THE INCORPORATIONOF PLASMAFREE FATTY ACIDS INTOPLASMATRIGLYCERIDES IN MAN*

By S. J. FRIEDBERG, R. F. KLEIN, D. L. TROUT, M. D. BOGDONOFFANDE. H. ESTES, JR.

(From the Central Reference Laboratory and Radioisotopes Unit, Veterans Administration Hos-pital, and Department of Medicine, Duke University Medical Center,

Durham, N. C.)

(Submitted for publication January 26, 1961; accepted June 8, 1961)

Plasma triglycerides (neutral fat) may be di-vided into 1) that portion which is present duringthe fasting state and 2) that which, after ingestionof fat, is added to and temporarily augments thefasting level of triglycerides.

The present investigation deals with the rela-tionship of plasma free fatty acids (plasma FFA)to fasting plasma triglycerides (plasma TG) inman. From these observations a quantitative as-sessment of the amount of plasma FFA convertedto plasma TG will be presented. The data alsopermit calculations which suggest that plasmaFFA may be the major source of fasting plasmaTG.

METHODS

Two types of studies were carried out in male patientswithout known abnormalities of fat metabolism.

1. Non-reinjection experiments. After an overnightfast of 15 hours, 9 subjects were given 0.004 to 0.008 mcalbumin-bound palmitic acid-i-C1' intravenously eitherby single injection or by 2-hour constant infusion. Twoindividuals received 0.00585 mc of albumin-bound oleicacid-i-C1' and another 0.00597 mc of stearic acid-1-C14.During the day of the study the subjects were permittedno breakfast, a nonfat lunch, and an unrestricted supper.Venous blood samples were drawn at appropriate in-tervals over a period of 24 hours after administrationof fatty acid C1' for determination of neutral fat activity.Two-ml aliquots of plasma were extracted by the methodof Dole (1). Eight ml of the upper phase, representing1.85 ml of plasma, was transferred to a separate tube.Ten ml of 0.1 N NaOHin 50 per cent ethanol was added,according to the method of Borgstrbm (2), in order to

* This investigation was supported in part by a grantfrom the Life Insurance Medical Research Fund; by aresearch grant H-4807; a training grant, HTS-5369,from the National Heart Institute, Public Health Service;a research grant, A-4535-MET, from the National In-stitutes of Health, Public Health Service; a researchgrant for July 1960-61, from the North Carolina HeartAssociation; and by the Regional Center for the Studyof Aging, Duke University.

remove fatty acids from the upper phase. The upperphase and its contents were then transferred quantitativelyto liquid scintillator vials, dried with a stream of warmair from a hair dryer, and redissolved in 5 ml of phosphorsolution for counting in a Packard Tri-Carb liquid scin-tillation spectrometer. Along with plasma glycerides,the extracts contained cholesterol, cholesterol esters anda small amount of phospholipid. These would also beexpected to become labeled; therefore, it was necessaryto determine the amount of activity in the various plasmalipids present in the extracts at various times after in-jection of C4-labeled fatty acids. This was done bymeans of silicic acid chromatography using the methodof Hirsch and Ahrens (3) without modification. Plasmapalmitic acid-l-C1 activity was measured as previouslydescribed (4).

The experimental plan required a method which wouldpermit easy and reproducible determinations on a largenumber of samples. The method selected for measuringplasma TG activity described in the preceding para-graph is based on the facts that: 1) the label in the ex-tracts appeared almost exclusively in TG in the earlyhours of the procedure, and 2) negligible activity waspresent in cholesterol, cholesterol esters and phospho-lipid until at least 4 hours had elapsed. Furthermore,although significant activity appeared in phospholipids,only a small fraction of this phospholipid activity waspresent in the upper phase extracts, and activity in freecholesterol was minimal. The data justifying themethod appear below.

Total plasma triglycerides were determined by themethod of Van Handel and Zilversmit (5), plasma FFAby the method of Dole (1) and esters of carboxylic acidsby the method of Rapport and Alonzo (6). Free cho-lesterol was precipitated as the digitonide and redissolvedin methanol for liquid scintillation counting. Samplesof expired air were analyzed for C1402 activity by trap-ping in Hyamine hydroxide according to the method ofFredrickson and Ono (7).

