The Relation between Protein Synthesis and Lipide Accumulation in L Strain Cells and Ehrlich Ascites Cells*

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1 Publshed Onlne: 1 May, 1959 Supp nfo: Downloaded from jcb.rupress.org on December 19, 2018 The Relaton between Proten Synthess and Lpde Accumulaton n L Stran Cells and Ehrlch Asctes Cells* By DONALD W. KNG, M.D., EDWARD L. SOCOLOW, M.D., and KLAUS G. BENSCH, M.D. (From the Department of Pathology, Yale Unversty School of Medcne, New Haven) (Receved for publcaton, August 18, 1958) ABSTRACT t has long been known that fat accumulates n old njured cells both n tssue culture and n many mammalan dsease states. The use of L cells grown n suspenson tssue culture permtted the opportunty to study condtons n whch lpde accumulaton could be retarded or accelerated. These cultures exhbt a three-phase growth curve whch s smlar to that prevously found wth bactera and conssts of a lag perod, logarthmc growth perod, and statonary perod. Daly alquots were removed from cultures gong through these phases and proten and cholesterol content correlated wth celt dvson. t was found that L cells gradually accumulated lpde n the cell concurrent wth retardaton of cell dvson and proten synthess. Conversely old lpde-laden cells, placed n fresh meda and encouraged to actve dvson wth net proten synthess progressed from a hgh to a low lpde/cell rato over a perod of 2 to 4 days. An amno acd analogue p-fluorophenylalanne and a mtotc nhbtor, colchcne, also markedly ncreased the lpde/cell rato. Smlar results were found n n vtro experments wth Ehrlch asctes cells. NTRODUCTON For many years pathologsts and cytologsts have observed the accumulaton of stanable lpde n cells that are ether old or njured. The old terms of fatty degeneraton (meanng ntracellular unmaskng of lpde) and fatty nfltraton (lpde deposted from exogenous sources) have been replaced by the term "fatty change." Both at autopsy and n expermental njury the lver has been a frequent ste of "fatty change." An early relatonshp was elucdated between detary defcences and lpde deposton n the lver. Dets low n proten (16, 22, 17, 15), contanng an amno acd mbalance (5), defcent n methonne (31) or tryptophane (29, 10, 1), have all produced fat n lver cells. njury to the lver by a varety of other means apparently accentuates lpde accumulaton (32). Studes on the deposton of lpde, partcularly cholesterol, on blood vessels have yelded * Ths work was supported by a grant from the Unted States Publc Health Servce C2928 (C). J. BOPl~Ysc. AND BOCHE~. CVTO,., 1959, VO. 5, NO smlar results. Dets defcent n methonne produce hypercholesterolema (14) and atheroscleross n rats (19) and monkeys (24). njury of some nature to the vessel also causes an ncrease n lpde accumulaton (7, 6, 30, 33). The lterature ndcates that lpdes appear n njured cells. There s lttle ndcaton as to why t appears or from whence t comes. The emphass n explanatons has been on a cellular degeneratve process assocated wth faulty breakdown of fat or a cellular membrane alteraton allowng ncreased accumulaton of abnormal lpdes from exogenous sources. n our laboratory mouse L stran fbroblasts are beng grown n suspenson cultures. t s possble wth ths system to evaluate the accumulaton of lpde n cells durng varous stages of growth and when subjected to dfferent forms of njury. Materals and Methods Stran L (Earle) cells were grown n suspenson tssue culture accordng to the procedure descrbed by Kuchler (21). Each 250 cc. Erlenmeyer flask contaned

