In vitro incorporation of palmitate-1-14C into several glycerolipids by normal and regenerating rat liver fractions

.ZKCHIVES

OF

In Vitro

BIOCHEM~HI‘IIY

Incorporation by Normal RALPH

Institute

of Nutrition

MW

115, 598~GO5 (196Gj

BIOPHYSICS

of Palmitate-1-14C and

into

Regenerating

M. JOHKSOK

and Food Technology,

AND

Rat Liver

Glycerolipids

Fractions’

,J. 1\1. LJ3DI~;RKRE:MER~

and Department Columbus,

University,

Received

Several

November

of Physiological

Chenkstry,

The Ohio

State

Ohio

24, 1965

A study has been made of the requirement,s for the incorporation of palmitate-l-IX in vitro into triglycerides, and phosphatidylcholinc, phosphatidylinositol, phosphatiylethanolamine, and phosphatidylserine by homogenates and mitochondria of normal and mitotic phase regenerat’ing liver. The data obt.ained indicate that the reactions of the individual lipids proceed independently of One anot,her, and that, the entry of palmitic acid into the lipids occurred via several pathways rather than via a common diglyceride precursor. The incorporation of palmitate into the lipids of regenerating liver enzymes homogenates and mitochondria generally occurred at, a faster rate than was observed with normal liver enzymes. The pattern of relat)ive uptakes of palmitate int,o the lipids of the regenerating liver preparat,ions differed from that found in normal liver.

In an earlier study in which rat, liver cell chondria (A\It,) from normal and part,ially fruct,ions were used as enzyme, the relat,ive hepatectomizcd rats were used as sources of uptakes of palmit’ate into three glyceroenzymes. lipids differed depending on the portion of the cell employed, and mhet,her normal or regenerating liver was used (1). This may Male albino Iloltzman rats were reared from have been due in part’ to differing medium weaning on a standard commercial pelleted diet, requirements, i.e., the incubation medium and water cld libitum. They were used for the employed may have hecn more adequate experiments at. (i-12 weeks of age. Partially for some of the enzyme preparations t,han hepalectomized animals were prepared as defor others. In t’he present study this is in- scribed previously (2) and were sacrificed 7%hours after hepatectomy. ‘I’hc rats were killed by a blow vestigated further by measuring the extent on the head :r~~d rapidly essanguitu~ied. The livers to which Ihe incorporation of paln~itntc-l-14C were excised! chilled, pulped by forcing ihrm into triglycerides, and ~~l~osphatitlyletl~a~~olthrough a perforaled I,ucite disc lo remove conamiw, ~~l~o~phatidyli~~osit~ol, phosphatidylxlpc( ive tissue, and homogellizcV1 ill the cold if1 au serine, and ~~hosph~~titlyl~~)lolit~edepends on all-glass Pot ler-l~lwhjcm lypr lromogenizcr. phospllatc, glllcose, I\lg, IWK-,* oc-(>I’, COA, IIomogetlatrs :LIIC~rrrirocholltlriu were prepared ant1 ATP. I,ivcr homogcnatcs and liver mitoin a E-is-KC1 bull’er, plI 7.1 (1). The washed %It pellet was suspended finally ill Tris-KC1 bull’er (pII 5.4) in an :tmoutll equivalrtil to 0.75 gm of original tissue prr milliliter. The nritocholldrial preparxtiotls from Ilormal at~l wgcnt~ralillg livers lvere free of tluclei :111d cellular tlcbris. They possessed only lregligiblc glycolylic activit,v and exhibired idenlical I’/0 and respiratory control ratios (3). 130th caxhibitcd slight, but quantita-

1 This work was supporfetl by research grwrt CM14i20 from the N:ttio~~:tl Illstitutcs of ITealth, L’nited Slnics Public TIealth Service. 2 Abbreviations used: L)I’N, diphosphopyridiIle nuc~lwt idc; Co:\, coenzyme ;2; a(:P, nz-a-glycerotriphosphatr; Tris, phosphale; ATP, adenosine t ris(hydrosymet ~r~l)amillon~~thane; MI, mitochondria. 598

