Phospholipid Exchange Between Plasma and

Erythrocytes in Man and the Dog

CLAUDEF. REEmwith the technical assistance of MARIONMuRPHYandGERALDINEROBERTS

From the Department of Medicine, University of Rochester School of Medicineand Dentistry, Rochester, NewYork 14620

A B S T R A C r The turnover of the four majorerythrocyte phospholipids has been studied with32P, both in vivo and in vitro, in man and the dog.Phosphatidyl serine and phosphatidyl ethanola-mine appeared to be stable erythrocyte lipids inboth species. Turnover of the phosphate moietyof lecithin and sphingomyelin in the circulatingerythrocytes of these two species seems entirelydue to an exchange of the whole molecule withthe corresponding plasma compound. Exchange-able and nonexchangeable pools of these twocellular lipids were found. In man about 60%o oferythrocyte lecithin is exchaigeable. The 12 hrfractional turnover of this pool is approximately13%. Only 30% of the sphingomyelin in humancells appeared exchangeable; this portion had a12 hr fractional turnover of about 14%o. Similarresults were obtained in the dog except that inthis species about 75%o of the erythrocyte sphingo-myelin was exchangeable. Inorganic 32P was notincorporated into any of the four major phospho-lipids in either species. The present findings aidin estimating quantitatively the effect of plasma-erythrocyte lipid exchange on red blood cellphospholipids.

INTRODUCTION

There is now substantial agreement about themajor classes and amounts of lipids present inhuman erythrocytes (2). While hemoglobin and

A preliminary report of this work has been presentedpreviously (1).

Received for publication 8 August 1967 and in revisedform 30 November 1967.

structural membrane protein are thought to bestable within the life-span of the circulating maturered blood cell (RBC) (3, 4), a number of reportshave indicated that some exchange of erythrocytelipid with corresponding plasma compounds occursin various species.

Hevesey and Hahn, using the rabbit, (5) werethe first to describe an exchange of phospholipidsbetween erythrocytes and plasma. These workers(6) also suggested that the process was probablya slow one, and that all of the erythrocyte phospho-lipids were not involved equally. British workershave presented suggestive in vitro evidence thatphospholipid exchange between cells and plasmaoccurs in human blood (7, 8). More recently suchan exchange has been described in the rat byseveral groups (9-11). In all of the above reportsthe description of the process has been primarilyqualitative, and actual rates of exchange for theindividual phospholipids have not been determined.

The present study describes the dynamic stateof the phosphate moiety of the four individualmajor phospholipids of circulating erythrocytes(sphingomyelin, lecithin, phosphatidyl ethanola-mine, and phosphatidyl serine) in quantitativeterms. The dog and man were studied by using32p as a label. The results of short-term in vitrostudies of plasma and erythrocyte phospholipidexchange are compared to results obtained frommore prolonged in vivo observations. It wasfound that erythrocyte lecithin and sphingomyelinexchange with the corresponding plasma com-pounds in both species, and it was possible todetermine the rate of these processes. In addition,exchangeable and nonexchangeable fractions of

The Journal of Clinical Investigation Volume 47 1968 749

erythrocyte sphingomyelin and lecithin were foundto exist. In contrast to these two lipids, phospha-tidyl ethanolamine and phosphatidyl serine ap-peared to be stable membrane lipids.

METHODS

Normal human donors, patients with uncomplicatedpolycythemia rubra vera who had received therapeuticorthophosphate-32P, and mongrel dogs were used as sub-jects. Blood was collected into Na2EDTA (1.25 mg/ml ofblood). The red blood cells were washed three times in 5volumes of cold 0.17 M NaCl and the buffy coat wascarefully removed each time. The white blood cellcount was less than 500 per cubic mmand platelets wereabsent from the washed cells. The erythrocyte and plasmalipids were extracted, separated, and quantitated bychromatography on silicic acid-impregnated paper aspreviously described (12). A modification in the prepa-ration of the silicic acid-impregnated papers was used:the papers were given a final wash in a 1/4% aqueoussolution of Na2EDTA (w/v), air dried, and then storedat 60'C for 24 hr. The presence of the retained Na2EDTAand the mild heating improved the separation and defini-tion of the phospholipid spots obtained after chromatog-raphy.

In the early experiments reported here, the total lipidextracts were directly chromatographed on paper. Laterin the study, the total lipid extracts were first separatedinto two fractions before paper chromatography. Thiswas accomplished as follows: about 10 mg of total lipidextract in benzene was applied to a silicic acid column(I.D. 8 mm, 1.5 g of 100 mesh silicic acid). The firstfraction was eluated with 20 ml of chloroform-methanol,4 :1 (v/v). This fraction contained the neutral lipids,phosphatidic acid, phosphatidyl ethanolamine, phospha-tidyl serine, phosphatidyl inositol, and, rarely, smallamounts of lecithin. The second fraction was eluted with20 ml of methanol-water, 99: 1 (v/v); it contained lyso-lecithin, sphingomyelin, and lecithin. The column chro-matography was carried out at 4°C under nitrogen. Thisinitial fractionation procedure facilitated the completeseparation of phosphatidyl inositol and sphingomyelin,and permitted final separation of larger amounts of phos-pholipid by paper chromatography than was possiblewhen the total extract was chromatographed directly.