2 422 PROTEN SYNTHESS AND LPDE ACCUMULATON t / =, \~, 280 aso ~ ::S 240~, 2ao~> 200 z 180 ~ 160 o. 140.j 120,, ~, 00 z D AYS m,----m CELL COUNT ~.~ ~ CELL PROTEN 'to 20 FG. 1. Cell growth of stran L cells. Eagle's basal medum (72 ml.) (12), horse serum (8 ml.), penclln (100 unts/ml.), and streptomycn (100 /zg/ml.). Cultures were gassed wth 8 per cent CO2 n ar. The flasks were then placed on a rotary shaker n an ncubator mantaned at 37.5 C. Each culture ntally contaned 200,000 cells/ml. Para-fluorophenylalanne and colchcne (Calforna Corporaton for Bochemcal Research) were ntroduced drectly nto the L stran cultures n a fnal concentraton of 8 X l0-4 M for para-fluorophenylalanne and 10-7 M for colchcne. Control cultures from the same homogeneous stock cultures were set up n an dentcal manner. The use of suspenson tssue culture permtted the removal of accurate daly samples for determnatons of cell number, cell proten, and lpde. The cell counts were done by countng ten squares n duplcate wth a standard hemocytometer. After removal of approprate alquots (1 ml. for proten, 15 ml. for lpdes) the cells were washed three tmes n 5 ml. of a modfed Krebs- Rnger phosphate soluton (3). Cell proten was determned by Oyama's modfcaton of the Lowry method (26). Cells taken for lpde analyss were frst extracted under a reflux condenser n a 60 C. water bath for 1 hour wth 25 ml. of an alcohol-ether soluton (1:1). Sutable alquots of ths extract were taken for determnaton of cholesterol by the Schoenhemer- Sperry method (28), phospholpdes by a modfed Fske-SubbaRow method (34), and fatty acds by the method of Stoddard and Drury as modfed by Man and Gldea (23). Ehrlch asctes cells were used n a seres of experments of short term duraton. The tumor was mantaned n Swss-Webster mce and harvested as descrbed by Klen (20). The pooled cells from the nonhemorrhagc asctc fud of 10 to 12 mce were washed three tmes n 50 ml. of cold Krebs-Rnger phosphate soluton (ph 7.4). Usually the cells were then resuspended n Eagle's basal medum wth horse serum, as prevously descrbed for stran L cells, although some cells were resuspended only n fresh Krebs-Rnger soluton. n all nstances a cell concentraton of approxmately 2 X l0 T cells/ml, was obtaned. Samples of 10 ml. volume were ncubated n 50 ml. stoppered Erlenmeyer flasks and placed on a rotary shaker n the 37.5 C. ncubator for perods of 3 to 4 hours. Paraflurophenylalanne was added to the non-control flasks n a fnal concentraton of 10-2 ~ and 10-3 M and colchcne was added to others n a fnal concentraton of 10 _3 ~t and 10-6 M. Alquots for proten and lpde determnatons were taken ntally and at the end of the ncubaton perod. After washng, the cells were analyzed n the manner descrbed for L stran cells. The cells were counted n a standard blood countng chamber before and after ncubaton. No change n cell number was noted and less than 5 per cent of the cells permtted the entrance of trypan blue nto ther nucle. OBSERVATONS Other workers have prevously shown a characterstc growth curve when stran L cells are grown n suspenson culture as shown n Fg. 1 (21). There s usually a 1 day lag perod before dvson commences. The cells only dvde when