2. Reinjection experiments. In order to establish withgreater certainty the validity of using the rate of disap-pearance of TG label observed during non-reinjection ex-periments to measure fasting plasma TG turnover rate,a patient, terminally ill with degenerative cerebellar dis-ease, was given 30 /Ac palmitic acid-i-C1' intravenously.Blood samples were drawn at intervals for determinationof triglyceride activity. One hour after injection of the

1846

INCORPORATIONOF PLASMAFREE FATTY ACIDS INTO PLASMATRIGLYCERIDES

aEL)

0

CL)

EE

0. 20

0

ELoI0

0c

E

0

E

cL

0

E0.

a-

at.

c ) 3 6 9 12 15 18 21 24Time in Hours

Time in Hours

0 3 6 9 12 15 18 21 24Time in Hours

FIG. 1. PLASMA TRIGLYCERIDE ACTIVITY AFTER INTRA-VENOUS INJECTION OF C -PALMITATE. Triglyceride ac-

tivity represented by ascending limb and initial straightdownslope only (see text).

radiopalmitate, 400 ml of blood was withdrawn andanalyzed for TG and FFA activity. The blood was col-lected in a standard evacuated blood donor set con-

taining acid citrate dextrose solution as anticoagulantand stored at 4° C. There is no evidence that this pro-

cedure does or does not denature or alter lipoproteins.A known quantity of plasma obtained from this bloodwas reinjected into another subject several days later.

This study was repeated using another subject whoserved both as donor and as recipient. Plasma neutralfat activity was determined in both donors and recipientsas described in the preceding section.

RESULTS

I. Non-reinjection experimentsA. Triglyceride activity.' The results in nine

normal persons are presented in graphs in whichthe logarithm of activity is plotted against time(Figure 1). After rapid intravenous administra-tion of albumin-bound palmitic acid-1-C14, therewas a delay of about 10 to 15 minutes before ac-

tivity began to appear. After the initial delay justdescribed, activity rose rapidly and reached a peakin 2 to 4 hours, a time when FFA activity was verysmall. In the constant infusion experiments, a

longer time was required to reach peak activity;otherwise these were not different from the singleinjection experiments (see Table I). (The pur-pose of the constant infusion studies was to sim-plify the kinetic problem by attempting to producea situation in which the specific activity of TG in-flux into plasma was constant. This was laterfound to be both unnecessary and impractical.)Thereafter activity declined relatively rapidly atfirst and then more slowly over the 24 hour pe-riod of observation. Initially there was a straightline exponential decline after attainment of peakplasma activity. There was some uncertaintyabout this in two studies (Figure 1C). In allcases there was a departure from linearity begin-ning, at the earliest, 6 hours after injection ofpalmitic acid-i-C14. Figure 9 shows activity inTG 1 and 2 hours after injection of FFA. At 1

30125(

E20(

v 15(10

51

30125

E 20

c 15105

o- TG.0-0 1 Subject may

i 2 hours after I V palmitic0 ocid-I-C'4

CE. FFAO G.

O ~~~Subject macO / hour after I V pa1irnitc

acid-I-C"4O0 LOWER

GLYCERIDEio ~~~FFA0 20 40 60 80 100 120

Tube Number140 160

METHANOL

FIG. 2. SILICIC ACID CHROMATOGRAMOF PLASMA, 1 AND2 HOURSAFTER INJECTION OF C1-PALMITATE BY GRADIENTELUTION METHOD.

1 The curves described are a measure of total activityin plasma extracts.

1847

Subject ACAR

SubjectHIC

t

tI

200-

too-80-60-

40-

FRIEDBERG, KLEIN, TROUT, BOGDONOFFANDESTES

TABLE I

Observed and calculated plasma triglyceride fatty acid contents and data used to obtain calculatedlevels in 9 subjects given palmitic acid-i-C'4 *

1 2 3 4 5 6 7 8 9 10 11 12 13

Fx K Fraction aF

Calcu- Plasma Fractional of FA Calcu-lated TG disappear- label lated Calcu-

Plasma plasma activity y z Average ance rate appear- plasma FFA latedTGFA TGFA Devia- at time At time At time FFA FFA of plasma ing in TG adminis- plasma

Subject content content tion L tl l conc. flux TG per hr plasma influx tered volume

mmoles/total cpm/1.85 ml plasma mmoles/L mmoles/hr mmoles/hr cPm mlplasma Vol

CARt 11.8 5.9 -50 145 93.0 238.0 670 27.6 0.217 0.046 0.425 6.7 X106 2,290THOt 6.5 5.9 - 9 94 118.5 212.5 700 29.0 0.232 0.047 0.458 5.86 X106 2,300BENt 12.0 9.8 -19 70 110.5 180.5 450 26.1 0.123 0.046 0.400 6.12 X106 2,900NUNt 30.1 20.5 -35 45 63.5 108.5 600 50.3 0.115 0.059 0.783 5.51 X106 4.670CLEt 15.3 24.0 +50 130 91.9 221.9 700 34.3 0.165 0.093 1.067 4.84 X106 3,270HIC 9.9 10.4 + 4.5 77 138.6 215.6 650 30.3 0.201 0.069 0.695 4.62 X106 2,950SHUt 15.5 13.1 -14 90 164.0 254.0 500 26.2 0.124 0.069 0.542 6.78 X106 2,910CORt 17.0 18.6 + 9 106 49.2 155.2 330 26.9 0.078 0.078 0.627 4.32 X106 3,400PET* 4.7 5.0 + 7 39 104.5 143.5 1,000 46.6 0.347 0.042 0.646 5.13 X106 2,330