4-10 ,g of lipid phosphorus was applied to each paperchromatogram. These were developed by ascending chro-matography at 4-6°C for 16-18 hr with a solvent con-sisting of 2,6-dimethyl-4-heptanone, n-butyl ether, aceticacid, and water, 20:20:20:3 (v/v). The phospholipidswere identified by their color under ultraviolet light af-ter they had been stained with Rhodamine 6-G, by theirreaction to ninhydrin and choline spot tests, and by theirrelative mobilities, as described previously for this sys-tem (12, 13). The individual lipid spots were cut out,and their radioactivity was measured using a low-back-ground, thin window, gas-flow detector system.' The

'-Model C 110-B, Nuclear-Chicago Corporation, DesPlains, Ill.

background was less than 2 cpm with this system whichallowed the determination of very low levels of labeling.The lipid phosphorus was eluted from the spots with 0.5N HCl at 65°C (12). The phosphorus content of theeluates was determined by the method of Bartlett (14).The lipid spots from four to six chromatograms wereused to determine each specific activity. Sufficient countswere accumulated so that the standard error of the meanspecific activities of sphingomyelin and lecithin (reportedbelow) determined in this way were 5% or less. At least500 counts were accumulated before deciding that a lipidwas not labeled at all. Corrections for physical decay ofthe "P were made every 24 hr.

Fig. 1 is a radioautograph of canine bone marrow phos-pholipids isolated after the in vivo administration of or-thophosphate-"P. All of the phospholipids have becomelabeled and the figure illustrates the separation achievedafter paper chromatography of a total lipid extract.

Incubations of RBC with serologically compatibleplasma or with dispersed plasma lipids were carried outat 37°C in siliconized or polyethylene flasks, using a

SOLVENT FRONT

POLYGLYCEROLPHOSPHATIDE

PHOSPHATIDYLETHANOLAMINE

PHOSPHATIDYL SERINE

LEGITHIN

SPHINGOMYELIN

INOSITOL PHOSPHATIDE

LYSOLE

ORIGIN

MARROW

- CITHIN

FIGURE 1 Radioautograph of labeled canine bone marrowphospholipids. Orthophosphate-82P (0.15 mc/kg) was ad-ministered intravenously, as a single injection, to a dog,and the lipids of the bone marrow cellular elements wereisolated 96 hr later. 4.5 ug of total lipid P where chro-matographed on silicic acid impregnated paper.

750 C. R. Reed

metabolic shaker, for periods of up to 12 hr. Sterile con-ditions were maintained and were confirmed by culture.Penicillin (100 units/ml) and streptomycin (0.1 mg/ml)were used in some of the incubation and had no effecton the results. Sufficient glucose was added to provide atotal concentration of 12 umoles/ml of blood.

Some of the incubations were carried out with plasmalipids dispersed in an aqueous buffer. The neutral lipidswere first removed by silicic acid chromatography andelution with chloroform. The phosphatides were re-covered from the column by elution with methanol, andthen suspended, with a tissue homogenizer, in a bufferconsisting of 5.9 X 10' M KH2PO4, 2.5 X 10' MNa2HPO4,1.4 X 10' M NaCl, and 1 X 10i M MgSO4, with a re-sulting pH of 7.4. This suspension of the plasma phos-phatides, usually 50 ml in volume, was then subjected toultrasonic irradiation for 20 min with a dipping probewhich generated energy at 20 kc/sec at a power outputof 120 watts. The beaker containing the suspension wassurrounded by ice water, and the temperature of thesuspension remained at 100C or less during the sonication,although it might have been higher locally, immediatelyadjacent to the probe. This treatment produced a water-clear solution of the phosphatides, and the concentra-tions of lecithin and sphingomyelin in the buffer were thesame before and after irradiation in each instance. Theuse of ultrasonic irradiation to produce stable, water-clear dispersions of phospholipids, without breakdown ofthe compounds, has been described, in detail, by Gam-mack, Perrin, and Saunders (15). It was not possible toobtain a clear lipid solution if the neutral lipids were notfirst removed. After the ultrasonic treatment the phos-pholipid solution was mixed with an equal volume of

40

30

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packed normal erythrocytes from which the buffy coathad been removed. The mixture was incubated for 3 minat room temperature and then centrifuged at 2,000 g for10 min at 4VC. The supernatant fluid containing the dis-persed plasma phospholipids was recovered and then usedin test incubations with additional normal erythrocytes.The preliminary absorption procedure served to removeany metallic particles introduced from the probe duringthe ultrasonic treatment, and to monitor the extent towhich the dispersed phospholipids might adhere immedi-ately, in a nonspecific manner, to the red blood cell sur-faces. When labeled plasma phospholipids were used noradioactivity was found in the phosphatides of the ad-sorbing erythrocytes after 3 min, nor in the lipids of thetest erythrocytes at time zero.

Plasma was dialyzed against 20-50 volumes of 0.17 MNaC1 for 48 hr at 4°C, with three changes of the dialy-sate. Packed cell volumes were determined by the micro-hematocrit technique.