3 D. W. KNG, E. L. SOCOLOW, AND K. G. BENSCH 423 A B --.- b. 0 a- W 75 " g. 50 '0 X g~ z5 -- () -J 20 s S ANALOGU s 33j / js s / S CONTROL ~ ANALOGUE..,j o z o 0 z 126 bj V ~loo ANALOGUE o CONTROL / - ~ CONTROL ANALOGUE 6, _ o FG. 2 A. Effects of p-fluorophenylalanne 8 )< 10-4 M on cholesterol n young stran L cells. B. Effects of p-fluorophenylalanne 8 >( 10 -~ ~t on cholesterol n old stran L cells. the ph of the medum has dropped to 7.1, but durng ths ntal lag perod there s a doublng of the cell proten. Later fgures wll show that durng ths same perod the cholesterol content of the cell begns to fall. The lag perod s followed by a logarthmc growth perod n whch the cells dvde every 24 to 26 hours untl a cell concentraton of approxmately cells/ml, s reached. Durng ths perod the lpde content of the cells s mnmal. Fnally the cells enter a phase where they reman unchanged n cell number for 2 or 3 days. Durng ths statonary phase the proten content/cell may vary and the lpde content of the cell ncreases greatly. n several ntal experments t was found that the phospho- lpdes, neutral fat, and cholesterol all ncreased n the older cells whch had ceased dvson. Cells n log growth requre a resdual volume of 50 ml. of medum when usng a 250 ml. Erlenmeyer flask. t s thus mpossble to do multple complete lpde analyses over a perod of several days. n order to follow the cells over a longer perod of tme and n order to obtan larger and more accurate samples, the cholesterol content was taken as an ndcator of the lpde content of the cells. Snce t was determned that the cholesterol ncreased n old cells n the statonary phase whch had ceased actve proten synthess, t was decded to accentuate ths process wth an amno

4 PROTEN SYNTHESS AND LPDE ACCUMULATON A B 5C \x\ COLCHCNE ~ L. / COLCHCNE. CNTROL COTROL.J o,.p o o - - ~-,,~ 2 =~==5 ~,, 4 _z CONTROL COLCHCNE lo G ,- COLCHCNE tlll [ll llll o La=k=g_z a l a_l o# VkYt DAY S FG. 3 A. Effect of colchcne 10.7 M on cholesterol n young stran L cells. B. Effect of colchcne 10-7 M on cholesterol n old stran L cells. acd analogue, para-fluorophenylalanne. After a wde range of dfferent doses, a concentraton of 8 X 10.4 ~t was found to cause no apparent ncrease or decrease n cell number, although a moderate ncrease n cell proten was noted over a perod of 3 to 4 days. Concentratons of 10 -~ ~t had no effect on logarthmc growth, whle concentratons of 10.2 M resulted n death and dssoluton of the majorty of cells n a 24 hour perod. The normal concentraton of phenylalanne n Eagle's basal medum s 1 X 1~ 4 M. Fg. 2 shows the effect of p-fluorophenylalanne on the cholesterol content of L stran cells. n Fg. 2 A, cells were taken from a culture n logarthmc growth, broken nto two dentcal cultures, and ntroduced nto fresh medum. One of the cultures contaned p-fluorophenylalanne ( M) the the other remaned as a control. The ntal cholesterol content per cell and per mg. proten s very low and remans so n the control cultures. n the analogue-treated culture, the cholesterol content rses rapdly, both on an ndvdual cell bass and n relaton to the cell proten. n the experment shown n Fg. 2 B, cells were obtaned from an old culture of cells n the statonary phase of the growth curve. Sudan-staned sectons showed the lpde content n these old cells to be very hgh when compared wth young cells