PALMITATE

TNCOJ:POI:ATION

lively similar, TPNH-cytochrome c reductnse activities,3 indicai.irrg that) they may have been contaminated by small but similar amounts of microsomal membranes. The enzyme preparation described herein as liver homogeuate consisted of whole homogenized liver suspended in the TrisKC1 buffer, such that one ml represented 0.5 gm of liver. Protein determittations were made on tissue suspensions by a modified biuret procedure (4). The itrcubat ion medium consisted of 0.5 ml Tris-KC1 buffer (pH i.3), 20 pmoles potassium phosphate (pII 7.4), 20 pmoles magnesium chloride 50 pmoles glucose, 1.5 pmoles sodium a-glycerophosphate, 10 pmoles disodium ATP, 5 Nmoles DPN, 1 pmolc CoA, 10 @moles potassium bicarbonate, 2 pmoles palmitic acid-l-‘% representing approximately400,OOO cpm, and 1 ml of enzyme. The final volume was 4 ml. Incubations were carried out in air in m-ml Erlenmeyer flasks for 20 minutes at 37” in a 1)ubnoff respirometer. At the end of the incubation period the reactions were slopped by adding 20 ml of methanol to each reaction flask. Control flasks were prepared itt each experiment in which methanol was added immediately after the addition of the enzyme to the reaction mixture. The flask contettts were heated to GO”, 10 ml of chloroform was added, atrd the flasks were kept at GO” for 10 minutes. After retrtrifugiug the lipid extracts were qwtntitat ively trattsferred to round-bottomed flasks. The residues were resuspended in 20 ml of ethanolethyl ether (3:l) attd extracted in a water bath at 50” for 10 minutes. The supcrnatant solutions, after centrifugation, were pooled with the original extracts, and the ethanol-ether extractions were repealed twice more. The pooled extracts were evaporat,ed at a reduced pressure to near dryness at JO”, in a rotary evaporator, and the lipids were taken up in 3-5 ml of chloroform-methanol (3:l) attd filtered through a plug of glasswool; the solvents were evaporaied in a water bath at 50” under Nz. The residues were redissolved in 0.5 ml of a solution of “cold” palmitic acid in petroleum ether (1 mg per milliliter). Twenty ml of anhydrous acetone was added and I he phospholipids were precipitated at 5” overnight. They were removed hy centrifugation, the supernatant solutions were collected, and the precipitated phospholipids were redissolved in 0.5 ml of the palmitic acid-petroleum ether solution attd were precipitated in the cold twice more by the addition of acetone. The supernatattt, fractions were pooled and evaporated IO near dryness; the neutral 3 Dr. David (:. McCormell kindly assayed mitochondrial preparations for I~PKH-cytochrome c reductase activity.