RESULTS

When orthophosphate-32P was administered invivo, the label appeared promptly in the plasmalipid phosphorus; it appeared more slowly and ata lower level in the RBCphospholipids. The gen-eral pattern observed in both humans and dogs isshown in Fig. 2. It can be seen that labeled plasmaand RBC can be harvested for in vitro studiesover a period of days after a single administrationof the isotope. Fig. 3 shows the typical labeling

PLASMA PHOSPHOLIPID

DAYSFIGURE 2 Appearance of radioactivity in human plasma and erythrocyte lipid P after 'P administra-tion. Orthophosphate-'P (0.15 mc/kg) was given as a single oral dose. The specific activity of thetotal plasma and erythrocyte lipid P was determined, as shown, over a period of 10 days.

Phospholipid Exchange between Plasma and Erythrocytes 751

DAYSFIGURE 3 Labeling of human plasma P containing compounds after '3P administration.Orthophosphate-'P (0.15 mc/kg) was given as a single oral dose. Solid circles = plasmanonlipid P; X's = plasma lecithin; open circles = plasma sphingomyelin.

pattern of plasmprincipal plasma85-90%o of thespecies), and ofabout 90%o of M

The interactiolipid phosphorus

RELATIVE S ARBC LIPID P

M%

la sphingomyelin and lecithin (the serologically compatible labeled plasma with un-phospholipids, which account for labeled RBC at a hematocrit value of 30-40%o.

plasma lipid phosphorus in both Fig. 4 shows that radioactivity appeared in thethe plasma nonlipid phosphorus, erythrocyte lipid phosphorus and that it increased

rhich can be removed by dialysis. at a nearly constant rate for a period of 12 hr,on between plasma and erythrocyte at 370C. Little radioactivity was found in thewas studied in vitro by incubating erythrocyte lipids when the incubations were car-

ried out at 4VC.The appearance of radioactivity in the individual

major erythrocyte phospholipids was determinedat the end of the 12 hr period in incubations car-

370 C ried out at 370C. The results of these determina-tions are shown in the first column of Table I.The degree of labeling of erythrocyte lecithin and

/ 4 C sphingomyelin is expressed as their "relative spe-D0 cific activities" (RSA). This is defined by the

0 4 8 12 ratio:HOURS

FIGURE 4 Appearance of labeled plasma P in erythro-cyte phospholipids during in vitro incubation. Human la-beled plasma was harvested 48 hr after the oral adminis-tration of s'P (0.15 mc/kg) and incubated with serologi-cally compatible normal red blood cells at a hematocritvalue of 35%. The specific activity of the total erythro-cyte lipid P was divided by the specific activity of theplasma lipid P to obtain the relative specific activity. Theresults at 370C are typical of eight such experiments.

RSAC(t)= (SAc(O))/(SAp) (1)whereSA = specific activity, net counts per minute

(cpm) pergag of phosphorust = duration of incubation (usually 12 hr)c = individual RBCphospholipid

SA1 = average SAof corresponding plasma phos-pholipid (p): (SAp(g) + SAp(O)) /2.

752 C. R. Reed

4

2

c

4

TABLE I

RSA of Human Erythrocyte Phospholipids After 12 Hr ofIncubation at 370C in Media Containing 32P*

Labeled medium

Plasmaphospho-

lipids Inorganicdispersed 32pby ultra- added to

Dialzed sonica- normalErythrocyte Plasma plasma tiont plasmaphospholipid (n =4) (n =4) (n =5) (n =4)

Sphingomyelin 4.0 4.0 4.0 0(3.3-5.2)§ (3.7-4.1) (3.0-4.4)

Lecithin 8.0 9.0 8.0 0(7.5-9.5) (8.5-9.4) (6.8-9.2)

Phosphatidylserine 0 0 0 0

Phosphatidylethanolamine 0 0 0 0

* Abbreviation: RSA, relative specific activity.At 6 hr, (n =3), lecithin RSA=4.0 (3.8-5.1), sphingomyelin RSA

=2.0 (1.2-2.2).§ Mean values, ranges in parentheses.

This ratio can be expressed as a percentage bymultiplying the RSA by 100. In each instance,SAC(o) was zero. The SA of plasma sphingomyelinand lecithin decreased very little during these ex-periments; they were approximately 95% of thetime zero values for both lipids in each case atthe end of 12 hr. Thus, SAp was virtually con-stant. Column 2 of Table I shows that priorremoval by dialysis of the plasma nonlipid phos-phorus had no effect on the labeling achieved byerythrocyte lecithin and sphingomyelin. Column 3of Table I shows that labeled plasma phospho-lipids, dispersed in an aqueous buffer at approxi-mately the same concentration as they were pres-ent in native plasma, produced the same degreeof labeling of the erythrocyte phospholipids as wasfound with whole plasma. Column 4 of the tableshows that when orthophosphate-32P was addedto the incubation mixtures as the only source ofisotope there was no incorporation of radioactivityin any of the four major erythrocyte phosphatidesover a period of 12 hr. All of these results indicatethat plasma dialyzable phosphorus and inorganicphosphorus play no part in the labeling of themajor erythrocyte phospholipids and that theplasma phosphatides are the sole immediate pre-cursors in the labeling of the corresponding eryth-rocyte compounds.