5 D. W. KNG, E. L. SOCOLOW, AND K. G. BENSCH " NTAL O0~ROL 1 4HR. CONTROL ~:~ ' '2--2 P-F 'o'~.---~ ~ cole -T T,o-~.,o-.. --T-- 'l t 'T'~ 4 HRo N NTAL CONTROL [ll P-F 0 "2M 798 m COL? 0 -SM F '; z P_ ~" 00 EXP. EXP. 2 Fro. 4. Total lpdes n Ehrlch asctes cells gven p-fluorophenylalanne and colchcne. taken from a culture n rapd growth. As shown n the graph, the ntal cholesterol content of the cells was very hgh also. The culture was broken nto two dentcal fractons and ntroduced nto fresh medum. The control culture gradually showed a reducton n cholesterol content concdent wth the begnnng of growth and rapd proten synthess. The culture treated wth the analogue retaned and appeared to show a slght ncrease n ts cholesterol content. Another seres of experments was made n whch colchcne was used nstead of an amno acd analogue (Fg. 3). Colchcne apparently only nterfered wth proten synthess ndrectly n

6 426 PROTEN SYNTHESS AND LPDE ACCUMULATON 150 U) / 00 ~E (.9" o ~! ~. 22 o o: ~! 16.2 =E ~ 15.4 ~- / 12.7 t-"'- O L.. s. J! EXP. EXP. 2 Fro. 5. Cholesterol n Ehrlch asctes cells gven p-fluorophenylalanne and colchcne. these cells when a concentraton of 10-7 M was used. Although there was no ncrease or decrease n cell number noted over a perod of several days, there was an ntal doublng of cell proten. Concentratons of ths order on these cells presumably nterfered wth some other essental process besde cell dvson. Although the effects wth colchcne do not appear as marked as those wth parafluorophenylalanne, the results are nevertheless very smlar. nterrupton of cell dvson, growth, and proten synthess by any one of several methods s assocated wth an ncrease n cholesterol. Ehrlch asctes tumor cells were used for another seres of experments. The cells were removed from

7 D. W. KNG, E. L. SOCOLOW, AND K. G. BENSCH J ~E "-' O Z a. "/'5,50 5Om 64 s~ 6.~0 ] 'A'5 ~E 33 2, EXP. EXPo 2 FG. 6. Phospholpdes n Ehrlch asctes cells gven p-fluorophenylalanne and colchcne. the mce, pooled, washed, and ncubated n vtro n varous meda as descrbed prevously. As wth the L stran fbroblasts much depends on the age and stage of growth of the tumor cells when used. Young cells from mce njected 2 to 3 days prevously are n the perod of rapd growth. The lpde per cell and per mg. proten shows very low values and the effect of nterferng wth proten synthess s very evdent. On the other hand, cells removed from mce 8 to ll days after njecton, and whch have ceased actve dvson, have very hgh ntal lpde values per cell and per mg. pro-

8 428 PROTEN SYNTHESS AND LPDE ACCUMULATON 300.J.. 0 zk v aoo 00 Z ll -- O a O 6_ ~--- (; EXPo ~XP. 2 FG. 7. Fatty acds n Ehrlch asctes cells gven p-fluorophenylalanne and colchcne. ten. Wth decreased mtotc ndces and proten synthess, the further nhbton of proten synthess wth analogues and colchcne produce less sgnfcant changes n the lpde values. The age of the cell s also of great mportance n regard to the amount of proten lost from the cell durng the 4 hour ncubaton. Contrary to other reports we sometmes fnd a very sgnfcant decrease n proten after ncubaton wth analogues n vtro. When ths occurs the lpde accumulaton s apprecable even when the cells are placed n Krebs-Rnger phosphate soluton wthout serum.