IX

172’RO

INTO

LI\‘ER

LIPIDS

590

lipids were redissolved in chloroform aud tram+ ferred to 5.ml volumetric flasks. 1:adio:rctivity counts were determined on the neutral lipid attd phospholipid preparatiotts by a scitrtillntion counting technique described below. The individual lipids were separated as follows. Nerttral lipids were separated by two-stage thin layer c~liromatogr:~p2i~- 011 Altm~iitrun Oxide t; as described earlier (5) This procedure pcrmi t s the preparaiiott of mixed triglycerides, fret of cotttamiilatiitg radioactively lat)eled fatty acid. After drvelopittg attd stainittg the ~hrom:ltogr:lnls, the spots of triglycerides were scraped at~tl the ntaterial was collected on (;lassiite paper and lrattsferred to a vial cotttainitrg the scitrtillator 15). The radioartivity was measured with a Packard TriCarb liqttid scintillation spectrometer, model 3211. LThe phospholipids u-ere prepared by twodimensional thin-layer chromatography OII Silica Gel H (Merck-Darmstadt), 250 p in thirknrss. The plates were activated in an oven at 110” for 30 minutes prior to their use. Chromatograms were developed in the first, direction from a misturc of 100 ml chloroform-mcthallol (60:35, v/v), atrd 5 ml concentrated aqueous ammonia water (2: 1, v/v). The plates were dried itt a current of air attd developed at right atigles to the ittitial separation in a solveitt mixture cotrsisting of clilorc~forrrimeth:tllol-glacial acetic acid-water (lio:30:2:1, v/v). A representative rhromat ogram is showtt i II Fig. 1. The amount of sample placed on each spot plate was equivalent to the lipids contained in either 20 or 30 mg of the original tissue, for homogenate and mitochondrial fractions, respectively. The phospholipids were located by comparison with chromat ograms of authentic samples of purified phospholipids, developed in au idrttt ical manner. Phosphat idylinositol used for this purpose X:LS a gift of I)r. Skipski of the Sloan-Kettering Institute. Phosphatidylseriiie was ohtaiucd from Geueral Hiochemicals, Chagrin Falls, Ohio. Phosphat idylcholinc and phosphatidylet hattolamine were purified from egg yolk by the methods of Rhodes and Lea (6) attd IIamthatt (~1cl. (7). They contained only traces of lgsopl~ospl~olipitIs (8) as found by thin-la?-er chrornat(~graPtlV. fhosphatidylserine was identified, attd the identity of the other phospholipids was demotrst ratrtl, following the hgdrolyt ic procedure of Skitlmore at~tl Eittenman (9) atrd a subsequent chronutt ography of the free bases by thin-layer chromatography. The plates were dryed in air attd ihe phospholipids were located by staining in I, vapor. The spots were outlined with a glass stylus, the iodine was allowed to evaporate, and the areas located

600

JOHNSON

AND

LEDERKREMER

FIG. 1. Thin-layer chromatogram of phospholipids of rat liver. Conditions are described in the text,. PE, phosphatidylcthallolarnine; PI, phosphatidylinositol; I’S, phosphatidylserine; PC, phosphntidylcholille; LPk:, LIP, and LPC are corresponding lyso compounds. TBBLE

I

INCORPORATION OF P,uxu~: AuI)-I-“C IXTO THE C~LY(~EIZOLIPII~SOF K\;omra~ AND REGENERATING 1i.i~ LIVEH 110~0GEi~h~ES AND MITY~~HONDILI.~" Liver

tissue

Normal homogenateb Regenerating homogenatec Normal mitochondriac Regenerating mitochondriac

Phosphatidylcholine

Triglyceride

269 524 35 99

zt + f f

54 78 6 1

n Incubations were at, 37” for 20 minutes with * Means and standard deviations for 8 livers. c Means and standard deviations for 6 livers.

95 54 2.7 85

f * xk +

19 20 0 23

the medium

were transferred to a scintillation rounting vial containing the mixture described elsewhere (5). For the sake of clarity in presentation, the data describing the uptake of palmitate-1.1% into the several glycerolipids are shown in bar graph form (Figs. 2-G). Data describing experiments with normal homogenates include 8 animals per group; all others include 6 animals. In presenting the data obtained, only those values that difer at a probability level of 0.05 or less are discussed.

Phosphatidylethanolsmine

Phosphatidyl inositol

23 5.i 4.2 63

23 34 0.3 80

* zt f f

described

9 0.5 0.7 12

f zt * It

fhosphatidylscrine

7 2 0.1 14

1F 31 0.5 107

f f f f

4 2 0.2 2

in the text.