The relative specific activities of erythrocytelecithin and sphingomyelin shown in Table I aresmall (less than 10%) and, therefore, theseproduct-precursor specific activity ratios are agood approximation of the actual fractional turn-over per 12 hr of the cell phospholipids in theexchange process (16). After 12 hr the recoveryof each of the two plasma and four erythrocytelipids measured averaged 100% ( with a rangeof 97-103%). Thus, there was no measurable netmovement of phospholipids between plasma anderythrocytes in these studies. Wehave previouslyreported that in the presence of adequate glucosenormal erythrocytes lose very little lipid when theyare incubated under the same circumstances ashere for periods up to 24 hr (17).2

Measurable radioactivity was not found inerythrocyte phosphatidyl serine and phosphatidylethanolamine, probably because these two lipidsare almost absent from the plasma. In two experi-ments, not included in column 3 of Table I,labeled plasma lecithin virtually free of sphingo-myelin was obtained by silicic acid chromatogra-phy. When this material was dispersed by ultra-sonication and incubated with RBC, the resultingRSA of the erythrocyte lecithin, after 12 hr, was

7.8% and 8.3%, a normal degree of labeling.Erythrocyte sphingomyelin, on the other hand,was not labeled at all in these two experiments.These results suggest that the plasma-erythrocytelipid exchange is, in fact, between correspondingcompounds, and that phosphoryl choline, presentin both lecithin and sphingomyelin, does not ex-change independently of the entire lipid moleculein which it is present.

The exchange of lecithin and sphingomye-lin between plasma and erythrocytes was alsostudied by incubating cells which had becomelabeled in vivo (harvested 3-6 days after the initialadministration of orthophosphate-32P, Fig. 2) withunlabeled plasma. In these experiments, labeling ofthe plasma lipid phosphorus increased at a con-

stant rate for 12 hr and this period was, again,2 Over a period of 12 hr and using Na2EDTA plasma

in nonwettable surfaces, we found no evidence for theactivity of the serum cholesterol-esterifying enzyme firstdescribed by Sperry (18). In the presence of glass sur-

faces and over longer periods of incubation a reductionof serum lecithin and an increased amount of esterifiedcholesterol results from the activation of this enzyme(18, 19).

Phospholipid Exchange between Plasma -and Erythrocytes 753

chosen for analyzing the SA of the individualphosphatides.

It was possible to calculate the movement ofRBC lecithin and sphingomyelin into the plasmafrom these experiments. The RSA's of the "prod-uct" compounds, plasma lecithin and sphingo-myelin, were determined and this gave the fractionof the plasma lipid derived from the correspondingerythrocyte lipids at 12 hr. This value was multi-plied by the total amount of the plasma lipid perml of incubation mixture which gave the absoluteamount of the lipid moving from the erythrocyte tothe plasma compartment. This product was thendivided by the amount of the lipid in the erythro-cyte compartment, and the resulting quotient gavethe fractional turnover of the erythrocyte lipid per12 hr, measured in the outward direction. Com-bining the three steps, the expression for thiscalculation is:

SAP(,)/SA, X ,gp X l/tgc (2)where

, = jig of individual plasma lipid phosphorusper ml of incubation mixture

Hgc = Ag of corresponding RBClipid phosphorusper ml of incubation mixture, and theother symbols as in expression 1.

SAP(O), again, was zero, and, as mentioned, ,ugpand yg, did not change. The results of these out-ward studies are shown in the first column ofTable II. It can be seen (Table II) that thefractional exchange rates and actual flows oferythrocyte sphingomyelin and lecithin calculated

from both types of in vitro experiments do notagree, and that the rates determined from theoutward studies are considerably larger.

No net movement of lipid phosphorus betweenthe erythrocyte and plasma compartments was dis-cernible in these experiments, however, and therates of actual lipid exchange should, therefore,be equal in both directions. The discrepancy be-tween the calculated inward and outward ratesmay be resolved by assuming that the radioactivityin human erythrocyte lecithin and sphingomyelin,labeled in vivo, was not distributed uniformlythroughout these lipid pools, but that it was dis-tributed in only 63%o of the lecithin and 29% ofthe sphingomyelin. If this is so, calculation of thefractional turnover from the outward experimentwould require that SAC of expression 2, (the SAof the total erythrocyte lecithin or sphingomyelin,which is what can be measured) be divided byapproximately 0.6 and 0.3, respectively, to yieldthe true SA of the erythrocyte molecules actuallyparticipating in the lipid movement. The calculatedoutward rate would then be equal to the observedinward rate. This hypothesis postulates that onlyabout 60% of human erythrocyte lecithin and 30%of the sphingomyelin are actually capable of ex-change with the corresponding plasma compounds.As will be shown below, results of the in vivostudies of lipid exchange over a period of dayssupport this view.

Parallel in vitro studies were carried out withcanine blood. The results are shown in Table III.The overall results were quite comparable to those

TABLE I I12-Hr Exchange Rates of Human Erythrocyte Sphingomyelin and Lecithin Determined In Vitro*

Outward labeled RBCand Inward$ labeled RBCandunlabeled plasma (n = 4) unlabeled plasma (n = 4)

Amoles per emoles per RatioFractional ml RBC Fractional ml RBC inward/

RBC lipid turnover X 102 turnover X 1O2 outward

Sphingomryelin 14.0 14.0 4.0 4.0 0.29(12.5-15.8)§ (3.3-5.2)

Lecithin 13.0 17.5 8.0 11.0 0.63(12.5-13.8) (7.5-9.5)

* Abbrevaition: RBC, red blood cell.t Values from column 1 of Table 1.§ Mean values, ranges in parentheses.