9 D. W. KNG, E. L. SOCO[,OW, AND K. G. BENSCH 429 Ths s clearly shown n Fg. 4, Experment 1 where the total amount of lpde s several tmes hgher than the ntal values, most pronounced n those cells treated ether wth p-fluorophenylalanne or colchcne. Experment 2 of Fg. 4 s a representatve experment where there was lttle decrease n total cell proten. Only the flask contanng the amno acd analogue showed a sgnfcant ncrease n net lpde accumulaton. The concentraton of analogue 10 -~ has been shown to prevent almost completely the ncorporaton of valne-lc 14 nto these cells n control experments. Fgs. 5 to 7 show the results when the lpdes are broken nto three fractons: cholesterol, phospholpdes, and fatty acds. Fg. 5 depcts a marked net cholesterol accumulaton n the cells regardless of whether the cells are merely placed n a new envronment, as n the Eagle's basal medum control fasks and the Krebs-Rnger soluton flasks, or whether the cells are actvely njured by an analogue and colchcne. Fgure 5, experment 2 represents results wth older cells; the ncrease s less marked except for the flasks contanng the analogue. n general, the pattern wth phospholpde analyss n Fgs. 6, Experments 1 and 2 parallels that shown wth cholesterol. The loss of proten n the experments shown n Fg. 6, Experment l makes the phospholpde/proten rato greatly elevated. Fg. 7, Experments 1 and 2 showng fatty acd content of the cells, descrbe results whch were n general smlar wth the excepton that those cells mantaned n Krebs-Rnger soluton appeared to lose large amounts of fatty acds along wth the loss of proten. Perhaps ths was assocated wth ncreased energy requrements of the cells resultng from the marked change n the cells' envronment. Agan, the ncrease n lpde n the cells was hghly dependent on the ntal pde content and age of the cells. DSCUSSON We have prevously mentoned the many reports n the lterature n whch nutrtonal defcences contrbuted to lpde accumulaton n varous cells of the ntact organsm. The experments reported here wth stran L cells orgnally solated from a mouse fbroblast (27) may have lttle relaton to the specalzed cells of the lver, kdney, and aorta. t s, however, nterestng that these three stes whch readly show fatty changes n the ntact organsm are n farly drect contact wth the plasma and present a somewhat analogous stuaton to cells cultvated n meda contanng serum. Also the basc ntereonversons of fat, carbohydrate, and proten have proven to be qualtatvely smlar n most all cells studed. Whether our studes have any bearng upon other cells has lttle relevance at present. These experments document the well known pcture of lpde accumulaton n old or njured cells frequently seen by workers n tssue culture. The accumulaton of cholesterol n old stran L cells s presumably assocated wth a defcency of some sort, or the accumulaton of an nhbtor, as ths process can be reversed when the cells are placed n fresh medum. f some essental substance s mssng, t s probably not a smple defcency of amno acds. n addton to substantal amounts of amno acds n the medum, workers n tssue culture have shown utlzaton of serum proten for celt growth (2, 13) and large excesses of proten are thus presumably present. Calleau et al. have prevously reported an ncrease n lpde content n stran L cells nhbted by other metabolc nhbtors, prncpally those affectng the energy-producng systems of the cell (8). t s known that p-fluorophenylalanne may be degraded to fluoroacetate and fluoroctrate. The latter compound s an nhbtor of acontase, and nhbton of ths enzyme could possbly prevent proten synthess by nterferng wth the producton of ATP (9). However, a concentraton of p-fluorophenylalanne of M had lttle effect on respraton, and we presume the drug acted as a compettve nhbtor of phenylalanne. t has also been reported that amno acd analogues are ncorporated nto the protens of growng cells (11), As prevously mentoned, we were able to get logarthmc growth when concentratons of 10-5 ~ para-fluorophenylalanne were used. t would be of nterest to determne how much of ths analogue was ncorporated nto the protens of stran L cells. The ncrease n lpde n Ehrlch asctes cells s most nterestng snce the ncubatons were of such short duratons. The mere removal of the young cells from ther normal envronment consttutes an njury of some sort and results n a net ncrease n lpde, but ths was markedly ncreased wth the analogue. Belkn and Wodnsky (4) have prevously shown that old, non-dvdng

