Concomitant Alterations of Sodium Flux and Membrane ...€¦ · fore incubation. After removal of a sample of cells for counting of blood elements and hematocrit determination, the

Journal of Clinical InvestigationVol. 46, No. 2, 1967

Concomitant Alterations of Sodium Flux and MembranePhospholipid Metabolism in Red Blood Cells: Studies

in Hereditary Spherocytosis *

HARRYS. JACOBt ANDMANFREDL. KARNOVSKY

(Front the Thorndike Memorial Laboratory and Second and Fourth [Harvard] MedicalServices, Boston City Hospital; the Departments of Medicine and BiologicalChemistry, Harvard Medical School; and St. Elizabeth's Hospital and Tufts

Medical School, Boston, Mass.)

Summary. The role of membrane phosphatides in transport processes hasbeen investigated in red cells from splenectomized patients with hereditaryspherocytosis (HS).

Incorporation of inorganic 32phosphate into the membrane phosphatides ofHS red cells was approximately twice normal, coinciding with the nearly two-fold increment in flux of sodium ions in the cells.

A consistent, inordinate increase in specific activity of a chromatographicfraction containing phosphatidylserine provided the bulk of the over-all in-crease in labeling of HS red cell phosphatides. The specific activity of phos-phatidic acid was increased but not consistently.

Radioactivity of the "acidic phosphatides" (phosphatidylserine and phos-phatidic acid fractions) decreased, in general, when the sodium flux waslow, i.e., when the cells were suspended in media of low sodium content.When the cation flux was elevated (hypotonic media), there was a marked(ca. 35%) increase in the labeling of phosphatidylserine fractions. Normalred cells whose permeability to cations was increased by exposure to 0.5 Nbutanol also exhibited increased labeling of acidic phosphatides.

Considerations of the stoichiometry of cation transport and phosphatidelabeling make it unlikely that phospholipids act directly as carrier moleculesfor cations in red cell membranes. On the other hand, the involvement ofthese lipid substances in cation movements is substantiated by correlatingseveral different states of sodium flux with the labeling of the phosphatidicacid and phosphatidylserine fractions.

Introduction

Phospholipids have been implicated in cationand other transport systems by the findings of nu-merous investigators (1-7). The possibility thatthese compounds might play a central role intransport phenomena stems partly from the facts

* Submitted for publication June 8, 1966; acceptedOctober 20, 1966.

Supported in part by grants HE-07652, T1-AM-5391,HE-10053, and AI-03260 from the National Institutes ofHealth.

tAddress requests for reprints to Dr. Harry S. Jacob,St. Elizabeth's Hospital, 736 Cambridge St., Brighton,Mass.

that they are important constituents of cellularmembranes and that certain members of the classhave considerable ability to bind cations (8-10).The phosphatides thet bear a net negative chargeat physiologic pH levels ("acidic" phosphatidessuch as phosphatidic acid, phosphatidylserine, andinositol phosphatides) are especially active in thelatter regard, and the extensive work of the Hokins(11) has demonstrated that the metabolism ofphosphatidic acid and phosphatidylinositol is mostmarkedly affected during transport processes.For example, when sodium transport in the aviannasal gland is stimulated by the action of acetyl-

173

HARRYS. JACOB AND MANFREDL. KARNOVSKY

choline, the Hokins have found that the metabolismof phosphatidic acid is greatly enhanced (11).These observations, and others, led to the postula-tion some years ago of a direct role of phospha-tidic acid as a "carrier" of the sodium ion, involv-ing its dephosphorylation by a phosphatase andre-formation from diglyceride by a specific kinaseand ATP (12, 13). If phosphatidic acid were acarrier of sodium ions, such a cycle would havethe over-all effect of linking sodium transport andATP utilization. Stimulation of sodium transportwould be expected to result in increased turnoverof the phosphate moiety of phosphatidic acid.However, when the kinetics of cation transportand phosphatidic acid turnover are examined invarious tissues, serious discrepancies are evident(14). Furthermore, it has been shown that threesodium ions are transported per ATP moleculeturned over (15, 16), whereas, if phosphatidicacid were indeed the carrier, the maximal numberof sodium ions transported per ATP would betwo. These discrepancies and the fact that in-corporation of radioactive phosphate into otheracidic phosphatides such as phosphatidylinositolhas also been found to respond to alterations in so-dium transport (17) have necessitated the formula-tion of other ideas concerning the role of complexphosphatides in cation transport (11). Finally,the altered phosphatide metabolism evoked bypharmacologic or physical stimulation of tissuesin vitro may not be linked specifically to changesin cation transport but instead may reflect otherunrelated phenomena induced by the stimulatingagent.

In the present studies, some aspects of mem-brane-lipid metabolism in red cells from patientswith hereditary spherocytosis (HS) have beeninvestigated by examining the incorporation ofinorganic 32phosphate (32P,) into various phospha-tide species under different experimental condi-tions. The membranes of HS red cells leak so-dium ions at inordinate rates (18, 19). Underoptimal conditions, as in the general circulation,HS red cells maintain normal cation distributionsand thus preserve their viability by nearly doublingtheir active pumping of sodium outward againstthe cation gradient (19, 20). Failure of this ac-celerated uphill transport results in accumulationof intracellular sodium and water, followed byosmotic swelling and ultimately hemolysis (19).

The survival of spherocytes becomes normal aftersplenectomy (21), even though they continueto manifest almost double the normal flux of so-dium (19). These cells, therefore, offer a uniqueopportunity to investigate the possible relationshipof membrane phospholipid metabolism and sodiummovements in red cells of normal mean age. Aminimum of experimental manipulation of the cellsis involved in such studies, and conditions of in-creased flux of sodium are maintained.

The results obtained with HS cells in this studyhave been strengthened by the following experi-mental maneuvers: Sodium movements were a)diminished and b) increased in both normal andHS red cells. Further, c) the intracellular levelsof sodium were raised acutely, and d) transportof cations was inhibited by ouabain. In all cases,the incorporation of 32PI into phosphatides wasdetermined. The results obtained provide sup-port for the idea that phospholipids are involved incation flux and also suggest mechanisms for thehemolytic process in HS itself. These resultshave been partially presented in preliminary formelsewhere (22).