754 C. R. Reed

TABLE II I12-Hr Exchange Rates of Canine Erythrocyte Sphingomyelin and Lecithin Determind In Vitro*

Outward labeled RBCand Inward labeled plasma andunlabeled plasma (n X 4) unlabeled RBC (n = 4)

pmoles per pmoles per RatioFractional ml RBC Fractional ml RBC inward/

RBClipid turnover X 102 turnover X 102 outward

Sphingomyelin 10.0 7.0 7.6 5.3 0.76(8.6-12.1)t (6.3-8.9)

Lecithin 10.0 18.0 6.2 11.2 0.62(8.8-12.0) (5.4-7.1)

* Abbreviations: RBC, red blood cell.t Mean values, ranges in parentheses.

obtained with the human material. Phosphatidylserine and phosphatidyl ethanolamine were stable;sphingomyelin and lecithin were found to undergoexchange between the plasma and erythrocytes.The major difference found was that the size of theexchangeable sphingomyelin erythrocyte pool waslarger in the dog. The distribution of sphingo-myelin between plasma and erythrocytes is thesame in both species, the plasma-erythrocyte molarratio being approximately 1: 1. The observed dif-ference may, therefore, be due to differences in

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the distribution and binding of sphingomyelinwithin the membrane in the two species.

The exchange process was also studied in vivo.Serial determinations of the SA of erythrocyte andplasma phospholipids were carried out in fivepatients and four dogs after the administrationof orthophosphate-32P. Four of the humans andone dog received a single dose of the isotope. Theother subjects received 32P in divided doses.

When the isotope was administered as an initialloading dose followed by small daily replacements,

PLASMA LECITHIN

4RBC LECITHIN

.-X----- -x--- -

1'RBC PHOSPHATIDYL SERINE

6 7 8 9

0.07 MC/DAY

DAYSFIGURE 5 Appearance of radioactivity in some individual phospholipids of canineblood after 'P administration. The isotope was administered intravenously (0.15mc/kg) in divided doses, as indicated.

Phospholipid Exchange between Plasma and Erythrocytes

I

#a55

the SA of plasma lecithin and sphingomyelin couldbe kept nearly constant at a relatively high levelfor a period of days. Fig. 5 shows the SA ofplasma lecithin and erythrocyte phosphatidylserine and lecithin observed in a typical experi-ment in the dog when the isotope was administeredin divided doses. The basic features of the ex-change process seen in the in vitro studies areapparent in this type of experiment. The SA ofplasma lecithin remained considerably higher thanthat of the RBCcompounds, and it remained rela-tively constant from the 2nd to the 9th day. TheSA of erythrocyte lecithin increased until aboutthe 6th day and achieved a level which was 50-60% of the plasma lecithin SA. These findingssuggest, qualitatively, that some fraction of thecirculating erythrocyte lecithin pool exchangesvery slowiy or not at all with plasma lecithin, aswas indicated by the in vitro studies. The SA ofphosphatidyl serine remained very low and wasnot measurable during the first 2 or 3 days. Thelabeling of this compound may be due to the influxof new cells from the bone marrow where phospho-lipid synthesis occurs (Fig. 1).

In all the in vivo experiments a quantitativeapproximation of the fraction of erythrocyte leci-thin or sphingomyelin which existed as labeledlipid on any given day was obtained by dividingthe SA of the erythrocyte lipid on that day by theweighted average of the SA of the correspondingplasma lipid, to that point, according to the fol-lowing expression:

YC(k) SAC(k)- (3)E SAP(i)i=O

kwhere

Yc(k) = fraction of individual RBClipid poolwhich exists as labeled lipid on thekth day

SAc(k) = specific activity of the RBC lipid onk the kth dayE SAp(i) = sum of the specific activities of thei=0 corresponding plasma lipid from day

zero to the kth day.

The fraction of the erythrocyte sphingomyelinor lecithin pools which are labeled (that is, thefraction of the erythrocyte lipids that was at onetime in the plasma) can also be predicted exactly

TABLE IVSummary of the Parameters of ErythrocytePhospholipid Exchange Determined In Vitro

Fractional size 12-Hr fractionalof exchangeable turnover of the

pool exchangeable pool(E)* (r)*

Erythrocytephospholipid Man Dog Man Dog

Lecithin 0.63 0.62 0.13 0.10Sphingomyelin 0.29 0.76 0.14 0.10

*"E" and "r" refer to the symbols in expression 4 of the text, andwere the values used in constructing the solid curves of Fig. 6.

from the fractional turnover and sizes of the ex-changeable erythrocyte pools determined in thein vitro studies, (Table IV), by using this expres-sion 3:

Y() = (1 - e-rt) X E (4)where

t = time (12 hr = 1 unit)YC~) = fraction of the total individual RBClipid

pool which exists as labeled lipid at time tr = 12 hr fractional turnover rate of the ex-

changeable pool of the RBClipid (TableIV)