10 430 PROTEN SYNTHESS AND LPDE ACCUMULATON Yoshda asctes tumor cells have large amounts of stanable lpde that s mmedately lost when old tumor cells are transferred nto new anmals. Ths lpde was retaned n the cells, however, f small amounts of podophylln and colchcne were njected ntrapertoneally along wth the cells. There appears to be lttle queston that expermentally n vtro and n vvo t s possble to produce an accumulaton of lpde n cells by nterferng wth growth and proten synthess. n some nstances, as n defcent meda cultures, ths phenomenon s completely reversble. n other nstances n whch the drug or metabolc nhbtor cannot be easly removed, t appears rreversble. We beleve the reversble cases may be analogous to the experments done wth ntrogen defcency n E. col by Holme and Palmsterna (18). n ther experments the cells accumulated large quanttes of glycogen nstead of lpde. When NH4C1 was gven to the cells, the glycogen was degraded both for energy and drectly for amno acd synthess. t does not appear unreasonable to suggest that an amno acd defcency, the use of analogues, energy nhbtors, or the presence of other condtons ncompatble wth the normal proten metabolsm of the partcular cell beng studed, accentuate or stmulate lpde accumulaton by less complex or perhaps less vulnerable pathways. Of specal nterest n ths regard s the observaton of Ors el al. (25) that nfecton of Ehrlch cells wth vruses resulted n a great ncrease n stanable lpde n the cell. The vrus presumably nterfered wth both nuclec acd and proten metabolsm. When cells are ncubated n serum t s, of course, probable that much of the lpde accumulates n the cell as a result of transport from extracellular sources. CONCLUSONS 1. L stran fbroblasts undergong dvson and actve synthess of proten have low concentratons of cholesterol/cell and per rag. proten. nterrupton of ths actve proten synthess as a result of exhausted meda, or drugs such as p-fluorophenylalanne and colchcne, causes a marked ncrease n the amount of cholesterol/cell and ncrease n cholesterol/proten rato. 2. Ehrlch tumor cells also have a low concentraton of lpde durng actve growth. Older cells that have ceased dvson have much hgher levels/cell. Short term njury by several methods ncludng a new envronment, colchcne, and para-fluorophenylalanne wll produce net accumulaton of lpde. Ths may or may not be assocated wth ncreased degradaton of proten. REFERENCES 1. Adamstone, F. B., and Spector, H., Tryptophan defcency n the rat. Hstologcal changes nduced by force feedng of an acd-hydrolyzed casen det, Arch. Path., 1950, 49, Bachtold, J. G., and Gebhardt, L. P., Utlzaton of serum protens by normal and polovrus nfected monkey kdney cells, Exp. Cell Research, 1957, 13, Barron, E. S. G., Meyer, J., and Mller, Z. B., The metabolsm of skn. Effect of vescant agents, J. nv. Dermat., 1948, 11, Belkn, M., and Wodnsky,., Cytochemcal studes on Yoshda rat asctes tumor.. Lpdglycogen behavor, Proc. Am. Assn. Cancer Research, 1955, 2, Beverdge, J. M. R., Lucas, C. C., and O'Grady, M., The effect of detary protens and amno acds on lver fat, J. Bol. Chem., 1945, 160, Boucek, R. J., and Noble, N. L., Connectve tssue. A technque for ts solaton and study, Arch. Path., 1955, 89, Boucek, R. J., and Noble, N. L., Bochemcal studes on cholesterol n n vvo cultvated connectve tssue, Crculaton Research, 1957, 1, Calleau, R., Moss, S., and Segel, B. V., Effect of some metabolc nhbtors on the growth and lpde composton of Earle's stran L cells, J. Nat. Cancer nst., 1956, 16, part 2, Cameron, G. R., New pathways n cellular pathology, London, Edward Arnold, Ltd., 1956, Cole, A. S., and Scott, P. P., Tssue changes n the adult tryptophan-defcent rat, Brt. J. Nutrton, 1954, 8, Cowe, D. B., and Cohen, G. N., Bosynthess by Eschercha col of actve altered protens contanng selenum nstead of sulfur, Bochlm. et Bophysca Acta, 1957, 26, Eagle, H., Oyama, V.., Levy, M., Horton, C. L., and Fleschman, R., The growth response of mammalan cells n tssue culture to/-glutamne and l-glutamc acd, J. Bol. Chem., 1956, 218, Evans, V. J., Bryant, J. C., Foramont, M. C., McQulkn, W. T., Sanford, K. K., and Earle, W. R., The change n concentraton of certan consttuents of the medum durng growth of the stran HeLa cells, Am. J. Hygene, 1955, 61, Fllos, L. C., and Mann, G. V., nfluence of sulfur amno acd defcency on cholesterol metabolsm, Metabolsm, 1954, 3, 16.