Methods

Incubation procedure. Five patients, all of whom hadbeen splenectomized in the past, served as donors of HSred cells. The patients, from four families, demonstratednormal routine blood counts (23), including reticulocytepercentages, at the time of the studies. The followingcharacteristics, considered typical of the disease (24),were present in every donor: a) A congenital hemolyticanemia affected at least two family members; b) com-plete clinical remission occurred after splenectomy; c)spherocytes were demonstrable on peripheral smear, andassociated with this, increased osmotic fragilities of freshand incubated blood were noted. Finally, d) an abnormaldegree of autohemolysis occurred after prolonged incu-bation; this was partially corrected by adding glucose.These five donors have been utilized in previous studiesof the pathogenesis of HS (19, 25). Normal cells wereobtained from healthy male volunteers.

Fresh blood drawn into solution' was centrifuged andthe buffy coat removed, and the cells were resuspendedin the incubation medium. After recentrifugation anda second removal of any remaining buffy coat, the redcells were resuspended in the incubation medium to givea hematocrit of 50Q%. Leukocytes in these cell suspen-sions numbered less than 1,000 and platelets less than

1 Abbott Laboratories, North Chicago, Ill. Each 10ml contains dextrose, 132 mg; sodium citrate, 250 mg;and citric acid, 80 mg.

174

MEMBRANEPHOSPHOLIPID METABOLISMIN HEREDITARYSPHEROCYTOSIS

TABLE I

Incorporation of inorganic phosphate (32Pi) into the phospholipids of red blood cells (RBC)*

Normal Hereditary spherocytosis (HS)Ratio of SA of

Experiment Phospholipid Radioactivity SA Phospholipid Radioactivity SA HS/normal

Jg P/ml RBC cpm/ml RBC cPm/pg P pg P/ml RBC cpm/ml RBC cpm/pg P1 108 34,800 322 107 102,000 953 2.962 101 29,600 292 100 65,100 651 2.233 100 25,000 250 88 49,800 567 2.264 112 94,500 844 134 162,000 1,210 1.445 96 191,000 1,990 106 393,000 3,710 1.866 102 220,000 2,160 106 376,000 3,550 1.65

Mean i SE 103 + 2 107 i 6 2.07t±0.23

* Washed red cell suspensions of the same red cell concentration from four normal and five HS patients were incu-bated in parallel in identical media for 4 hours at 370 C with 32Pj (approximately 100 juc per ml). The phosphate con-centration of the suspending media varied as follows: experiments 1 to 3, 30 mmoles per L; experiment 4, 12 mmolesper L; experiments 5 and 6, 6 mmoles per L.

t p value for difference between HS and normal cells < 0.005.

20,000 per mm3. The incubation medium, describedpreviously (19), was, unless otherwise stated, a buffered5% dialyzed human serum albumin2 solution containingthe following constituents: K+, 3.5 mmoles per L; Na+,160 mmoles per L; P04, 30 mmoles per L; HCOs-, 25mmoles per L; and Cl-, balance of anions. Glucose wasadded after dialysis to a final concentration of 22 mmolesper L. The medium was isosmolal with normal plasmaas determined by freezing point depression.

The cell suspension was equilibrated to a pH of 7.4with a 95% oxygen and 5% carbon dioxide mixture be-fore incubation. After removal of a sample of cells forcounting of blood elements and hematocrit determination,the remaining cell suspension was divided into 5-ml ali-(luots. To these was added carrier-free 3 'Pt (approxi-mately 100 uc per ml red cells), and the stoppered flaskswvere incubated with shaking at 37° C. During the incu-bation the pH of the system dropped by 0.1 to 0.2 U (cf.19).

At various intervals, the cell suspensions were re-moved from the incubation flasks, and the incorporationof label into cellular phospholipids was determined. Inall experiments, the phospholipid metabolism of HS redcells was compared to that of red cells from normal sub-jects incubated at the same time under identicalconditions.

2 Normal human serum albumin was obtained from theM\assachusetts Public Health Biologic Laboratories, as a25% solution, through the courtesy of Dr. R. Pennell.After dialysis against appropriate buffer, the solutionwas diluted to a final concentration of 5% in buffer.This medium was used rather than plasma because previ-ous studies (26, 27) indicated that many of the phos-pholipids present in plasma are rapidly (but not pre-dictably) exchangeable with those of the membranes ofred cells incubated therein.

3 Available from IsoServe, Cambridge, Mass., asH3,PO4 in 0.1 N HCl; the solution was adjusted to neu-tral pH with 0.5 N NaOH.

Extraction and analysis of red cell lipids. After incu-bation, the red cell suspensions were washed three timeswith large volumes of ice cold phosphate-buffered iso-tonic saline solution (final P04 concentration, 40 mmolesper L) and again made to original volume. Lipids wereextracted from the cell suspension by procedure III ofWays and Hanahan (28). Although this procedure hasbeen shown to yield lipid virtually free of nonlipid con-taminants, it was modified in a minor way by increasingthe number of aqueous washes of the final lipid extractfrom one to three. This assured even more fully theabsence of contamination of lipid by inorganic phosphateand other water-soluble phosphorylated compounds. Toreduce the heme-catalyzed oxidation of lipids (29), weperformed the initial extraction steps in an atmosphereof dry nitrogen.4 Samples of lipid extract were analyzedfor phosphorus by the method of Lowry and co-workers(30) scaled up to a convenient level and were countedafter drying on aluminum planchets in a Nuclear Chicagogas flow counter (model 186A). Duplicate planchets werecounted under such conditions as to maintain a countingerror no greater than 1%. Agreement between dupli-cate samples was within +2%o. The remaining lipidextract in small volumes of chloroform-methanol (2: 1)was spotted on specially cut, long, narrow (2- X 16-inch)glass plates coated with silica gel G as described byStahl (31), and ascending thin layer chromatographicseparation of the individual phosphatides was performedwith the solvent system, chloroform: methanol: aceticacid: water (500: 300: 80: 40, vol: vol) as described bySkipski, Peterson, and Barclay (32). The separatedphosphatides were localized with iodine or sulfuric acidspray and were identified by comparison with referencecompounds,5 by group-specific sprays such as Ninhydrin

4Good results were obtained by extracting under ni-trogen in glove boxes available from Instruments forResearch, Cheltenham, Pa.

5 The authors are grateful to Dr. E. P. Kennedy, whogenerously provided phosphatidylinositol-3H and phos-

175

HARRYS. JACOB ANDMANFREDL. KARNOVSKY

a.

EL

200 AD/ /

0 2 3 4HOURS

FIG. 1. THE INCORPORATIONOF INORGANI

('PI) INTO THE PHOSPHATIDES OF HEREDII

CYTOSIS (HS) AND NORMALRED CELLS. WIat 370 C in identical media with "P, the p1

washed HS red cells incorporated more Lthose of normal cells at all time intervalsdepicted is representative of six performedcells from five HS and four normal donors.

for a-amino groups and the Dragendorffcholine, and by deacylation and chromatogiter-soluble products on paper with the phacetic acid (100: 12: 10) solvent systemDawson, Hemington, and Davenport (33).