E = fractional size of the exchangeable poolof the RBClipid-

e = base of the natural logarithms.The observed results for YC(k) and the predicted

values were compared at each point in the fivehuman subjects and the four dogs studied forperiods of up to 15 days. All of these results areshown in Fig. 6. It can be seen that the observeddata agree well with the solid curves of the figure.These solid curves indicate the expected labeling ofthe erythrocyte lipids if the existence and sizeof exchangeable lecithin and sphingomyelin poolsand their fractional turnover rate, determined invitro and given in Table IV, are correct. Thebroken curves, as described in the legend of Fig 6,show the labeling of the erythrocyte compounds

3 In expression 3 1 day (k) is used as the unit of timebecause the SA of the plasma and RBClipids were deter-mined daily in the in vivo studies; 12 hr (t) is used inexpression 4 because "r" was determined over this pe-riod in vitro. Expressions 3 and 4 are minor adaptions ofthe calculation of turnover from a precursor with con-stant SA given by Zilversmit (16). Expression 3 will givea good approximation of Yc( ) if SA, is relatively con-stant and if SA0 does not decrease over the time intervalstudied.

756 C. R. Reed

HUMANSPHINGOMYELIN

).22

01.0

HUMANLECITHIN).8_--

).4 ,1

_ _._2

D2 4 6 8DAYS

FIGURE 6 Comparison of the observed in vivo labeling of erythrocyte sphingomyelin and lecithin withthat predicted from the in vitro data. The solid curves were constructed from expression 4 of the textwith the values for "E" and "r' given in Table IV. The broken curves were contructed from, simply,the expression Y,<*) = 1 - e-t, where "r" is the fractional influxes observed in vitro and given in Ta-bles II and III. This calculation assumes that the cellular lipids are entirely exchangeable. Bracketedsolid circles mean 2 SE of the observed labeling, obtained from expression 3 of the text, in five hu-mans and four dogs.

that would have been obtained in these experi-ments if all the cellular lecithin and sphingomyelinwere exchangeable. The presence of exchangeablepools of erythrocyte lecithin and sphingomyelinwill affect the observed labeling of these com-

pounds primarily by altering the loss back to theplasma of newly acquired label during a giventime interval. This loss of acquired label will begreater if exchangeable and nonexchangeable poolsof a given lipid exist. When the RSA of thecellular lipids is low (less than 20%o) this greaterloss is a small factor, as indicated in Fig. 6 by theclose coincidence, for 3 to 4 days, of the brokenand solid curves. After this period, the greaterloss of newly acquired label when a nonexchange-able pool is present becomes increasingly signifi-cant and the two curves diverge progressively. Thegood agreement beyond 4 days of the observedlabeling with the solid curves of Fig. 6 stronglysuggests, therefore, that exchangeable and non-

exchangeable pools, of the approximate sizes pos-

tulated from the in vitro results, do, in fact, exist.The in vivo results also support the validity of

the fractional turnover rates determined in vitro.It should be mentioned, however, that althoughthe above hypotheses satisfactorily account for allthe results, this does not completely exclude thepossibility that more than two membrane com-

partments actually exist for each phospholipid;however, the overall labeling of the system isreasonably well predicted by the two compartmentmembrane model.

Two factors, in addition to phospholipid ex-

change, will - affect the labeling of erythrocytesphingomyelin and lecithin in vivo. (a) The influxof new erythrocytes into the circulation. Thesenew cells will contain lipids synthesized fromsimple precursors (including the administered in-organic 32P) in the bone marrow. After 3-4 daysthis mechanism will tend to raise the observedlabeling of erythrocyte lecithin and sphingomyelinabove the value predicted from exchange alone.In one experiment in the dog, the bone marrow

cellular elements were isolated 3 days after theadministration of 82p. Identical SA's were foundin the four major phospholipids. This suggests

Phospholipid Exchange between Plasma and Erythrocytes 757

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that the low SA observed in phosphatidyl serineshown in Fig. 5 may serve as a rough guide tothe effect of new cell synthesis, and that this isnegligible for 6-9 days. (b) Removal of senescenterythrocytes from the circulation. About 1% perday of the circulating RBCmass will be removedthis way in both man and the dog. This will tendto lower the observed labeling of circulating eryth-rocyte lecithin and sphingomyelin, but this effectshould not be noticeable for 6-10 days. The factthat there was little deviation from the observedvalues and those predicted on the basis of ex-change alone for as long as 15 days (Fig. 6)suggests that the opposite effects of new cell pro-duction and red cell senescence may tend to canceleach other.

DISCUSSION

In the present work some of the quantitativeaspects of the exchange between erythrocyte leci-thin and sphingomyelin with the correspondingplasma compounds have been described in manand the dog. These findings are summarized inTable IV. Exchangeable and nonexchangeablefractions of each of these two cellular lipids werefound in both species. Rowe (8) has previouslysuggested that blood cellular lecithin, in humans,does not exchange completely with plasma lecithin.

It can be seen from Table IV that the turnovertimes of the exchangeable pools of erythrocytesphingomyelin and lecithin, in both species, areapproximately 5 days. Thus, alterations in thefatty acid composition of the plasma compoundscan affect, in a very significant manner, the cor-responding erythrocyte membrane lipids duringthe life span of a given red blood cell. The actualamounts of lipid involved in the exchange process,however, are quite small. In the case of lecithin,in both species studied here, the exchange is about9 x 10-3 mmoles/liter of erythrocytes per hour.This is in contrast to the active transport of mono-valent cations across the human erythrocyte mem-brane, for example, which is about 3 mmoles/literof erythrocytes per hour.