11 D. W. KNG, E. L. SOCOLOW, AND K. G. BENSCH Harper, A. E., Monson, W. J., Benton, D. A., Wnje, M. E., and Elvehjem, C. A., Factors other than cholne whch affect the deposton of lver fat, J. Bol. Chem., 1954, 206, Harper, A. E., Benton, D. A., Wnje, M. E., and Elvehjem, C. A., On the lpotropc acton of protens, J. Bol. Chem., 1954, 209, Harper, A. E., Monson, W. J., Benton, D. A., and Elvehjem, C. A., The nfluence of proten and certan amno acds partcularly threonne on the deposton of fat n the lver of the rat, J. Nutrton, 1953, 50, Holme, T., and Palmsterna, H., Changes n glycogen and ntrogen-contanng compounds n Eschercha col B durng growth n defcent meda, Acta chem. Scand., 1956, 10, Jones, R. J., Wssler, R. W., and Huffman, S., Certan detary effects on the serum cholesterol and atherogeness n the rat, Arch. Path., 1957, 63, Klen, G., and Klen, E., The transformaton of a sold transplantable mouse carcnoma nto an "asctes tumor," Cancer Research, 1951, 11, Kuchler, R. J., and Merchant, D. J., Propagaton of stran L (Earle) cells n agtated flud suspenson cultures (22620), Proc. Soc. Exp. Bol. and Med., 1956, 92, Lucas, C. C., and Rdout, J. H., The lpotropc actvty of proten, Canadan J. Bochem. Physol., 1955, 33, Man, E. B., and Gldea, E. F., The effect of the ngeston of a large amount of fat and of a balanced meal on the blood lpds of normal man, J. Bol. Chem., 1932, 99, Mann, G. V., Andrus, S. B., McNally, A., and Stare, F. J., Expermental atheroscleross n Cebus monkeys, J. Exp. Med., 1953, 98, Ors, E. V., Love, R., and Koprowsk, H., Cytologcal changes n the Ehrlch asctes tumor followng nfecton wth three vruses, Cancer Research, 1957, 17, Oyama, V.., and Eagle, H., Measurement of cell growth n tssue culture wth a phenol reagent (Foln-Cocaltean), Proc. Soc. Exp. Bol. and Med., 1956, 91, Sanford, K. K., Earle, W. R., and Lkely, G. D., The growth n vtro of sngle solated tssue cells, J. Nat. Cancer nst., 1948, 9, Schoenhemer, R., and Sperry, W. M., A mcro method for the determnaton of free and combned cholesterol, J. Bol. Chem., 1934, 106, Scott, E. B., Hstopathology of amno acd defcences. V. soleucne, Proc. Soc. Exp. Bol. and Med., 1956, 92, Strong, J. P., McGll, H. C., and Nyman, M. A., Deposton of lpd n calf aorta mantaned n vtro, Crculaton Research, 1957, 8, Tucker, H. F., and Ecksten, H. C., The effect of supplementary methonne and cystne on the producton of fatty lvers by det, J. Bol. Chem., 1937, 121, Werthessen, N. T., and Schwenk, E., Bosynthess of cholesterol. Factors regulatng bosynthess of cholesterol-lke substances n solated whole lvers, Am. J. Physol., 1952, 171, Werthessen, N. T., Mlch, L. J., Redmond, R. F., Smth, L. L., and Smth, E. C., Bosynthess and concentraton of cholesterol by the ntact survvng bovne aorta n vtro, Am. J. Physol., 1954, 178, Youngsburg, G. E., and Youngsburg, M. V., Phosphorus metabolsm.. A system of blood phosphorus analyss, J. Lab. and Cln. Med., 1930, 16, 158.