Thin layer chromatography on long glasduced excellent separation of the originaland permitted automated scanning of the defor radioactivity on a Baird Atomic scanner363. After development, scanning, and ,

area of silica gel containing each phosphamoved from the plate, and a weighed sat

gested and analyzed for phosphorus by mc

the methods of Lowry and co-workers (30blum and Chain (34) as follows: Digestiformed in perchloric acid and color develoyto Lowry and co-workers (30). After itsthe blue phosphomolybdous complex was

tracted into 4 ml of isobutanol (34) and thesity determined at 790 mu on a sample aft(tion.6 A sample of the isobutanol solution w-

phatidylserine-14C, and to Dr. John Law iphosphatidic acid and phosphatidylethanolaias standard reference compounds.

6 This method of dealing with thin layeraphy fractions was kindly made availableA. W. Shafer.

to aluminum planchets for counting of radioactivity asdescribed above. From these data specific activities

HEREDITARY (counts per minute per microgram phosphorus) of the;PHEROCYTOSIS individual phosphatides could be calculated.

Results

The uptake of 32p, into the phospholipids of HSand normal red blood cells.

As shown in Table I the total quantity of phos-pholipid that was extractable from HS red cellsdid not differ in any consistent fashion from that

NORMAL of normal cells. On the other hand, the incorpora-tion of label into phospholipid from added 32p, wassignificantly greater in HS red cells than in normalcells in all six experiments. After 4 hours of incu-bation, the specific activity of HS red cell phos-pholipid averaged slightly more than twice thatin the normal red cells, which had been incubatedsimultaneously under identical conditions. The

IC PHOSPHATE numbers of leukocytes and platelets in the cell sus-rARY SPHERO- pensions in these experiments varied randomly andhen incubated did not correlate with the results shown.-eosphatides of An increased rate of incorporation of 32PI into

The study HS red cell phospholipid was manifest at all in-utilizing red tervals studied, the shortest being 30 minutes, as

shown in Figure 1. This increased labeling oflipids was not associated with an abnormally rapid

reagent for influx of inorganic phosphate itself into HS cells;Lenofethanol the over-all uptake of radioactivity (32P5) fromdescribed by the incubation media was determined to be no dif-

ferent in HS cells from that in normal red cell;s plates pro- suspensions, in confirmation of previous results

phosphatides of others (35).veloped plate

r model RSCspraying, the The labeling of individual phosphatides in HSandLtide was re- normal red cellsmple was di-difications of The lower portion of Figure 2 demonstrates the) and Beren- silica gel chromatographic separation of red cellion was per- phosphatides on long glass plates. The materialped according present in each numbered zone was identified asdevelopment, flos

readily ex- follows:e optical den- Zone 1 contains material remaining at the ori-er centrifuga- gin after chromatographic development, includingas transferred contaminating inorganic phosphate or other water-for providing soluble, nonlipid phosphorylated compounds.mine for use Polyphosphoinositides may also be present in this

r chromatog- zone (36).to us by D~r. Zone 2 material was identified as sphingomyelin

by agreement with Rf values reported by Skipski

176


-*---T-.-74~~ ~ ~ ~ ~ ~ - -

: ~4_ . ! 1;.-.t:: -- -',,,,......

FIw.1-H C~f,1 . -1z5jj riiise _______,. ..... . .* _'....*_r-

6 .... 5 -4 3

...... ....... . . ..._...K.Y._

._...

._..__wEi *w.,^ , .......... .... ....... A ..............

#3 1*,x, .: :: .:

FIG. 2. THIN LAYER CHROMATOGRAPHYOF 3P-LABELED PHOSPHATIDESFROMRED CELLS.The extracted phosphatides from red cells incubated with 3P2, are chromatographically sep-arated into six zones on 2- x 16-inch glass plates coated with silica gel. The zones are re-vealed by spraying with H2S04 and heating (bottom). For identification of individualphosphatides, see text. By radioautography (middle), origin material (zone 1), phospha-tidylserine (zone 4), and phosphatidic acid (zone 6) are heavily labeled. This is also dem-onstrated in the top portion by automated radioactivity scanning (see text).

and associates (32), by comparison with authenticsamples in this laboratory, by its positive reactionfor choline when sprayed with the Dragendorffreagent, and by the extent of its occurrence in thetotal phosphatide pool, which corresponds withpreviously published (28, 37) values for sphingo-myelin in red cells.

Zone 3 was identified as phosphatidylcholine bythe kinds of criteria noted for zone 2. It had achromatographic mobility identical to that of asample of authentic egg lecithin, in the same sys-tem. Deacylation of material in this zone by mildalkaline hydrolysis followed by rechromatographyon paper (33) yielded a compound running withthe same Rf as glycerophosphorylcholine obtainedfrom standard synthetic lecithin.7

Zone 4 was identified as phosphatidylserine bythe coincidence of its Rf value with referencestandard material under the same chromatographicconditions. The compound is Ninhydrin positive,and after deacylation and rechromatography on

Generously provided by Dr. Erich Baer.

paper (33), a Ninhydrin-positive compound wasobtained with mobility identical to that of stand-ard glycerophosphorylserine derived from syn-thetic phosphatidylserine. After prolonged stor-age at 40 C, spontaneous partial deacylation of thematerial from red cells yielded a Ninhydrin-positivecompound that upon rechromatography on silicagel ran coincidentally with a product (lysophos-phatidylserine) formed in the same way from au-thentic phosphatidylserine. The presence of avery small quantity of phosphatidylinositol in thiszone is a possibility that cannot be completelyexcluded.

Zone 5 was identified as phosphatidylethanola-mine by its Rf value, which was identical to thatof reference standard material 5 during simultane-ous silica gel chromatography. The compound isNinhydrin positive and yielded a water-solubleNinhydrin-positive substance with the mobility ofstandard glycerophosphorylethanolamine derivedfrom phosphatidylethanolamine after mild alkalinehydrolysis and rechromatography on paper (33).

177

..1.4


Zone 6 contains neutral lipids and phosphatidicacid, the latter identified by the matching of itsRf with that of reference standard material 5 dur-ing silica gel chromatography. Cardiolipin, whosepresence in red cells is not yet certain (28, 33, 37),might also be present in this zone.

