon the reference unit selected; the verge rte of cette-i-c14 oxidtion to C142 ws not significntly different between lipom nd norml dipose tissue, regrdless of the reference unit used for clcultion. The proportion of the newly synthesized mixed lipid rdioctivity trnsferred to the medium by lipom nd norml dipose tissue rnged from 1 to 34 per cent nd the men rtio of lipom: norml ws 1.5. It ws found tht n incresing mount of the mixed lipid rdioctivity ppered in the medium with time but the proportion of this, reltive to the totl rdioctivity in the mixed lipids, remined constnt. Composition of norml subcutneous dipose tissue nd lipom. Gs-liquid chromtogrphic nlysis of the ftty cids from norml dipose tissue nd lipom reveled tht oleic nd plmitic cids predominted (Figure 2 nd Tble III). There were no qulittive or significnt quntittive differences in the ftty cid composition of lipom compred with norml ft. The seven ftty cids indicted in Tble III ccounted for bout 9 per cent of ll the ftty cids present in these tissues. Twenty-six ftty cids, in ddition to those listed in Tble III, were identified in the ft of the tissues here studied in cpillry column gs-liquid chromtogrphic nlysis.2 These ftty cids, however, comprise less thn 2 per cent of the totl ftty cid composition. No qulittive differences in these minor (by criterion of weight) ftty cid components were noted between norml dipose tissue nd lipom. The stndrd error of the men shown for ech ftty cid in Tble III indictes the smll vrition noted between smples from different subjects. A sttisticl comprison of the vrition of ftty cid composition within sources nd between sources led to the conclusion tht there ws no greter vrition in the ftty cid composition between subjects thn in the sme subject. Distribution of C'4 in lipid components of tissue nd medium. More thn 9 per cent of the tissue lipid rdioctivity ws present in the triglyceride nd no more thn 1.5 per cent ws in the free ftty cids of the tissue (Tbles IV nd V). In the medium, on the other hnd, the rdioctivity ws found predominntly in the free ftty cid frction. Hydrolysis of the tissue triglycerides reveled tht per cent of the rdioctivity ws in the ftty cids. Although no significnt difference in the distribution of the incorported ce- 2 We wish to cknowledge the ssistnce of Dr. S. R. Lipsky, Yle University School of Medicine, in this nlysis nd the generous dvice given by him, Dr. Evn Horning nd Dr. Chrles C. Sweeley, Ntionl Hert Institute, Bethesd, Md., nd Dr. Dvid Turner, Sini Hospitl, Bltimore, Md., in our studies with gs-liquid chromtogrphy. FIG. 2. GAS-LIQUID CHROMATOGRAPHIC RECORDS OF METHYL ESTERS OF FATTY ACIDS ISOLATED FROM LIPOMA AND NORMAL ADIPOSE TISSUE. The designtions bove ech pek identify the ftty cid ccording to crbon chin length (e.g., C12 dodecnoic cid) nd number of unsturted bonds (e.g., monoenoic C16-1 nd dienoic C1-2). The numbers under ech pek refer to the percentge of the totl ftty cids represented by the prticulr ftty cid (e g., C1-1, 9-octdecenoic cid or oleir cid, comprises 52 per cent of the totl ftty cids in the lipom here shown).