* For explanation of x, y and z, see text. Columns 1 and 2 give measured and calculated triglyceride concentrations; columns 4-13 give dataused to obtain calculated levels in column 2 by methods described in the text.

t Constant infusion studies.$ Single injection studies.

hour no activity was detected in cholesterol es-

ters, and at 2 hours 1.5 per cent of the total ac-

tivity was found in this fraction.B. CO, activity. Figure 3 illustrates the ap-

pearance of CO., activity in expired air in relationto palmitic acid and TG activity during a con-

stant infusion experiment and also illustrates thefact that peak expired CO2 labeling occurs beforeplasma TG peak activity.

C. Phospholipids. Table II shows the amountof activity in phospholipids at various intervalsafter the beginning of the studies. These deter-minations were performed on separate samples ofplasma by silicic acid chromatography using

Duration SUBJECT CARof PA-C"4Infusion

200- Triglyceride Activity per

\/.85 m/. Plasm a

I0-

a.~ f

2 3 4 5 6 7 8TIME IN HOURS

FIG. 3. APPEARANCEOF CO2 ACTIVITY IN EXPIRED AIR

IN RELATION TO PLASMAFREE FATTY ACID AND TRIGLYCER-

IDE ACTIVITY.

methanol: chloroform (1: 2, vol/vol) for extrac-tion of plasma.

D. Cholesterol esters. Figure 4, a representa-tive example performed as a separate study, showsthe appearance of label in cholesterol esters. Ac-tivity is initially small, but in 24 hours is re-sponsible for a significant portion of the activityof the extracts. This can be ascertained by com-paring the amount of cholesterol ester activity inFigure 4 with the residual total activity in Fig-ure 1, A and B.

E. Free cholesterol. Activity in free cholesterolconstituted only a fraction of background activity.

TABLE II

Appearance of phospholipid activity in plasma after injectionof palmitic acid-i-C'4

Time afterpalmitic

acid-1-C'4 Radio-Subject administration activity

hrs cpm per 1.85ml plasma

CAR 1 20.42.5 395.5 41

THO 1 193 205.5 26

22.5 40

CLE 1 53 175.5 60

1848

INCORPORATIONOF PLASMA FREE FATTY ACIDS INTO PLASMATRIGLYCERIDES

E 30-en0

a.E 20-E

LO

- 10

E

Cl)

E

iL

0~C-)

0 2 4 6 8 10 12 14 16 18202224Hours

FIG. 4. APPEARANCEOF CHOLESTEROLESTER ACTIVITYAFTER INTRAVENOUSPALMITIC ACID-1-C4.

F. Oleic acid. The early part of the curves ob-tained (Figure 5 is a representative example) re-

sembled the curves seen in the palmitic acid stud-ies. The fractional turnover rates for the earlyTG portions of the curves were 0.225 and 0.314per hour, respectively. Later the form of thecurve was different. Duplicate determinations on

separate samples of plasma revealed identical re-

sults. It is also quite clear from inspection thatthe amount of oleate label converted to TGFAlabel and the rate of disappearance are in the same

range as these values for palmitate. Silicic acidchromatography of plasma obtained at 9 hoursrevealed that the difference in appearance be-tween this curve and the palmitic acid curve isaccounted for by a greater amount of activity incholesterol esters. This is consistent with theknown fact that cholesterol esters contain greateramounts of unsaturated acids (8).

G. Stearic acid. The form of the stearic acidcurve resembled those for palmitic acid. The de-cline of radioactivity after attainment of peak ac-

0 3 6 9 12 15 18 21 24TIME IN HOURS

FIG. 5. PLASMA TRIGLYCERIDE ACTIVITY AFTER INJEC-TION OF C"4-OLEATE. Triglyceride activity represented byascending limb and initial straight downslope only (seetext) .

tivity was a .straight line on the semilogarithmicplot and the fractional turnover rate was 0.146 perhour, a value which fell within the range of valuesobtained for palmitic acid. The curve was dif-ferent in that maximal activity was only 51 cpmper 1.85 ml plasma, a value which was consider-ably lower than any obtained for palmitic acid(see Figure 1).

II. Reinjection experimentsThe results of the two reinjection studies are

summarized in Table III. In the first study, re-

sults indicated that the volume of distribution ofthe reinjected triglycerides was 25 per cent greaterthan the calculated plasma volume based on 40ml plasma per kg body weight. The rate of disap-pearance of TG activity in the recipient was very

BLE III

Plasma triglyceride fractional disappearance rates in reinjection studies and related data

KFractional Actual

disappearance Estimated Expected extrapolatedrate of plasma No. of cpm plasma activity at activity atTG per hour injected volume* zero time zero time

Expt. 1:Donor 0.422Recipient 0.402 26,300 3,000 43.8 cpmi per 35 cpm per

5 ml plasma 5 ml plasmaExpt. 2:DonorRecipient 0.282 17,050 2,200 77.5 cpm per 59 cpm per

10 ml plasma 10 ml plasma* Estimated plasma -volume based on 40 ml per kg body weight.