Radioautography of labeled and chromatographi-cally separated phosphatides from red cells incu-bated with 32P1 for 4 hours is shown in the middleportion of Figure 2 (the corresponding stainedplate is shown in the lower portion of the Figure).Origin material (zone 1), phosphatidylserine (andpossibly phosphatidylinositol) (zone 4), andphosphatidic acid (zone 6) are labeled. Sphingo-myelin (zone 2), lecithin (zone 3), and phos-phatidylethanolamine (zone 5), although compris-ing approximately 85% of the total phospholipidcontent of red cells (28, 37), had not significantlyincorporated 32Pi during the time interval of thisincubation and under the conditions of the detec-tion techniques employed. The upper portion ofFigure 2 shows the radioactive scan of the thinlayer plate portrayed in the lower portion. Thesymmetry of the peaks suggests the homogeneityof radioactive compounds.

Phosphatidylinositol, whose presence in red cellsis controversial (28, 33), behaves similarly tophosphatidylserine in the chromatographic sys-

tem utilized in Figure 2. We attempted, as fol-lows, to determine whether labeled inositide mightindeed be present in zone 4: The material fromthis zone was eluted (32), deacylated by mild alka-line treatment (33), and mixed with standard,nonradioactive glycerophosphorylserine (GPS)and radioactive glycerophosphorylinositol (GPI)

tritiated in the inositol moiety.5 After rechroma-tography on paper (33), a homogeneous 32p_labeled peak was found to be exactly coincidentwith the Ninhydrin-positive, standard GPS spot.A homogeneous peak of radioactivity due to tritiumalone 8 ran more slowly with an Rf two-thirds thatof the peak containing 32p. We tentatively con-

cluded, therefore, that phosphatidylserine is theonly labeled compound significantly present inzone 4 of our thin layer chromatograms. In viewof the faint element of doubt that remains, how-ever, this zone will be referred to as the "phos-

phatidylserine fraction."The over-all proportions of individual phospho-

lipids in HS red cells were found to be the same

as in normal cells, confirming the results of others(38, 39) .9 However, the incorporation of 32P,into the various membrane phosphatides of thesetwo cell types was clearly different. As shown inTable II, red cell phosphatidic acid was highly la-beled after 4 hours of incubation with 32P1. Inthree of six experiments, the specific activity ofthis compound was the same in HS and in normalred cells, whereas in the remaining experiments,its activity was higher in spherocytes.

The specific activity of the phosphatidylserinefraction sometimes exceeded that of phosphatidicacid, but more importantly, was consistently in-

8 Counted as paper strips 1 cm X 2X cm in a liquidscintillation spectrometer, Packard Instrument Co., LaGrange, Ill.

9 The mean percentage distribution of phosphatidesin HS and normal red cells was as follows: phospha-tidylcholine (lecithin), 32%; phosphatidylethanolamine,25o; sphingomyelin, 25o; phosphatidylserine, 10%o; phos-

phatidic acid, 3%; unknown, 5%,.

TABLE II

Incorporation of 32Pi into the "acidic" phosphatides of red blood cells*

Phosphatidic acid fraction Phosphatidylserine fraction

Experiment Normal HS HS/normal Normal HS HS/normal

cpm/ jg P cpm/pg P

1 6,830 7,020 1.02 4,900 7,550 1.542 4,270 10,200 2.39 1,900 4,080 2.053 3,470 9,390 2.70 1,450 2,220 1.534 9,950 9,970 1.00 5,020 13,500 2.695 23,200 22,900 0.99 17,000 42,200 2.486 10,400 34,300 3.30 16,000 36,200 2.26

Mean i SE 1.90 i 0.42 2.09 ±t 0.20p = 0.10 p = <0.005

* The conditions of incubation are described in Table I.

178


creased in HS red cells relative to normal cells(Table II), and this was so at all time intervalsstudied (Figure 3).

Effect of decreased concentration of sodium on redcell phospholipid metabolism

When red cells are suspended in media of lowsodium content, and the passive diffusion of thision into the cells is thereby diminished, the re-

quirement for active transport of sodium in theopposite direction is reduced. Previous studieshave shown that incubation in media in whichcholine replaces sodium decreases the abnormalosmotic swelling and autohemolysis that occur inincubated HS red cells (19). As shown in TableIII, the incorporation of 32P, into phosphatidicacid was reduced by an average of 35% in threeexperiments in which HS and normal red cellswere suspended in media containing cholinerather than sodium. The labeling of the phos-phatidylserine fraction was also significantly di-minished in the one experiment in which HS cellswere suspended in choline media; in normal cellsthis effect was not consistently observed.

Effects of increased flux of cations on red cellphospholipid metabolism

Suspension of red cells in mildly hypotonic me-

dium produces osmotic swelling and increasedmembrane permeability to a variety of small mole-cules. In response, active transport of cations bythese cells accelerates (40). Previous studies,both in red cells (19) and in brain tissue (41),have indicated that by such treatment an ouabain

OL

0

a-Co

0

NORMAL

0 1 2 3 4HOURS

FIG. 3. THE INCORPORATION OF s2P, INTO THE PHOS-

PHATIDYLSERINE FRACTION OF HS AND NORMALRED CELLS.

The specific activity of this phosphatidylserine fractionfrom HS red cells exceeds that from normal cells at alltime intervals. The conditions of incubation are given inthe legend to Figure 1.

inhibitable ATPase of the cell membrane is stimu-lated. As shown in Table IV, the labeling of thephosphatidylserine fraction is accelerated by sus-

pension of red cells in hypotonic media that do notcause hemolysis. In the five experiments per-

formed, the effect was more pronounced in nor-

mal red cells (three experiments) than in HS redcells (two experiments) whose cation permeabilityand phosphatide metabolism are already higherthan normal. In these experiments, the incorpora-tion of 32P1 into red cell phosphatides other thanthose of the phosphatidylserine fraction was notaltered by hypotonicity in any consistent fashion.

TABLE III

Effect of extracellular sodium on the labeling of red cell phosphatides*

Sodiumin

Red cells medium Phosphatidic acid fraction Phosphatidylserine fraction

cPm/jsg P %change cPm/pg P %changeHS + 22,900 42,200

18,900 -17 27,900 -34

Normal + 654 6,750390 -40 5,500 -19

Normal + 23,200 17,00012,100 -48 17,700 +4

* Two aliquots of the same red cells were incubated in parallel with 32Pi for 4 hours at 370 C in identical suspendingmedia except for sodium content. In sodium-free media, an equivalent amount of choline chloride replaced sodiumchloride.