In the present study, by using 32P to evaluateturnover, it was found that erythrocyte phospha-tidyl serine was completely stable and phosphatidylethanolamine nearly so. As mentioned above, thismay simply result from the virtual absence ofthese two phospholipids from the plasma of man

and the dog. Erythrocyte cephalins have beenfound to be stable in exchange studies with 32Pin the rat (10) where these compounds are alsonearly absent from plasma. The different ratesand degree of exchangeability found for sphingo-myelin and lecithin, particularly in the human, onthe other hand, suggest that, in addition to plasmaconcentration, the position of a lipid molecule inthe membrane and the nature of its bond to mem-brane protein (ionic vs. hydrophobic) may alsoinfluence its exchange. Cholesterol, for example,is thought to be bound in many membranes onlyto the hydrophobic portion of phospholipids, pri-marily by the relatively weak van der Waalsforces (20). This erythrocyte lipid has been foundto exchange very rapidly with plasma free choles-terol (turnover time less than 12 hr) in both manand the dog (21-23). This is in contrast to themuch slower rate of phospholipid exchange de-scribed here. The latter compounds probably arebound to membrane protein by charge-chargeinteractions, in large part (20).

Inorganic 32p was not incorporated into any ofthe four major erythrocyte phospholipids. In thesecompounds, the phosphorus group exists as a di-ester and is internally placed in the molecule.Incorporation of inorganic 32P would, therefore,require enzymatic breakdown and resynthesis ofthe lipid. There is a substantial body of evidence,summarized by van Deenen and de Gier (2), thatmature nonnucleated erythrocytes are incapable ofcarrying out such synthetic lipid processes. Forthis reason, and because, as mentioned above,phosphoryl choline from lecithin did not appearin erythrocyte sphingomyelin, we believe that theexchange of lipid phosphorus herein describedprobably involves the entire lipid molecule.

Very small amounts of lysolecithin were foundin the plasma and erythrocytes of both speciesstudied. This compound was weakly labeled andits participation in the exchange process could notbe evaluated. In the rat, on the other hand, sub-stantial amounts of lysolecithin have been re-

ported in the plasma and this compound is ex-

changed actively (9-11). Mulder and coworkers(24, 25) and, more recently, Tarlov (26) haveindicated that reversible acylation by plasma fattyacids of membrane lysolecithin, a process knownto occur in human red blood cells (27, 28), ac-

counts for a substantial proportion of lecithin turn-

758 C. R. Reed

over in rat erythrocytes. The exact degree towhich such transacylation of erythrocyte phospho-lipids occurs in human is not known at present.As suggested by Farquahar and Ahrens (29),additional studies using phospholipids labeled withboth 32P and 14C are needed to compare the con-tributions of transacylation and exchange of entiremolecules to the dynamic state of the erythrocytephospholipids.

The nonsymmetrical exchange of erythrocytephospholipids described here is in contrast to theprocess of cellular lipid loss which has been re-ported during the course of in vivo ageing (30,31), during in vitro incubations of abnormal cellsfor 24 hr (17), and in normal cells incubated formore prolonged periods (32). In these instances,the loss of lipid has been found to involve all lipidcompounds equally. The concomitant formation ofsmall membrane fragments of varying sizes alsoobserved in some of these studies (33) providedan explanation for the symmetrical nature of thelipid loss. The process of lipid exchange may,however, prove important in the pathogenesis oferythrocyte abnormalities associated with altera-tions in plasma lipids, as has been suggested pre-viously (2, 26, 34). It is hoped that the quan-titative data on phospholipid exchange given inthe present study may prove helpful in furtherexploration of this possibility.

ACKNOWLEDGMENTThis investigation was supported by the U. S. ArmyMedical Research and Development Command, Depart-ment of the Army, under research contract MD-2656, andby a Public Health Service Research DevelopmentAward (HE-6234) from the Heart Institute.

REFERENCES1. Reed, C. F. 1959. Studies of in vivo and in vitro ex-

change of erythrocyte and plasma phospholipids. J.Clin. Invest. 38: 1032. (Abstr.)

2. van Deenen, L. L. M., and J. de Gier. 1964. Chemicalcomposition and metabolism of lipids in red cells ofvarious animal species. In The Red Blood Cell. C.Bishop and D. M. Surgenor, editors. Academic Press,Inc., New York. 7: 243.

3. London, I. M. 1960. The metabolism of the erythro-cyte. Harvey Lectures. 56: 151.

4. Muir, H. M., A. Neuberger, and J. C. Perrone. 1952.Further isotopic studies on haemoglobin formationin the rat and rabbit. Biochem, J. 52: 87.

5. Hevesy, G., and L. Hahn. 1940. Turnover of lecithin,cephalin and sphingomyelin. Kgl. Danske Videnskab.Selskab. Biol. Med. 15: 1.

6. Hahn, L., and G. Hevesey. 1939. Interaction betweenthe phosphatides of the plasma and corpuscles.Nature. 144: 72.

7. Lovelock, J. E., A. T. James, and C. E. Rowe. 1960.Lipids of whole blood. 2. The exchange of lipids be-tween the cellular constituents and the lipoproteinsof human blood. Biochem. J. 74: 137.