1849

FRIEDBERG, KLEIN, TROUT, BOGDONOFFAND ESTES

800'600'

400'

200

E 100'E 800 60'

40'

20'

-15 0 15 30 45 60 75 90 105 120Time in Minutes

FIG. 6. PLASMA TRIGLYCERIDE ACTIVITY IN REINJECTIONSTUDY.

close to the initial rate of TG disappearance afterattainment of peak activity in the donor (Figure6), but in both donor and recipient was more

rapid than in the non-reinjection studies. Whenthe experiment was repeated using the same indi-vidual as donor and recipient, only the rate ofdecay of the reinjected material could be observed,and confirmed the previous findings that the vol-ume of distribution corresponds rather closely (25per cent greater) to the plasma volume and thatthe rate of disappearance was of the same orderof magnitude observed in the non-reinjection ex-

periments. Uptake of labeled TG during the pe-riod of infusion and possible equilibration withtissue TG are factors which may have been re-

sponsible for an overestimation of the size of theTG pool. In our first reinjection experiment thefractional disappearance rate was 0.402 per hour,a rate which was close to but higher than thosefound in our non-reinjection studies. However,in the second reinjection experiment, the frac-tional disappearance rate was 0.282 per hour, a

value which fell within the range of the non-re-

injection studies.

INTERPRETATION

I. Background

The studies in man of Fredrickson and Gordon(9) demonstrated that a portion of intravenouslyadministered C14-labeled fatty acids is later foundin plasma as labeled neutral fat. There is goodevidence that this conversion is effected in liver.

Thus Stein and Shapiro (10), and Laurell (11)found that this organ removed intravenously ad-ministered radiopalmitate and that activity wasfound in liver triglycerides and then in plasmatriglycerides. In the experiments of Laurell, thespecific activity of liver and plasma triglyceridesappeared to be the same so that these findingswere thought to be compatible with the conceptthat fasting plasma triglycerides might be entirelyderived from liver triglycerides. On the otherhand, Stein and Shapiro believed that liver tri-glycerides mix with a larger triglyceride poolfrom depots (10).

II. Discussion of results

A. Reinjection experiments. The observationsin man made in this study differ from those in therat in at least two obvious particulars. First, therate of disappearance in the non-reinjection stud-ies of Laurell is more rapid than that in man.Second, the rate of disappearance in the rat ofreinjected fasting triglycerides is much morerapid than in the non-reinjection experiments(11). In our own studies this difference waseither not observed or remained in doubt (seeFigure 6 and compare K values in Tables II andIII). However, unpublished observations in thislaboratory in dogs resemble those made in the ratby Laurell. From our own reinjection studies weconclude that the rate of disappearance resembleda simple first order decay and that the volume ofdistribution of the reinjected TG approximatedthe calculated plasma volume.

B. Non-reinjection experiments. The activityin an extract containing triglycerides, cholesterolesters, free cholesterol and some phospholipids isconsidered, on the basis of silic acid chromato-graphic studies, to represent triglyceride activityalone during the ascending portion of the curveand during the initial descending portion of thecurve which forms a straight line on the semilog-arithmic plot. Furthermore, the straight loga-rithmic downslope is considered to be representa-tive of the disappearance of a single substance.This would be valid except in the unusual circum-stance in which two or more different substanceshappened to be disappearing at the same fractionalrate. The departure from linearity which occursseveral hours after injection of labeled fatty acid

SubjectfiCtK=422/hr.

rGac, ,,~"Duration of 5 mi piPOftinfusion ofreadministered K- .402/hr.n~l n---

1850

=I--

INCORPORATIONOF PLASMAFREE FATTY ACIDS INTO PLASMATRIGLYCERIDES

can be explained either by recirculation of la-beled triglyceride or by the appearance of anotherlabeled material. The results, in fact, demonstratethe late appearance of activity augmenting theresidual TG activity, both in cholesterol estersand in phospholipids.

III. Current hypothesis

The purpose of the studies on the relationshipof C14-labeled fatty acids to plasma C14-labeledtriglycerides was to evaluate the quantitative sig-nificance of this conversion in man.