179


TABLE IV

Effect of osmotic swelling on labeling of the phosphatidyl-serine fraction in red cells*

Medium %effect

Isotonic Hypotonic b -a 100Experiment (a) (b) a

Cpm/pIg PHS 1 13,500 16,800 +24HS2 42,200 43,400 + 3Normal 1 5,020 8,870 +77Normal 2 17,000 22,700 +34Normal 3 1,720 2,370 +38

35 i 12t

* Two aliquots of the same red cell suspension were in-cubated in parallel with 32p- for 4 hours at 370 C in mediathat differed only in respect to tonicity. Isotonic mediawere 300, and hypotonic media 200 milliosmolal. HSrepre-sents experiments with spherocytes from two donors.Normal red cells were obtained from two donors.

t p value for difference between experiments in isotonicand hypotonic media = 0.03.

For example, in the case of phosphatidic acid, ef-fects due to hypotonicity varied from a decreaseof 50%o to an increase of 200% in five experiments.The effects with respect to total phosphatide werefrom - 30% to + 25%o.

Effects of Na loading on red cell phospholipidmetabolism

A reversible increase in membrane permeabilitycan be produced in red cells by exposing them forbrief periods to cold 0.5 N butanol (42, 43).During this period of increased permeability, red

TABLE V

Effect of Na loading on labeling of RBCphospholipids*

"Control" Na+-IoadedRBC RBC

pcm/psg P

Total 1,125 1,436phospholipid

Phosphatidyl- 3,383 4,440serine fraction

Phosphatidic 253 1,300acid fraction

* Two aliquots of packed, washed red cells were sus-

pended in equal volumes of n-butanol diluted to 0.5 moleper L in either 0.15 M NaCl (Na-loaded RBC) or KCI("control" RBC). The cells were incubated for 20 minutesat 100 C. Thereafter Na-loaded cells were washed and in-cubated for 4 hours at 370 C with 32pi in the usual albuminmedium of high Na concentration. "Control" cells were

incubated similarly in medium of identical compositionexcept for replacement of all btut 20 mEqper L Na+ by K+.The final intracellular Na+ content of "control" RBCwas

12 mEq per L of RBC; that of Na+-loaded cells was 17mEqper L of RBC.

cells rapidly incorporate cations from the externalmedium into their intracellular space. After re-moval of butanol and washing of the cells, mem-brane permeability becomes normal, and cellsloaded with extracellular cations are thereby ob-tained (44). The labeling of phospholipids fromNa-loaded red cells, which had been prepared byexposure to butanol while suspended in media ofhigh Na+ concentration, was compared to that ofsimilarly treated "control" cells whose Na con-tent was normal after exposure to butanol in mediaof low Na (high K) concentration. As shown inTable V, the specific activity of the total phospho-lipid pool and of the phosphatidylserine fractionwas increased by about 30% in Na-loaded redcells relative to control cells with normal Na con-tent. In a further experiment restricted to mea-

TABLE VI

Effect of ouabain on the specific activity of phosphatidesin HSred cells*

Ouabain Phosphatidic Phosphatidyl-in acid serine

Experiment medium fraction fraction

cpm/pg P cPm/Ipg P

1 - 9,970 13,500+ 14,800 15,400

2 - 34,300 36,200+ 36,500 39,500

* Two aliquots of the same red cell suspensions were in-cubated in parallel with 32p; for 4 hours at 370 C in identicalsuspending media except for the presence of 5 X 10- MIouabain. Experiments 1 and 2 represent cells from two HSdonors.

surements on total phosphatides, a stimulation of30%o due to Na+ loading was obtained. Labelingof phosphatidic acid was even more markedlyenhanced in the Na-loaded cells.

Effect of ouabain on phospholipid metabolism inred cells

The cardiac glycoside, ouabain, depresses ac-tive pumping of cations in biological membranesby depressing membrane ATPase activity (2).This is the result of the inhibition by ouabain ofthe dephosphorylation of intermediates that wereoriginally phosphorylated through the agency ofATP (45-47). This dephosphorylation step ap-pears essential to ATPase activity and also to thetransport process. One might predict that block-ade of the dephosphorylation step by ouabain

180


might lead to an increased level of radioactivity inthe phosphate moiety of intermediates involved inactive transport. The specific activity of bothphosphatidic acid and phosphatidylserine fractionsin HS red cells incubated with 32P, was slightlyenhanced by ouabain as shown in Table VI. Simi-lar effects on normal red cells and other tissueshave been previously reported (11, 48-50).

Discussion

The molecular basis of active "uphill" transportof cations through biological membranes has beenintensively investigated and recently reviewed(1, 2). It has been conclusively demonstratedthat this process in red cells is energized by ATP(51-54), through an ATPase system which, inturn, is regulated by the concentrations of intra-cellular sodium and extracellular potassium ions(15, 55). It is also generally accepted that cationsare transported across membranes through theagency of carrier molecules, which bind them onone side of the membrane, ferry them to the op-posite side, and there discharge them (1). Thepossible involvement of phospholipids in this proc-ess has been repeatedly suggested because they areconstituents of membranes and because they areable to bind cations in the lipid milieu of mem-branes (1, 2-4, 8-10). The results of the pres-ent studies, which demonstrate alterations in phos-pholipid metabolism concomitant with sodiumtransport in red cells, are consistent with this sug-gestion. Indeed, the incorporation of 32P, intomembrane phosphatides has been found to be en-hanced in a variety of tissues in which transportof cations (11), as well as hormones (6), en-zymes, and other proteins (56), or particulatematter (7) has been transiently provoked. In ourstudies incorporation of 32P, into the phosphatidesof HS red cells was roughly twice normal, whichcoincides with the nearly twofold increment inactive transport of sodium ions in these cells (19,20). The acidic phosphatides, in the phosphatidyl-serine fraction, and less consistently, the phospha-tidic acid fraction, were mainly involved in thishypermetabolism. The present studies also dem-onstrated that when alterations in the flux ofsodium were artificially induced in red cells, la-beling of membrane phosphatides was concomi-tantly altered. Thus, the radioactivity of the phos-

phatidylserine and phosphatidic acid fractions wasdecreased toward normal in HS red cells whosesodium fluxes had been reduced by suspendingthem in media of low sodium content (Table III).Osmotically swollen red cells, whose flux of ca-tions is increased, exhibited an increased labelingin their phosphatidylserine (but not their phos-phatidic acid) fraction (Table IV). Further-more, Na-loaded red cells, which have previouslybeen shown to have increased ATPase (44) andNa-pumping activities (43), incorporated in-creased amounts of 32P1 into their membranephosphatides (Table V).