RESULTS

Data describing the upt,ake of palmitate1J4C in the glycerolipids under consideration, using the complete media, are shown in Table I. :\lost’ of t,he incorporated rudioactivit,y wax found in the triglycerides when either normal homogenate, normal Rlt, or regenerating homogenate was employed. When regenerating I\‘lt were used, however,

PALMITATE

INCORPOl?ATION

I

I&

m 0 IllILl

VITllO

INTO

LIVl3~

LIPIDS

601

RAT LIVER HOMOGENATE REGENERATING RAT LIVER HOMOGENATE RAT LIVER MITOCHONDRIA REGENERATING RAT LIVER MlTOCHONDRlA

TRIGLYCERIDES

COMPLETE

-(Z(GP

-

-DPN

-CoA

-ATP

FIG. 2. The incorporation in vitro of palmitate-l-C14 into triglycerides of normal and regenerating rat liver preparations. The graph shows the incorporation in the absence, individually, of glucose, phosphate, (uGP, 346r++, DPN, Cob, and ATP. The components of the complete medium are given in the test.

the upt,ake in the phospholipids greatly exceeded that’ in the triglycerides (Table I). Regenerat’ing liver homogenates wwe more active than the normal homogenates in incorporating pnln~itnt~c-l-14C into 1riglycrri(les, phoPphxtidylinoaito1, and phosphatidylserine, but less :Ave in incorporating the radioactivity into phosI-‘ll:ltitlylcl-lolinc and plios~~l~atitlyletl~nr~olamir~c. Kormal l\It were a relatively poor sourw of enzyme for carrying out the mcorporation rea&ons being measured, but the Ylt, isolatrd from Icgerierating liver were c~oiiipnrativoly highly active in this respect, especially ill the case of phosphntidylserill(l, pIloti~~liatidylii~oi;itol, xd phosphatitlyletha~lolamine (Table I). When gluco~c was omitted from the mcdium, there was a decreased incorporat,ion of paln~itatc~-l-14C into the triglyccridcs of only

the regenerating liver Mt, and there was no change in it#s incorporation into the phosphatidylinositols in any of t,he enzyme preparations. Its incorporation irito phospl-Iatidyletharlolanline by either normal or rcgcnerating liver homogrnate or regenerating mitochondria was depressed, and there was a diniinislit~tl incorporation into phosphatitlylcholine when either normal homogenate or rcgeneratiiig mitochondrin were employed in the absence of glucose. Nso, there was less uptake in the pl~osphntidylscrincs wh(ln either liomogenatc or mkocliondria from rcgc~nernting liver:: ww: used (k?gs. 2-6). The omission of phospllatc from the medium did not lower the incorporation of radioactivity into t’he triglycerides in any of the cxpcriment’s, nor was a requirement for phosphate indicated in tjhc case of any of the

602

JOHNSON

AND LEDERKREMER

m IIII 0

RAT LIVER HOMOGENATE REGENERATING RAT LIVER RAT LIVER MlltXHONDRlA

HOMOGENATE

m

REGENERATING

MITOCHONDRIA

RAT LIVER

PHOSPHATIDYLETHANOLAMINE

PHOSPHATIDYLCHOLLNE

FIG. 3 (top). The incorporation in vitro of palmitate-1-W into phosphatidylethanolamine of normal and regenerating rat liver preparations. See Fig. 1 for explanation. FIG. 4 (bottom). The incorporation in vitro of palmitate-1-14C into phosphatidylcholine of normal and regenerating rat liver preparations. See Fig. 1 for explanation.