As stated previously, it is believed, from thedata of Laurell (11), Stein and Shapiro (10), andHillyard, Cornelius and Chaikoff (12), that fattyacid is converted to triglyceride in the liver andthat plasma TG may be in equilibrium with liverTG (11). However, in the present investigation,since only plasma levels were measured, it will benecessary to limit the interpretation arbitrarily toevents occurring in the plasma pool even thoughthis pool might be only a fraction of a larger TGpool with which TG equilibrates rapidly. Thefractional rate of disappearance of TG-C14 cal-culated from the TG activity curves is interpretedto represent the rate of disappearance of fastingplasma triglycerides in general. It should bestated in this regard that TGmolecules in blood arecomposed of a mixture of fatty acids (13) and arebelieved to be removed intact by adipose tissue invitro (14). Another point in support of the hy-pothesis that TG is removed intact is the observa-tion in this study that both oleic and palmitic acids,the two most abundant compounds in this class, be-haved in the same way and fell into the samerange of disappearance rates from plasma. It isalso known that lower glycerides do not normallyappear in plasma (3). Since TG molecules arecomposed of a mixture of fatty acids, one wouldanticipate that lower glycerides would be presentin plasma if the rate of disappearance were morerapid for one species of fatty acid than for another.Futhermore, it is assumed that the calculated frac-tional rate is also the rate of disappearance for TGthroughout the course of the study, especially dur-ing the time of increasing triglyceride activity.

Another important assumption, relating to themethod of interpretation which follows, involvesthe concept that once peak plasma triglyceride ac-

tivity has been reached and the straight exponen-tial downward decay begins, no further net addi-tion of TG label into the TG pool takes place.The word "net" is emphasized since a straightline could be observed if plasma TG equilibratesrapidly with other TG pools, i.e., if plasma TGwere part of a larger TGpool from which TG dis-appearance proceeded at a uniform rate. Thequestion that this investigation attempts to answerdoes not concern the total magnitude of conversionof FFA to TGFA, but whether enough FFA be-comes TGFA to account for the observed TGconcentration in plasma. The concept of "no fur-ther net addition" is justified by the followingarguments. First, further net entry of label wouldalter linearity. Second, a model which fits the ob-served data was based on the observed rate ofTG decay and a rate of entry into plasma also ap-proximated from the data. The following equa-tion was derived:

X = k2z- (e-klt - e-k2t)k2-kiwhere x is plasma TG activity, ki is the rate ofdisappearance from the pool emptying intoplasma, k2 is the plasma TG fractional disap-pearance rate, and zo is the amount of TG labelreleased or equilibrating into plasma (15).Plotting this equation with values approximatingthose obtained experimentally gives a curve re-sembling the experimental curves. This equa-tion, furthermore, is based on the assumptionthat release of TG label into plasma is exponen-tial with time, an assumption which is consistentwith the form of the increasing limb of the tri-glyceride activity curves. Third, inspection ofthe curves indicates that label equilibrates veryrapidly at first and that this rapid rate of entryslows promptly and, therefore, the net influx oflabel would be expected to become rapidly in-significant and approach zero. The consequenceof this would be that only the rate of disappear-ance of label would be manifest and that thispicture would be uncomplicated by further addi-tion of labeled material.

In addition to the foregoing, two other factsare pertinent to the discussion. First, plasmaFFA is composed of about 50 per cent oleic acid,30 per cent palmitic acid, 8 per cent stearic acidand several per cent of various other fatty acids

1851

FRIEDBERG, KLEIN, TROUT, BOGDONOFFAND ESTES

LOGx

(i,x )

TI ME

FIG. 7. DIAGRAMMATICREPRESENTATIONOF DATA ILLUS-

TRATING METHODOF CALCULATION. Explanation in text.

(16). Second, the fatty acid composition of tri-glycerides in plasma is very similar approxi-mately 40 per cent oleic, 30 per cent palmitic, 10per cent linoleic, 5 per cent stearic, 5 per centpalmitoleic and varying smaller amounts of otherfatty acids (8).

The presentation which follows is not intendedto represent an accurate description of the kinet-ics of the individual studies but serves to indicatein a general way the magnitude of the conversionof FFA to plasma TGand to suggest that plasmaFFA are the major source of plasma TG. Thefractional disappearance rate and volume ofdistribution of plasma FFA, the plasma volume,and the values derived from these quantitieswere not experimentally determined but are

representative of known normal values and may

be the source of considerable error in individualstudies. The interpretation is presented below.

In order to take into account the likely situa-tion that plasma TG may be in part of a largerTG pool probably including liver TG, it is nec-

essary to interpret the data in a manner which isindependent of pool size. The following modelis pictured. FFA label is converted to TGFAlabel which then equilibrates rapidly with allparts of the TG pool including plasma. Thispool may be the same size or larger than theplasma pool. Disappearance then proceeds ata uniform rate from the entire pool.