Although these studies demonstrate that 32P, in-corporation into the acidic phosphatides of redcells can be roughly correlated with flux ofcations, they do not allow conclusions regard-ing the mechanism or mechanisms by whichphosphatides may be involved in transport phe-nomena. It seems unlikely that phosphatidylserineor phosphatidic acid acts directly as a cation car-rier in biological membranes. The metabolic se-quence of incorporation and loss of phosphate inphosphatidylserine of red cells is not specificallyknown, and detailed calculations of turnover ratesof the phosphate moiety in this compound cannotbe made. Nevertheless, the uptake of phosphateby this fraction (roughly 10 umoles per L of cellsper hour) seems orders of magnitude removedfrom that required if this phosphate group wereutilized for direct cation binding and transfer.For instance, it has been estimated that roughly1,000 umoles of phosphate per L of cells per hourmust be turned over to support the establishedflux of 3 mEq of sodium ions per L red cells perhour (16). This serious discrepancy in thestoichiometry of cation transport and phospholipidmetabolism has been noted also in other tissuesand has led to the suggestion (11) that acidicphosphatides, rather than directly carrying cations,alter the conformation, and hence the cation avid-ity, of other molecules, perhaps proteins (57, 58).which act as the actual cation carriers. This sug-gestion is strengthened by recent evidence ofTosteson, Cook, and Blount (59) that ATPaseactivity involved in cation transport can be foundin a proteinaceous fraction from sonically dis-rupted red cell membranes. This fraction con-tains the bulk of membrane phosphatides. Re-gardless of what mechanism might be involved, it

181


would appear from the present studies that acidicphosphatides do have a role in the movementof cations in red cells. A similar conclusion hasbeen reached recently by Ohnishi and Kawamurafrom evidence that red cell ATPase that has beeninhibited by a snake venom phospholipase can bereactivated specifically and solely by replacementof phosphatidylserine (60). On the other hand,it should be noted that in some systems other thanred cells, such as the toad bladder (61) and theelectric organ of the eel (62), the metabolism ofacidic phosphatides does not correlate with cationtransport. In this respect, our studies do notallow us to conclude whether changes in phos-phatide metabolism are linked to active or to pas-sive transfer of cations, or to both. In each of thecircumstances in which increased labeling of phos-phatides was noted, both passive flux and activetransport of cations were increased.

Our finding that 32P, of the external mediumwas incorporated into the membrane phosphatidyl-serine fraction to a significant degree is at variancewith some previous reports (63, 64) and in agree-ment with others (65). Differences in incubationprocedures, activities of 32Pi, and especially thechoice of suspending media may explain these dis-crepancies. We used buffered human albuminsolutions as suspending media to circumvent varia-tions in the behavior of different plasma samples(66) and to eliminate such phenomena as plasma-red cell exchange of phosphatides (26, 27). Theover-all incorporation of 32P1 into red cell phos-phatides was suppressed nearly tenfold when weused plasma as the incubation medium ratherthan buffered human albumin.

It is also possible that other phosphatides, be-sides phosphatidylserine and phosphatidic acid,might be present in tiny amounts in red cells andbe of high radioactivity, yet not be recognized byour chromatographic techniques. We have es-pecially sought for phosphatidylinositides sincethe turnover of this class in various tissues hasbeen shown to be altered concomitantly with al-terations in membrane function (17). RecentlyKirschner and Barker (67) have offered evidencein swine red cells that a phosphatide-probably apolyphosphoinositide-incorporates 32P, markedlyfrom suspending media and contaminates the phos-phatidic acid separated by column chromatography.Using inositol-labeled phosphatidylinositol as a

reference chromatographic standard, we have beenunable to document labeling of monophosphatidyl-inositol from 32P1 in red cells under our conditions.Indeed the presence of this compound in humanred cells is conjectural (28, 33). Little attentionhas been paid to zone 1, in which polyphospho-inositides would be found, because this zone didnot respond in any striking way to various ex-perimental conditions.

With our chromatographic techniques, the iden-tification of the highly labeled phosphatides as be-ing solely phosphatidylserine and phosphatidicacid should thus not be considered as conclusive.Further, because the amount of phosphatidic acidin red cells is small (28), contamination with otherphosphorus-containing lipids, such as polyglycerolphosphatides, might affect the observed specificactivity of the phosphatidic acid fraction mark-edly. The absolute specific activities of phos-phatidic acid in these studies thus remain in doubt.On the other hand, the changes in specific activityof the phosphatidic acid fraction with altered cationflux for any given batch of cells whose lipidswere extracted and separated under rigidly stand-ardized conditions are, in our belief, secure.

HS red cells manifest increased glucose (19)and ATP (68-70) catabolism, which has beenshown to relate to their heightened active transportof sodium (19). It is possible that the increasedmetabolism of phospholipids we observed in thesecells reflects simply this general hypermetabolism.This possibility is thought unlikely for the follow-ing reasons: 1) All membrane phosphatides inHS red cells are not of increased radioactivity.In fact, phosphatidic acid, whose phosphate groupis probably derived directly from ATP (71), wasnot labeled excessively in HS red cells in three ofsix experiments (Table II) despite increased la-beling of the total phosphatide pool of these cells.2) The increased metabolism of the acidic phos-phatides of HS cells is further enhanced afterexposure to ouabain (Table VI) despite the factthat this glycoside renders these cells eumetabolic(19, 70). 3) Osmotic swelling of red cells, whichincreases their glucose catabolism (19), consist-ently increased the labeling of the phosphatidyl-serine fraction (Table IV), yet often diminishedthe uptake of 32P, into the total red cell phospho-lipid pool.

In our preliminary communication (22), we sug-

182


gested that the increased phospholipid metabolismof HS red cell membranes might be involved bothin the abnormal shape and the propensity to un-dergo osmotic hemolysis. If abnormally rapidturnover of HS phospholipids is associated withdecreased avidity of their attachment to the mem-brane structure, an accelerated loss of surface lipidmaterial from these cells might ensue. The re-sulting diminution in surface area with constantcellular volume would necessarily result in a morespheroidal cell. Indeed, studies by Prankerd (68)and more recently by Weed, Bowdler, and Reed(72) have demonstrated losses of membrane lipidsfrom HS red cells during incubation in vitro.Jacob has confirmed these observations and haspresented evidence that this lipid instability is re-lated to the accelerated flux of sodium in HSred cells, for it is completely prevented whenflux of sodium is made normal (73). Thusthe defect in HS membranes that increases thepermeability of the red cell to sodium might notonly jeopardize the integrity of the cell by directlyincreasing the tendency for osmotic swelling andlysis, but also might increase the tendency tospheroidicity by provoking the depletion of mem-brane phospholipids (73). This depletion isconceivably linked to the increased labeling ofphosphatides observed in the experiments re-ported above, but no mechanistic explanation isyet available.