phospholipids. On the contrary, the data suggested, but did not prove, an actual stimulation of incorporation of palmitate-lCl4 in several experiments when phosphate was not present in the medium. Omitting CIGP resulted in a decreased incorporation of palmitate-lJ4C into all of the lipids when regenerating liver mitochondria were employed as enzyme. Palmitate incorporation into the lipids of either normal or regenerating homogenate was not affected by the absence of ~YGP from the medium, while incorporation into all four phospholipids of normal mitochondria seemed definitely enhanced by omitting it (Figs. 3-6). The uptake of radioactivity into the triglycerides was Mg++-dependent when either regenerating liver homogenate or normal Mt were used. However, there were marked reductions in incorporation of palmitate-l-I% into all the phospholipids of normal liver

homogenates and regenerating liver Mt when Mg++ was omitted from t#he medium. When the regenerating liver homogenate was employed, there was a reduced incorporation in the case of both phosphatidylcholines and phosphatidylethanolamines. When homogenates of either normal or regenerating livers were employed in the absence of DPN, there was a decreased incorporation of palmitat.e-lJ4C into triglycerides, and no change in that of any of the phospholipids. No evidence was seen for a DPN requirement for the incorporation of the acid into any of the glycerolipids by either type of mitochondria (Figs. 2-6). Omitting CoA from incubation media employing either normal rat liver homogenate or mitochondria resulted in a decreased uptake of radioactivity in the triglyceride fraction, and no change in that of any of the phospholipids. When regenerating liver

PALMITATE

150

INCORPORATION

RAT LIVER HOMOGENATE REGENERATING RAT LIVER RAT LNER MITOCHONDRIA REGENERATING RAT LIVER

ISIJ 13 UIIO

IA’ VITRO

INTO

LIVER

603

LIPIDS

HOMOGENATE MITOCHONDRIA

r

PHOSPHATIDYLI z E 0 g

150 125

E” 100 8 : s ;

75 50 25

COMPLETE

-GLUCOSE

-CoA

-DPN

-o( GP

-PHOSPHATE

-ATP

5 (lop). The incorporation in vitro of palmitate-1-W into phosphatidylserine and regenerating rat liver preparations. See Fig. 1 for explanation. FIG. 6 (bottom). The incorporation in vitro of palmitate-1-W into phosphatidylinositol of normal and regenerating rat liver preparations. See Fig. 1 for explanat,ion.

of

FIG.

normal

TABLE MEDIUM

REQUIREMENTS

A summary

Glucose Phosphat,e aGP hlg++ DPN CoA ATP

FOR THE INCORPORATION in Vitro OF PALMITIC ACID-~-W NORMAL AND REGENERATING RAT LIVER FRACTIONS

INTO LIPIDS

OF

of the data of Figs. 2-6.

SH”

RH

NM

RM

-b ---+ + + +

-

-

+ -

-++ +--++ +++

Phosphatidylinositol

Phosphatidylethanolamine

Triglycerides

Medium component

II

NR

RH

+--+ - - + + ---- -t+++

KM

RR1

NH

RH

NM

RM

++-+ - -+ + + -

+

---+ + + + ---- -++ + + +

A-H

RH

NMC

Phosphatidylserine RM

---+ +

---+ f ------+ + +

NH

RH

NMC

RM

-

+

-

+

+

+

---+ +-++

+

+

---+ ++++

a NH, Normal rat liver homogenate; RH, regenerating rat liver homogenate; mitochondria; RM, regenerating rat liver mitochondria. b + indicates a requirement for the compound; - indicates no requirement the compound. c Control values were so low the data are relatively meaningless.

NM,

normal

rat liver

was demonstrated

for

60-1

JOHNSON

AND

mitochondris were used, the omission of CoA resulted in marked reduct,ions in radioactivity uptake in all of the lipids. Adenosinc triphosphate was required for the incorporation of palmitate-l-14C into all of the lipids studied, when all four tissues were employed as enzyme. DISCUSSION