In Figure 7, the logarithm of plasma TGactiv-ity, x, is plotted against time, t. Let z = totalTGFAactivity (TGFA*) which has equilibratedwith the plasma compartment, i.e., the totalamount of administered fatty acid label (FA*),which appeared in plasma as TGFA*. Let y =

amount of TGFA* which has left plasma fromtime t = 0 to t = ti; t1 is any point in time on thestraight downlimb of the semilogarithmic plot.Let M= amount of FA label administered. LetF = fraction of FA label converted to plasmaTGFA*. F equals z AM. Let a = endogenousFFA turnover rate or flux. Let k = fractionaldisappearance rate of plasma TG. Then at anytime t, on the straight downlimb of the semi-logarithmic plot, z = x - y; dy, the amount ofTGFA* which has left plasma during an intervalof time, dt, = kx dt, and the total amount ofTFGA*, y, which has left the

rtiplasma from t = 0 to t = t = f kxdt

tj~~~~~t

and z = x - k x dt.

to~~~~~~t

But F = z/Ml1 = x- k x dt]/M.

The amount of TGFA formed per hour whichenters plasma equals the fraction of FA labelconverted to plasma TGFA* times the endogen-ous FFA turnover rate. This equals Fa, which

equals a/M x - k x dt]

If TGFAenters plasma at the rate of Fa milli-inoles per hour and leaves at the fractional disap-pearance rate k, the anticipated plasma TGFAcontent which will be reached can be expressedby Fa/k (1 -ekt) (17).

When I= x, plasma TFGA content will

equal Eu,/k; or, substituting Fa = a/M [x - k

X x dt], plasma TGFA content = a/u Mk

X [x-k Ixdt] = a/AI [x/k- b xdt]

The integral x dt is the area under theto

shaded portion of the curve. This area wasdetermined in these studies by the trapezoidrule. The area depicted in the diagram is, ofcourse, not correct since it is drawn under semi-logarithmic plot. The correct area would berepresented by the equivalent area drawn underan arithmetic plot. In practice the semilog-arithmic diagram was used to facilitate the

1852

INCORPORATIONOF PLASMAFREE FATTY ACIDS INTO PLASMATRIGLYCERIDES

measurement of k and to insure that xi is mneas-ured on the straight semilogarithmic downslope.

Note that when ti = oc, x = 0 and plasmart

TGFA = a/ M J x dt. The expression 1/'Mrti

x x dt equals the area under the TGFA

curve from t = 0 to t = c divided by the totalamount of FA label administered.2

In calculating the data with the equations pre-sented, the following factors were taken into ac-count: 1) Plasma volumes were calculated on thebasis of 40 ml plasma per kg body weight (18).2) It was assumed that the fraction of palmiticacid-i-C14 converted to TGFA* is representative

2 Since the writing of this paper an alternative method ofanalyzing the data has been proposed. The derivationwhich follows is more general in nature, and under certaincircumstances requires no knowledge concerning the rate ofdisappearance of TGFA.

Let w = time of introduction of labeled FA. Let M=total dose of labeled FA. Let f (t -) = fraction of agiven quantity of FFA which enters plasma at time andwhich is to be found with plasma TGFApool at time t (asteady state is assumed). Let m (w) = plasma FFA flux(rate of entry of FFA into plasma). Then, the quantity ofFFA which entered plasma during time interval dw ism(w)dco.

At time t from the time this quantity, m(co)dco, entered,the quantityf(t -w)m(co)dw would be found in the plasmaTGFApool, or adding up for all previous time intervals weobtain:

total plasma TGFA = ff(t -)m(w)dw

The substitution Q = t- co transforms this into theequivalent expression:

total plasma TGFA = f m(t - ) f(Q)dQ

If the rate of entry of FFA into the plasma is constant, saym(w) = a, then the quantity of TGFA present at time twill be:

total TGFA a f'f(Q)dQThe integral f(Q)dQ is the total area under the plasma

TGFA curve divided by the total initial dose of labeledfatty acid. It can be seen immediately that the equation

TGFA = a f f(0)d0 is identical with the equation pre-

sented in the main text when t = oo.The alternative equation for practical application re-

quires extrapolation of the straight semilogarithmic down-limb of the TGFA* curves to infinity in order to measurethe required area.

of fatty acids as a whole. Whereas this is notstrictly true it is a very reasonable approximation,since the fatty acid composition of plasma FFAand plasma TGFA is almost identical (8, 16) andsince oleic and palmitic acids behaved similarly.3) Endogenous hourly FFA turnover rate, a, wasestimated from the average plasma FFA level dur-ing the period of TGFA* formation by using afractional disappearance rate of 0.3 per minute(9). The plasma volume was taken as the vol-ume of distribution of FFA, although the lattervolume may be somewhat greater than the former(4, 9). 4) Extraction efficiency of 87 per centfor FFA and 95 per cent for TG was taken intoaccount.