6 93 ALFRED GELLHORN AND PAUL A. MARKS TABLE V Distribution of rdioctivity in lipid frctions Mixed lipid triglyceride/mixed lipid Mixed lipid free ftty cid/mixed lipid Triglyceride ftty cid/triglyceride Norml [6]* Tissue Medium Lipom [3] Tissue Medium * The numbers in brckets indicte the number of experiments from which the men vlues hve been clculted. tte-i-c14 ws found between the norml tissue nd the lipom, it is pprent tht in the dipose tissues of mn s in the rodent (15) there is smll free ftty cid intrcellulr pool nd lrge triglyceride pool. The lipid which is trnsferred cross the cell membrne is predominntly in the form of the free ftty cid. All of the ftty cids isolted from dipose tissue incubted with cette-i-c'4 were lbeled (Tble VI). The rte of C14 incorportion into the sturted ftty cids is greter thn into the unsturted ftty cids. Although the bsolute mount of rdioctivity ws greter in the individul ftty cids isolted from lipom thn from norml, the distribution of the rdioctivity mong the ftty cids s shown by the reltive specific ctivity ws essentilly the sme in lipom nd in norml dipose tissue. The percentge of the totl rdioctivity recovered in the myristic cid (C-14) of lipom ws high in one experiment nd comprble to tht of norml in the other. This ccounts for the higher men reltive specific TABLE VI The specific ctivities of ftty cids of the mixed lipids of norml dipose tissue nd lipom following incubtion with cette-1-c'4 * Per cent of Per cent of totl ftty totl cpm Reltive sp. cids recovered ct.* Ftty cidt N L N L N L C C C C C C C * Results of 2 experiments; ll nlyses were determined t lest in duplicte. N =norml, L =lipom. t Designtion of ftty cids s in Figure 2. The reltive specific ctivity represents: % of totl cpm recovered % of totl ftty cids ctivity of the myristic cid of the lipom thn of the norml. The finding of smll mounts of rdioctivity in the essentil ftty cid, linoleic (Ci-2), ws unexpected. This could represent contmintion from other lbeled ftty cids; isoltion nd degrdtion of the linoleic cid will be required for interprettion of the result. DISCUSSION This study demonstrtes tht norml dipose tissue of mn cn incorporte cette into lipids in vitro. The rte of cette incorportion per grm norml dipose tissue per hour mong the subjects studied rnged from.1 to 13 m/amoles. This my be compred to 34 nd 23 mu~moles cette incorportion per grm wet weight per hour of humn leukocytes nd pltelets, respectively (16). Although the medin in vitro rte of bout 1 mfumole cette incorported per grm dipose tissue per hour does not seem high, recognition tht this tissue constitutes 2 per cent of the body weight provides better indiction of its importnce in ft metbolism. In the humn subjects whose subcutneous dipose tissue ws studied, there ws gret vrition in the in vitro rte of cette incorportion into the mixed lipids. The epididyml ft pds from inbred strins of rts mintined under constnt lbortory conditions nd on stndrd diet hve less vrible in vitro rte of ft biosynthesis when the tissue from nimls of the sme ge re studied. With ging, this rte declines from men of 7 /Amoles per g epididyml dipose tissue in nimls weighing g, to.7 Mmoles in 3 g nimls.' In the present studies of humn dipose tissue, no correltion between ft synthesis nd ge, sex, or ethnic origin could be demonstrted.

7 LIPID SYNTHESIS IN HUMAN ADIPOSE TISSUES In contrst to the vribility of the rtes of lipid synthesis from one humn subject to nother, ws the smll devition in the ftty cid composition of the dipose tissue from one person to the next. Observtions of Moore nd Cook on humn subjects in Scotlnd showed essentilly the sme ftty cid composition s tht reported here (17); Hirsch nd co-workers hve lso emphsized the constncy of the ftty cid pttern in humn dipose tissue (1). The uniformity of the dipose tissue ftty cid composition my reflect n overll similrity of the dietry ft intke mong the subjects studied. The ptients were, however, unselected nd cme from ll economic groups s well s from different rcil