The experiments described here have concerned themselves with three aspects of lipid metabolism: (1) rcquircments for the incorporation ifa vitro of palmitatc-lJ4C into several different glycerolipids; (w) a comparison of mitochondria with whole homogcnate in the catalysis of Ohis reaction; and (5’) a comparison of mitotic phase regenerat,ing liver with normal liver. The data will bc discussed relative t.o t,hese aspects. lcor the sake of simplicity the data of Figs. 2-6 have been summarized in Table II t,o indicate the observed requirement, for each of the medium constituents. Glucose is not ordinarily a constituent of supported media in expcriment)s of the type reported here, and it, was therefore intercsting that it,s presence was necessary for maximum incorporation of t)he fatty acid into several of bhe glycerolipids. Eaton and Steinberg (10) reported that glucose in the medium stimulated glyceride synthesis from palmitate by skeletal muscle irk v&-o, and Vaughn rt al. (11) observed that glucose stimulated the incorporatlion of palmitatel-14C into glycerides by adipose tissue in vitro. Neither the experiments cited nor those reported here provide information relative to the role of glucose in the reactions. Added CoA was required for the ncylation of the triglycerides in all the t,iseucs employed but was not generally required, cxcept notably in regrnerat,ing liver filt. Presumably the greatly increased incorporation of palmit,atc-lJ4C in the phospholipids of regenerating Rlt was the basis for the Coil, requirement. It can be calculated from the data in Table I that approximately 10 times as much radioactivity is incorporated into the total lipids (mostly into phospholipids) of regenerat’ing RIt than of normal Mt. -4pparently there is sufficient CoA in whole tissue to permit optimum acylation of the phospholipids, but the amount of Co,4 in

I~l2DEI1KREME:I~

rcgenerntling liver Mt is not sufficient, to support the high metabolic activit)y in the prescncc of increased mitotic activity. Tzur cl al. (1”) co~~cludetl that- L-a-glyccrophospliat~e was t,he doniinutilig f:ttt!y :Ic*id acceptor in cslerifying systems c:tt,alyzcd by AIt and mi(wsonws of rat liwr. Thwc conclusions n-w’ bawd upon observnlions on mixed phospholil)ids and on glywritlo fractions. Whrn on(’ c~xaminw i~~tlivitlual phospholipids, however, t,his iti not always the c:iw. I:or exanipl(~, the 111 prcparaf ions from normal rat liver mlqnestion:ibly (10 ml wquirc cr-gl~ceropl1osphat,c~for t,lic illcorporal-ion of pxlniil:~tc-l-‘4C~ illto ;uIy of tlrc phospholipids (Figs. 4 and 5); OIL the contrary, the dat’n suggwt, but do not prove, that these reactions might even tw irillibitctl by this substance. To the, example just cited (wi b(> ntltletl dwc~riptions of other cases which, in the light of the present’ tlnta, demontitrntc~ the h;rznr(l in at tempting to draw nieariillgful conclusions from obecrvations matlc on Inixed phospholipids. l‘or exampl(a, the palmtatc-1-14C uptaltc iI1 the mixed phospholipitls in regencruting liver nb w\‘ns 335f 51 m~molcs/100 mg protein, but tllat in normal homogenates was mu& lcw, 157 f 39 ni~nioles/100 nig prot’cin. Wlicn individual phospholipids MC c~xaminctl, l~owcvcr, out observes that there is no diffcrcnw betn-ecll the two tissues in the incorporation of radionct’ivity int)o thy lecithins of thtw two tissues (‘3.5 f 23 :mtl 95 f 19 Il~pnlolcs,/100 gr11 protein, rwpectiwly). Similar anomalies might be pointed out, although not as st,rikmg, by comparing homogcnatw from rcgenerat~ing and normal livers. While nrt will incorporate f:r tty acid into glycerolipids (l&15), it is rwognizcd that in most tissues ihcy probably do not amount for a major portion of this act’ivity going on wit(hin the cell. This is corroborated in the present expcrinlentJs in t,he c’ase of normal liver Rlt, which are relatively inactive as a cat,alyst in the reactions measured. nlitochondria from regenerating liver, however, while relatively inactive in the incorporation of palmitate-lJ4C with triglycerides, arc :a11 important site of its incorporation into the phospholipids (Table I). For example, it c’an be calculated from t#he data in Table I, con-