The data and method of calculations outlinedabove were used to estimate an anticipated plasmatriglyceride content for each subject based on arate of TG formation calculated from the isotopedata. The calculated levels would represent aminimal estimate if recirculation of TGFAoccurs.These anticipated or theoretically expected levelsderived from the tracer data were compared withthe actual TGFA plasma contents in each sub-ject. The actual TG contents with which thecalculated levels were compared were the meansof several levels obtained during the period offormation of labeled TGFA. During this timethere was little fluctuation in TG concentration.The data and calculations appear in Table I.Plasma TG content was calculated from the de-terminations by -using a mol wt of 900. It can beseen (columns 1, 2 and 3) that the calculated ap-proximations range from 50 per cent less to 50per cent more than the measured levels and thatsix fall within 19 per cent of the measured levels.The same results can be obtained in a somewhatdifferent manner: the plasma TGFA efflux isthe product of k and- TGFA content and thisefflux can be compared with aF, the calculatedrate of entry of plasma TGFA. Under these cir-cumstances the results are unaltered. Since cal-culated TGFA flux into plasma approximates en-dogenous TGFA flux out of plasma, it would ap-pear from these calculations that all three fattyacids of triglyceride turn over and that the ma-jority of plasma TG originated from FFA. Thatthe major fatty acids of TG turn over at the samerate is strongly suggested not only from a com-parison of the results with palmitic acid-i-C'4

1853

FRIEDBERG, KLEIN, TROUT, BOGDONOFFAND ESTES

and oleic acid-1-C14, but also from data of Rodbellshowing that TGis removed intact by adipose tis-sue in vitro (14).

A point which should be emphasized is that theresults, suggesting that plasma FFA conversionto plasma TGFAis sufficient to account for plasmaTG concentration, would be the same if thesecalculations were based on a unit volume of plasma;i.e., the interpretation is independent of pool size.Furthermore, the value obtained for the fractionof radiopalmitate converted to plasma TGFAap-plies only to the initial equilibration of label intoplasma. The actual fraction of FFA convertedto TGFAmight be considerably in excess of thisamount if one considers, as Laurell claims (11),that plasma TG specific activity might well bethe same as that in liver and that liver TG prob-ably greatly exceeds plasma TG.

DISCUSSION AND CONCLUSIONS

An important concomitant of the present inter-pretation is the concept that one of the factors re-lating directly to the plasma TG level is the mag-nitude of plasma FFA turnover rate. This ideafinds support in observations relating plasma FFAconcentration to plasma TG concentration. Forexample, Havel (19) found that administration ofglucose, which causes a fall in plasma FFA, alsocauses a fall in plasma TG concentration. Duryand Treadwell (20) showed that epinephrinewhich raises plasma FFA concentration also raisesplasma TGconcentration. Both diabetes and star-vation produce a rise in plasma FFA and plasmaTG.

Another point which should be made is that thepresent study does not distinguish actual triglycer-ide formation from a process which exchanges oneTGFA for another from FFA. There is, how-ever, suggestive evidence from the studies of othersthat FFA may not be released from the liver. Thelarge positive FFA arteriovenous difference ofthe blood perfusing liver (21) suggests that suchan exchange may not be the only mechanism con-cerned. Stein and Shapiro were unable to findsignificant amounts of FFA label after uptake ofC14-triglycerides by rat liver (22). This lastpoint is, however, difficult to evaluate becauseliver FFA is rapidly diluted by a large FFA fluxfrom plasma. It has also been shown that plasma

triglycerides are removed by adipose tissue in vitro(14, 23), and that only a fraction of administered,8-lipoprotein triglyceride is taken up by liver (22).If plasma triglyceride undergoes a fate other thanrecirculation to liver and if, as has been shown inthese isotopic studies, the majority, and perhapsall plasma TG originates from liver, it would beunlikely that incorporation of FFA into TGFAwould be entirely an exchange phenomenon.However, proof of this contention is not possibleat present.

It is also necessary to point out evidence con-cerning the importance of the liver in the incor-poration of FFA into TGFAand the lack of par-ticipation of extrahepatic tissues in contributingto the fasting triglyceride via an FFA precursor.Such evidence is forthcoming not only from ani-mal studies already mentioned but also from therecent observation by Havel and Goldfien (24)that hepatectomy in dogs abolished the conversionof FFA to TGFA.

It is concluded from the data presented andfrom the interpretation of these data that plasmaFFA is a major source of fasting plasma tri-glyceride. The calculations, furthermore, stronglysuggest that FFA is involved in a process in whichall three fatty acids of triglyceride turn over andthat FFA is therefore the preponderant source offasting TGFA.

SUMMARY

Observations have been made on the incorpora-tion of injected C14-labeled fatty acids into tri-glycerides, cholesterol esters, free cholesterol andphospholipids.

An estimate was made of the quantitative as-pect of the formation of triglyceride label. Theresults of this estimate are consistent with thehypothesis that a major fraction of fasting plasmatriglyceride is derived from plasma fatty acids.