References

1. Hokin, L. E., and M. R. Hokin. Biological trans-port. Ann. Rev. Biochem. 1963, 32, 553.

2. Skou, J. C. Enzymatic basis for active transport ofNa+ and K+ across cell membrane. Physiol. Rev.1965, 45, 596.

3. Solomon, A. K., F. Lionetti, and P. F. Curran. Pos-sible cation-carrier substances in blood. Nature(Lond.) 1956, 178, 582.

4. Kirschner, L. B. The cation content of phospho-lipides from swine erythrocytes. J. gen. Physiol.1958, 42, 231.

5. Larrabee, M. G., J. D. Klingman, and W. S. Leicht.Effects of temperature, calcium and activity onphospholipid metabolism in a sympathetic ganglion.J. Neurochem. 1963, 10, 549.

6. Freinkel, N. Pathways of thyroidal phosphorus me-tabolism: the effect of pituitary thyrotropin uponthe phospholipids of the sheep thyroid gland. En-docrinology 1957, 61, 448.

7. Karnovsky, M. L., and D. F. H. Wallach. Themetabolic basis of phagocytosis. III. Incorpora-

tion of inorganic phosphate into various classes ofphosphatides during phagocytosis. J. biol. Chem.1961, 236, 1895.

8. Christensen, H. N., and A. B. Hastings. Phospha-tides and inorganic salts. J. biol. Chem. 1940, 136,387.

9. Abramson, M. B., R. Katzman, C. E. Wilson, andH. P. Gregor. Ionic properties of aqueous dis-persions of phosphatidic acid. J. biol. Chem. 1964,239, 4066.

10. Garvin, J. E., and M. L. Karnovsky. The titrationof some phosphatides and related compounds in anon-aqueous medium. J. biol. Chem. 1956, 221, 211.

11. Hokin, L. E., and M. R. Hokin. Phosphatidic acidmetabolism and active transport of sodium. Fed.Proc. 1963, 22, 8.

12. Hokin, L. E., M. R. Hokin, and D. Mathison.Phosphatidic acid phosphatase in the erythrocytemembrane. Biochim. biophys. Acta (Amst.) 1963,67, 485.

13. Hokin, L. E., and M. R. Hokin. Diglyceride kinaseand other pathways for phosphatidic acid syn-thesis in the erythrocyte membrane. Biochim. bio-phys. Acta (Amst.) 1963, 67, 470.

14. Jarnefelt, J. Some aspects of the physiological sig-nificance of the adenosinetriphosphatase of brainmicrosomes. Biochim. biophys. Acta (Amst.)1962, 59, 655.

15. Glynn, I. M. Activation of adenosinetriphosphataseactivity in a cell membrane by external potassiumand internal sodium. J. Physiol. (Lond.) 1962,160, 18P.

16. Sen, A. K., and R. L. Post. Stoichiometry andlocalization of adenosine triphosphate-dependentsodium and potassium transport in the erythrocyte.J. biol. Chem. 1964, 239, 345.

17. Hokin, M. R. Phosphatidylinositol and phospha-tidic acid interconversion in the triggering byacetylcholine of NaCl secretion in the salt gland.Fed. Proc. 1965, 24, 294.

18. Bertles, J. F. Sodium transport across the surfacemembrane of red blood cells in hereditary sphero-cytosis. J. clin. Invest. 1957, 36, 816.

19. Jacob, H. S., and J. H. Jandl. Increased cell mem-brane permeability in the pathogenesis of heredi-tary spherocytosis. J. clin. Invest. 1964, 43, 1704.

20. Harris, E. J., and T. A. J. Prankerd. The rate ofsodium extrusion from human erythrocytes. J.Physiol. (Lond.) 1953, 121, 470.

21. Emerson, C. P. The influence of the spleen on theosmotic behavior and the longevity of red cells inhereditary spherocytosis (congenital hemolyticjaundice) : a case study. Boston med. Quart. 1954,5, 65.

22. Jacob, H. S., and M. L. Karnovsky. Concomitantincrease of membrane phosphatide metabolism andsodium transport in hereditary spherocytosis (HS)(abstract). J. clin. Invest. 1965, 44, 1062.

23. Ham, T. H. A Syllabus of Laboratory Examinationsin Clinical Diagnoses. Critical Evaluation of Lab-

183


oratory Procedures in the Study of the Patient,rev. ed., L. B. Page and P. J. Culver, Eds. Cam-bridge, Harvard University Press, 1960.

24. Jandl, J. H. Hereditary spherocytosis in The Meta-bolic Basis of Inherited Disease, J. B. Stanbury,J. B. Wyngaarden, and D. S. Fredrickson, Eds.New York, McGraw-Hill, 1966, p. 1035.

25. Jacob, H. S. Hereditary spherocytosis: a disease ofthe red cell membrane. Seminars Hemat. 1965, 2,139.

26. Sakagami, T., 0. Minari, and T. Orii. Behavior ofplasma lipoproteins during exchange of phospho-lipids between plasma and erythrocytes. Biochim.biophys. Acta (Amst.) 1965, 98, 111.

27. Sakagami, T., 0. Minari, and T. Orii. Interaction ofindividual phospholipids between rat plasma anderythrocytes in vitro. Biochim. biophys. Acta(Amst.) 1965, 98, 356.

28. Ways, P., and D. Hanahan. Characterization andquantification of red cell lipids in normal man. J.Lipid Res. 1964, 5, 318.

29. Tarladgis, B. G. An hypothesis for the mechanismof the heme catalyzed lipid oxidation in animaltissues. J. Amer. Oil chem. Soc. 1961, 38, 479.

30. Lowry, 0. H., N. R. Roberts, K. Y. Leiner, M. L.Wu, and A. L. Farr. The quantitative histochem-istry of brain. I. Chemical methods. J. biol.Chem. 1954, 207, 1.

31. Stahl, E. in Dunnschicht-chromatographie. E. Stahl,Ed. Berlin, Springer-Verlag, 1962, p. 5.

32. Skipski, V. P., R. F. Peterson, and M. Barclay.Quantitative analysis of phospholipids by thin-layer chromatography. Biochem. J. 1964, 90, 374.

33. Dawson, R. M. C., N. Hemington, and J. B. Daven-port. Improvements in the method of determiningindividual phospholipids in a complex mixture bysuccessive chemical hydrolyses. Biochem. J. 1962,84, 497.

34. Berenblum, I., and E. Chain. An improved methodfor the colorimetric determination of phosphate.Biochem. J. 1938, 32, 295.