8. Rowe, C. E. 1960. The phospholipids of human-bloodplasma and their exchange with the cells. Biochem. J.76: 471.

9. Polonovski, J., and M. Paysant. 1963. Metabolismphospholipidique du sang. VIII. tchange des phos-pholipides marques entre globules et plasma sanguinin vitro. Bull. Soc. Chim. Biol. 45: 339.

10. Sakagami, T., 0. Minari, and T. Orii. 1965. Inter-action of individual phospholipids between rat plasmaand erythrocytes in vitro. Biochim. Biophys. Acta.98: 356.

11. Mulder, E., and L. L. M. van Deenen. 1965. Metabo-lism of red-cell lipids. III. Pathways for phospholipidrenewal. Biochim. Biophys. Acta. 106: 348.

12. Reed, C. F., S. N. Swisher, G. V. Marinetti, andE. G. Eden. 1960. Studies of the lipids of the erythro-cyte. I. Quantitative analysis of the lipids of normalhuman red cells. J. Lab. Clin. Med. 56: 281.

13. Marinetti, G. V., J. Erbland, and J. Kochen. 1957.Quantitative chromatography of phosphatides. Fed.Proc. 16: 837.

14. Bartlett, G. R. 1959. Phosphorus assay in columnchromatography. J. Biol. Chem. 234: 466.

15. Gammack, D. B., J. H. Perrin, and L. Saunders. 1964.The dispersion of cerebral lipids in aqueous media byultrasonic irradiation. Biochim. Biophys. Acta. 84: 576.

16. Zilversmit, D. B. 1960. The design and analysis ofisotope experiments. Am. J. Med. 29: 832.

17. Reed, C. F., and S. N. Swisher. 1966. Erythrocytelipid loss in hereditary spherocytosis. J. Clin. Invest.45: 777.

18. Sperry, W. M. 1935. Cholesterol esterase in blood.J. Biol. Chem. 111: 467.

19. Glomset, J. A., F. Parker, M. Tjaden, and R. H.Williams. 1962. The esterification in vitro of freecholesterol in human and rat plasma. Biochim. Bio-phys. Acta. 58: 398.

20. van Deenen, L. L. M. 1965. Phospholipids and bio-membranes. In Progress in the Chemistry of Fats andOther Lipids. R. T. Holman, editor. Pergamon PressLtd., Oxford. 8(Pt. 1): 1.

21. Hagerman, J. S., and R. G. Gould. 1951. The in vitrointerchange of cholesterol between plasma and redcells. Proc. Soc. Exptl. Biol. Med. 78: 329.

22. London, I. M., and H. J. Schwarz. 1953. Erythrocytemetabolism. The metabolic behavior of the cholesterolof human erythrocytes. J. Clin. Invest. 32: 1248.

23. Eckles, N. E., C. B. Taylor, D. J. Campbell, and R.G. Gould. 1955. The origin of plasma cholesterol andthe rates of equilibration of liver, plasma, and eryth-rocyte cholesterol. J. Lab. Clin. Med. 46: 359.

24. Mulder, E., and L. L. M. van Deenen. 1965. Metabo-lism of red-cell lipids. I. Incorporation in vitro of

Phospholipid Exchange between Plasma and Erythrocytes 759

fatty acids into phospholipids from mature erythro-cytes. Biochim. Biophys. Acta. 106: 106.

25. Mulder, E., J. W. 0. vandenBerg, and L. L. M. vanDeenen. 1965. Metabolism of red-cell lipids. II. Con-versions of lysophosphoglycerides. Biochim. Biophys.Acta. 106: 118.

26. Tarlov, A. R. 1966. Lecithin and lysolecithin metabo-lism in rat erythrocyte membranes. Blood. 28: 990.(Abstr.)

27. Oliveira, M. M., and M. Vaughan. 1964. Incorporationof fatty acids into phospholipids of erythrocyte mem-branes. J. Lipid Res. 5: 156.

28. Robertson, A. F., and W. E. M. Lands. 1964. Metabo-lism of phospholipids in normal and spherocytichuman erythrocytes. J. Lipid Res. 5: 88.

29. Farquhar, J. W., and E. H. Ahrens, Jr. 1963. Effectsof dietary fats on human erythrocyte fatty acid pat-terns. J. Clin. Invest. 42: 675.

30. Westerman, M. P., L. E. Pierce, and W. N. Jensen.1963. Erythrocyte lipids: a comparison of normalyoung and normal old populations. J. Lab. Clin.Med. 62: 394.

31. van Gastel, C., D. vandenBerg, J. de Gier, and L. L.M. van Deenen. 1965. Some lipid characteristics ofnormal red blood cells of different age. Brit. J.Haemat. 11: 193.

32. Weed, R. I., and C. F. Reed. 1966. Membrane altera-tions leading to red cell destruction. Am. J. Med. 41:681.

33. Weed, R. I., and A. J. Bowdler. 1966. Metabolic de-pendence of the critical hemolytic volume of humanerythrocytes: relationship to osmotic fragility andautohemolysis in hereditary spherocytosis and normalred cells. J. Clin. Invest. 45: 1137.

34. Ways, P., and C. F. Reed. 1965 The nature anddynamic behavior of red cell lipids. Scand. J. Haematol.10: 34.

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