nd ntionl ggregtes within metropolitn New York. Therefore, lthough not specificlly determined, it is resonble to ssume considerble differences in dietry hbits. If, s seems likely, this lso implies differences in the composition of the ft intke, then ctive physiologicl regultion of the dipose tissue triglyceride composition must exist. Cellulr control mechnisms for mintining the constncy of the dipose tissue ftty cid composition re not known. One spect of this my, however, be reveled in the present experiments. The il vitro studies with humn dipose tissue hve demonstrted tht the synthesized lipid which ppers in the medium is unesterified. These dt re in ccord with the observtions of Reshef, Shfrir nd Shpiro who found tht in rt dipose tissue, unesterified plmitic cid is trnsferred from the tissue to the medium (19). In mn, Dole (2), Gordon nd Cherkes (21), Spitzer nd Miller (22), nd Lurell (23) hve observed chnges in the serum free ftty cids under conditions of ft mobiliztion from dipose tissue which indicte tht ftty cids, not triglycerides, re relesed. The present results lso demonstrte tht there is less thn 1.5 per cent of newly synthesized lipid present s free ftty cid in the cell nd the reminder is esterified. In the studies of rt mesenteric dipose tissue, Shpiro, Chowers nd Rose found tht sterte-1-c14 ws incorported into the cellulr triglycerides; only smll proportion ws present s the free cid (15). From these observtions, it is suggested tht ftty cid synthesized in the cytoplsm is immeditely esterified nd stored in the intrcellulr triglyceride reservoir or trnsferred to the extrcellulr fluids This rpid disposition to one or nother comprtment provides flexibility which my fcilitte the mintennce of constnt ftty cid composition. It hs been stted nd generlly ccepted th't lipoms do not prticipte in the body economy when there is demnd for ft mobiliztion through strvtion or innition. The clinicl observtion on which this llegtion is bsed hs been questioned (24) nd the in vitro studies which hve shown tht rdioctive lipids formed during incubtion do pper in the medium lso indicte tht the potentil mechnisms for mobiliztion of lipom lipid exist. At the outset of this study, severl hypotheses were formulted which might chrcterize the fundmentl difference between the lipom nd the norml dipose tissue from which it rises. Thus, loclized ccumultion of ft could occur if the cells involved hd smller thn norml cpcity to oxidize ftty cids, or if there were impirment in the pthwy for the mobiliztion of ft from the cells, or if there were greter rte of ft synthesis in the tumor thn in the norml dipose tissue. One other possibility considered ws tht there might be qulittive difference in the biosynthetic pthwy which would led to the formtion of ft with different chemicl composition from tht of the norml dipose tissue. It is recognized tht in vivo there re mny fctors influencing lipid synthesis which cnnot be evluted in the in vitro system. Nevertheless, the finding tht the lipoms generlly incorporte cette into ftty cids t greter rte thn the norml dipose tissue from the sme subject, wheres there ws no consistent difference between these two tissues in the rtes of cette oxidtion to CO,, the proportion of newly synthesized lipid trnsferred to the medium, or the ftty cid composition, suggests tht disturbnce in lipid synthesis is mjor fctor leding to ft ccumultion in the lipom. SUMMARY The rte of cette-1-c14 incorportion in vitro into mixed lipids of lipoms nd norml humn subcutneous dipose tissue from the sme subject ws greter in the tumor thn in the norml tissue in 1 of 2 experiments. No significnt difference in the rte of cette-1-c14 incorportion into C14., or in the proportion of isotopiclly l-