ACKNOWLEDGMENTS

The technical assistance of Mrs. Anna B. Ferguson,Miss Helen L. Hilderman, Mrs. Gray D. Long, Mrs. SueH. Peters, Miss Corinna Thomas, Mrs. Gitta W. Jacksonand Mr. Albert Eaton is gratefully acknowledged. Inaddition, we would like to thank Dr. Thomas M. Gallie,Jr., Department of Mathematics, Duke University, forhis invaluable assistance in reviewing the mathematicalproblems discussed.

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INCORPORATIONOF PLASMAFREE FATTY ACIDS INTO PLASMATRIGLYCERIDES

REFERENCES

1. Dole, V. P. A relation between non-esterified fattyacids in plasma and the metabolism of glucose.J. clin. Invest. 1956, 35, 150.

2. Borgstrbm, B. Investigation of lipid separation meth-ods. Separation of phospholipids from neutral fatand fatty acids. Acta physiol. scand. 1952, 25, 101.

3. Hirsch, J., and Ahrens, E. H., Jr. The separationof complex lipide mixtures by the use of silicic acidchromatography. J. biol. Chem. 1958, 233, 311.

4. Friedberg, S. J., Klein, R. F., Trout, D. L., Bog-donoff, M. D., and Estes, E. H., Jr. The charac-teristics of the peripheral transport of C'4-labeledpalmitic acid. J. clin. Invest. 1960, 39, 1511.

5. Van Handel, E., and Zilversmit, D. B. Micromethodfor the determination of serum triglycerides. J.Lab. clin. Med. 1957, 50, 152.

6. Rapport, M. M., and Alonzo, N. Photometric de-termination of fatty acid ester groups in phospho-lipides. J. biol. Chem. 1955, 217, 193.

7. Fredrickson, D. S., and Ono, K. An improved tech-nique for assay of C"402 in expired air using theliquid scintillator counter. J. Lab. clin. Med. 1958,51, 147.

8. Hallgren, B., Stenhagen, S., Svanborg, A., andSvennerholm, L. Gas chromatographic analysisof the fatty acid composition of the plasma lipids innormal and diabetic subjects. J. clin. Invest. 1960,39, 1424.

9. Fredrickson, D. S., and Gordon, R. S., Jr. The me-tabolism of albumin bound C'4-labeled unesterifiedfatty acids in normal human subj ects. J. clin.Invest. 1958, 37, 1504.

10. Stein, Y., and Shapiro, B. Assimilation and dissimi-lation of fatty acids by the rat liver. Amer. J.Physiol. 1959, 196, 1238.

11. Laurell, S. Recycling of intravenously injectedpalmitic acid-1-C'4 as esterified fatty acid in theplasma of rats and turnover rate of plasma tri-glycerides. Acta physiol. scand. 1959, 47, 218.

12. Hillyard, L. A., Cornelius, C. E., and Chaikoff, I. L.Removal by the isolated rat liver of palmitate-l-C"4bound to albumin and of palmitate-1-C4 and cho-lesterol-4-C4 in chylomicrons from perfusion fluid.J. biol. Chem. 1959, 234, 2240.

13. Reiser, R., Bryson, M. J., Carr, M. J., and Kuiken,K. A. The intestinal absorption of triglycerides.J. biol. Chem. 1952, 194, 131.

14. Rodbell, M. The removal and metabolism of chylo-microns by adipose tissue in vitro. J. biol. Chem.1960, 235, 1613.

15. Zilversmit, D. B. The design and analysis of isotopeexperiments. Amer. J. Med. 1960, 29, 832.

16. Dole, V. P. Fractionation of plasma non-esterifiedfatty acids. Proc. Soc. exp. Biol. (N. Y.) 1956,93, 532.

17. Jokipii, S. G., and Turpeinen, 0. Kinetics of elimi-nation of glucose from the blood during and aftera continuous intravenous injection. J. clin. Invest.1954, 33, 452.

18. Storaasli, J. P., Krieger, H., Friedell, H. L., andHolden, W. L. Use of radioactive iodinated plasmaprotein in study of blood volume. Surg. Gynec.Obstet. 1950, 91, 458.

19. Havel, R. J. Early effects of fasting and of carbo-hydrate ingestion on lipids and lipoproteins of se-rum in man. J. clin. Invest. 1957, 36, 855.

20. Dury, A., and Treadwell, C. R. Effect of epinephrineon plasma lipid components and interrelationshipsin normal and epileptic humans. J. clin. Endocr.1955, 15, 818.

21. Gordon, R. S., Jr., and Cherkes, A. Unesterifiedfatty acid in human blood plasma. J. clin. Invest.1956, 35, 206.

22. Stein, Y., and Shapiro, B. Uptake and metabolismof triglycerides by the rat liver. J. Lip. Res. 1960,1, 326.

23. Stein, I., and Shapiro, B. The transport of lipidsinto adipose tissue. Metabolism 1954, 3, 539.

24. Havel, R., and Goldfien, A. Role of liver and extra-hepatic tissues in plasma fatty acid metabolism.Clin. Res. 1960, 8, 141.

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