PALMITATE

IA’ VITRO

INCORPOKATIO~

sidering 14.3 mg of regenerating liver Rlt protein per gram of liver and 53 mg of homogenate protein per gram of regenerat’ing liver, that, t,he incorporation of palmit)ate into the phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine by I\lt of regenerating liver is 27, 190,39, and 5S % of that, in corresponding regenerating homogenate phospholipids, respect#ively. A generalization that can be drawn after examining Table II is t,hat a multiplicity of pathways is operative in the incorporation of palmitate-1-14C into a glycerolipid, depending on the chemical nature of the glycerolipid, the nature of the enzyme source (whole homogenate vs. Mt), and the physiological state of the tissue (normal liver vs. mitotic phase regenerating liver). This may have been predicted, also, from the data of Merkl and Lands (16) and Lands and Hart (17). While their observations are made on acyl CoA-phospholipid acyltransferases, whose catalytic activity undoubtedly account for some of the differences observed here, ot,her pathways of formation of the glycerolipid have been described which must be considered as possible explanations of the differences observed in the present experiment,s ( 1S-20). ACKNOWLEDGMENT The technical assistance gratefully acknowledged.

of Elvira

de Castro

is

REFERE?JCES It. >I., AND KERUR, Biophys. Ada 70, 152 (1963).

1. JOHXSON,

L.,

Biochem.

ISTO

LIVER

LIPIDS

GO.5

1:. M., .QGD ALBEIW, S., ll~ch. Ijiothem. Biophqs. 36, 340 (1952). 3. ITO, T., xw JOHSSON, 11. M., J. Viol. Chew

2. JOHNSON,

239, 3201 (1964). 4. GORN.tLL, 8. C;.,

BAKU.\WLL, c. J., .1x1) L)nwD, M. M., J. Biol. Chem. 177,751 (1949). 5. LEDERICREMER, J. ?\I., AND JOHNSOS, 1I.. iXl.,

J. Lipid Res. 6, 5i2 (1965). 6. II,HODE~, I). K., .khw LE.1, C. II., Hiochen~. J. 65, 526 (195ij. D. J., DITTMEK, J. C., .\ND WAHAS7. IIANAHAN, HINA, H. J., J. Biol. Chem. 228, 685 (1957). Ii. F., SANDERS, 8. SICIPSICI, 1:. P., PETERSON, J., .\ND B.\RcL.~Y, M., J. J,ipid Res. 4, 227 (1963). W. D., AND ENTENBI.~N, C., J. 9. SKIDMORE, Lipid Kes. 3, 471 (1962). 10. EATON, P., AND STEINBERG, D., J. Lipid Res. 2, 376 (1961). 11. VAUGHN, &I., STEINBERG, D., AND PITTMAN, It., Biochim. Biophys. Acta 89, 154 (1964). 12. TZUR, K., TAL, E., AND SHAPIRO, B., Biochiv~. Biophys. L4cta 89, 18 (196-l). 13. STEIN, Y., AND SHAPIRO, B., Am. J. Physiol. 196, 1238 (1959). 14. STEIN, Y., AND SHAPIRO, ES., J. Lipid Res. 1, 326 (1960). 15. STEIN, Y., TIETZ, A., AND SHAPIRO, U., Biochim. Biophys. Acla 26, 286 (1957). 16. MERIW, I., AND LANDS, W. E. M., J. Biol. Chem. 238, 905 (1963). 17. LANDS, W. E. M., ANI) HART, l’., J. Biol. Chem. 240, 1905 (1965). R. A., AND HOKIN, L. E., J. Biol. 18. PIERINGER, Chem. 237, 653 (1962). S., J. Biol. 19. WILSOX, J. I)., AND CDEXFHIEND, Chem. 236, 673 (1961). K. J., J. 20. SENIOR, J. Ii., AND IssELB2Yc~ER, Biol. Chem. 237, 1454 (1902).