8 932 ALFRED GELLHORN AND PAUL A. MARKS beled lipid trnsferred to the medium ws found between the two types of dipose tissue. 2. Mrked vrition ws observed in the rte of cette incorportion into mixed lipids by subcutneous dipose tissue mong the 2 subjects studied. This could not be correlted with ge, sex or rce. 3. In contrst to the vribility of lipid synthesis s mesured by cette incorportion into,.the mixed lipids, no significnt vrition ws found in the ftty cid composition of the subcutneous ft between subjects or between the norml dipose tissue ft nd tht of lipoms. 4. Studies of the distribution of rdioctivity in lipid components of dipose tissue following incubtion with cette-i-c14 demonstrted bout 1 per cent of the totl in the tissue free ftty cids nd the reminder in the triglycerides. Eighty per cent of the triglyceride rdioctivity ws recovered in the ftty cids. Similr studies of the lipids in the medium showed recovery of bout 9 per cent of the rdioctivity in the free ftty cids nd the reminder in triglycerides. The rte of ccumultion of C14 ws greter in the sturted ftty cids thn in the unsturted cids in the norml dipose tissue nd lipom. REFERENCES 1. Wertheimer, E., nd Shpiro, B. The physiology of dipose tissue. Physiol. Rev. 194, 2, Shpiro, B., nd Wertheimer, E. The metbolic ctivity of dipose tissue. A review. Metbolism 1956, 5, Husberger, F. X., Milstein, S. W., nd Rutmn, R. J. The influence of insulin on glucose utiliztion in dipose nd heptic tissues in vitro. J. biol. Chem. 1954, 2, Winegrd, A. I., nd Renold, A. E. Studies on rt dipose tissue in vitro. I. Effects of insulin on metbolism of glucose, pyruvte nd cette. J. biol. Chem. 195, 233, Bll, E. G., Mrtin, D. B., nd Cooper,. Studies on the metbolism of dipose tissue. I. Effect of insulin on glucose utliztion s mesured by mnometric determintion of crbon dioxide output. J. biol. Chem. 1959, 234, Colowick, S. P., nd Kpln, N.. Methods in Enzymology. New York, Acdemic Press, 1955, vol. 1, p Umbreit, W. W., Burris, R. H., nd Stuffer, J. F. Mnometric Techniques nd Relted Methods for the Study of Tissue Metbolism. Minnepolis, Burgess, Dvidson, J. D., nd Feigelson, P. Prcticl spects of internl-smple liquid-scintilltion counting. Int. J. ppl. Rdit. 1957, 2, Folch, J., Lees, M., nd Slone Stnley, G. H. A simple method for the isoltion nd purifiction of totl lipides from niml tissues. J. biol. Chem. 1957, 226, Borgstr6m, B. Investigtion on lipid seprtion methods. III. Seprtion of tri-, di-, 1-mono- nd 2-mono-glycerides. Act physiol. scnd. 1954, 3, Jmes, A. T., nd Mrtin, A. J. P. Gs-liquid chromtogrphy: The seprtion nd identifiction of the methyl esters of sturted nd unsturted cids from formic cid to n-octdecnoic cid. Biochem. J. 1956, 63, Lipsky, S. R., Lndowne, R. A., nd Godet, M. R. The effects of vrying the chemicl composition of the sttionry liquid on the resolution of the long chin sturted nd unsturted ftty cid esters by gs-liquid chromtogrphy. Biochim. biophys. Act 1959, 31, Crig, B. M., nd Murty, N. L. The seprtion of sturted nd unsturted ftty cid esters by gsliquid chromtogrphy. Cnd. J. Chem. 195, 36, Krmen, A. Gs chromtogrphy nd lipid metbolism. Amer. Hert J. 196, 59, Shpiro, B., Chowers, I., nd Rose, G. Ftty cid uptke nd esterifiction in dipose tissue. Biochim. biophys. Act 1957, 23, Mrks, P. A., Gellhorn, A., nd Kidson, C. Lipid synthesis in humn leukocytes, pltelets, nd erythrocytes. J. biol. Chem. 196, 235, Moore, C. H., nd Cook, R. P. Humn dipose tissue (bstrct). Biochem. J. 1959, 73, 43p. 1. Hirsch, J., Frquhr, J. W., Ahrens, E. H., Jr., Peterson, M. L., nd Stoffel, W. Studies of dipose tissue in mn: A microtechnic for smpling nd nlysis. Amer. J. clin. Nutr. 196,, Reshef, L., Shfrir, E., nd Shpiro, B. In vitro relese of unesterified ftty cids by dipose tissue. Metbolism 195, 7, Dole, V. P. A reltion between non-esterified ftty cids in plsm nd the metbolism of glucose. J. clin. Invest. 1956, 35, Gordon, R. S., Jr., nd Cherkes, A. Unesterified ftty cid in humn blood plsm. J. clin. Invest. 1956, 35, Spitzer, J. J., nd Miller, H. I. Unesterified ftty cids nd lipid trnsport in dogs. Proc. Soc. exp. Biol. (N. Y.) 1956, 92, Lurell, S. Plsm free ftty cids in dibetic cidosis nd strvtion. Scnd. J. clin. Lb. Invest. 1956,, Wells, H. G. Adipose tissue, neglected subject. J. Amer. med. Ass. 194, 114, 2177.