35. Altman, K. I., H. Tabechian, and L. E. Young.Some aspects of the metabolism of red blood cellsfrom patients with hemolytic anemias. Ann. N. Y.Acad. Sci. 1958, 75, 142.

36. Shafer, A. W. Personal communication.37. Reed, C. F., S. N. Swisher, G. V. Marinetti, and

E. G. Eden. Studies of the lipids of the erythro-cyte. I. Quantitative analysis of the lipids of nor-mal human red blood cells. J. Lab. clin. Med.1960, 56, 281.

38. DeGier, J., L. L. M. van Deenen, R. A. Geerdink,K. Punt, and M. C. Verloop. Phosphatide patternsof normal, spherocytic, and elliptocytic red bloodcells. Biochim. biophys. Acta (Amst.) 1961, 50,383.

39. Phillips, G. B., and N. S. Roome. Quantitative chro-matographic analysis of the phospholipids of ab-normal human red blood cells. Proc. Soc. exp.Biol. (N. Y.) 1962, 109, 360.

40. Ponder, E. Hemolysis and Related Phenomena.New York, Grune & Stratton, 1948.

41. Yoshida, H., and H. Fujisawa. Influence of sub-cellular structures on the activity of Na+, K+-ac-tivated adenosine triphosphatase in brain. Bio-chim. biophys. Acta (Amst.) 1962, 60, 443.

42. Parpart, A. K., and J. W. Green. Potassium andsodium exchanges in rabbit red cells treated withnt-butyl alcohol. J. cell. comp. Physiol. 1951, 38,347.

43. Green, J. W., and G. Bond. Cation fluxes in butanoltreated erythrocytes. Fed. Proc. 1961, 20, 143.

44. Laris, P. C., and P. E. Letchworth. Cation influ-ence on inorganic phosphate production in humanerythrocytes. J. cell. comp. Physiol. 1962, 60, 229.

45. Ahmed, K., and J. D. Judah. Identification of activephosphoprotein in a cation-activated adenosinetriphosphatase. Biochim. biophys. Acta (Amst.)1965, 104, 112.

46. Charnock, J. S., and R. L. Post. Evidence on themechanism of ouabain inhibition of cation acti-vated adenosine triphosphatase. Nature (Lond.)1963, 199, 910.

47. Gibbs, R., P. M. Roddy, and E. Titus. Preparation,assay, and properties of an Na+- and K+-requiringadenosine triphosphatase from beef brain. J. biol.Chem. 1965, 240, 2181.

48. Nagano, K., and M. Nakao. Cation carrier in theerythrocyte membrane. J. Biochem. (Tokyo) 1962,52, 99.

49. Nicholls, D., J. Kanfer, and E. Titus. The effectof ouabain on the incorporation of inorganic P' intophospholipid. J. biol. Chem. 1962, 237, 1043.

50. Yoshida, H., T. Nukada, and H. Fujisawa. Effect ofouabain on ion transport and metabolic turnoverof phospholipid of brain slices. Biochim. biophys.Acta (Amst.) 1961, 48, 614.

51. Gardos, G. Akkumulation der Kaliumionen durchmenschliche Blutkorperchen. Acta physiol. Acad.Sci. hung. 1954, 6, 191.

52. Dunham, E. T. Linkage of active cation transport toATP utilization. Physiologist 1957, 1, 23.

53. Whittam, R. Potassium movements and ATP inhuman red cells. J. Physiol. (Lond.) 1958, 140,479.

54. Hoffman, J. F. Cation transport and structure ofthe red-cell plasma membrane. Circulation 1962,26, 1201.

55. Whittam, R. The asymmetrical stimulation of amembrane adenosine triphosphatase in relationto active cation transport. Biochem. J. 1962, 84,110.

56. Hokin, L. E., and A. L. Sherwin. Protein secretionand phosphate turnover in the phospholipids insalivary glands in vitro. J. Physiol. (Lond.) 1957,135, 18.

57. Judah, J. D., K. Ahmed, and A. E. M. McLean.Ion transport and phosphoproteins in humarred cells. Biochim. biophys. Acta (Amst.) 1962,65, 472.

184


58. Ahmed, K., and J. D. Judah. Preparation of lipo-proteins containing cation-dependent ATPase. Bio-chim. biophys. Acta (Amst.) 1964, 93, 603.

59. Tosteson, D. C., P. Cook, and R. Blount. Separationof adenosine triphosphatase of HK and LK sheepred cell membranes by density gradient centrifuga-tion. J. gen. Physiol. 1965, 48, 1125.

60. Ohnishi, T., and H. Kawamura. R6le des phos-phatides dans l'adenosine triphosphatase sensitive 'al'ouabain localisee dans les membranes d'eryth-rocyte. J. Biochem. (Tokyo) 1964, 56, 377.

61. De Graeff, J., E. F. Dempsey, L. D. F. Lameyer,and A. Leaf. Phospholipids and active sodiumtransport in toad bladder. Biochim. biophys. Acta(Amst.) 1965, 106, 155.

62. Glynn, I. M., C. W. Slayman, J. Eichberg, and R.M. C. Dawson. The adenosine-triphosphatase sys-tem responsible for cation transport in electric or-gan: exclusion of phospholipids as intermediates.Biochem. J. 1965, 94, 692.

63. Reed, C. F. Studies of in vivo and in vitro exchangeof erythrocyte and plasma phospholipids (ab-stract). J. clin. Invest. 1959, 38, 1032.

64. Westerman, M. P., and W. N. Jensen. In vitro in-corporation of radiophosphorus into the phospha-tides of normal human blood cells. Proc. Soc. exp.Biol. (N. Y.) 1965, 118, 315.

65. Rowe, C. E. The biosynthesis of phospholipids byhuman blood cells. Biochem. J. 1959, 73, 438.

66. Murphy, J. R. Erythrocyte metabolism. III. Re-lationship of energy metabolism and serum fac-tors to the osmotic fragility following incubation.J. Lab. clin. Med. 1962, 60, 86.

67. Kirschner, L. B., and J. Barker. Turnover of phos-phatidic acid and sodium extrusion from mam-malian erythrocytes. J. gen. Physiol. 1964, 47,1061.

68. Prankerd, T. A. J. Studies on the pathogenesis ofhaemolysis in hereditary spherocytosis. Quart.J. Med. 1960, 29, 199.

69. Robinson, M. A., P. B. Loder, and G. C. de Gruchy.Red-cell metabolism in non-spherocytic congenitalhaemolytic anaemia. Brit. J. Haemat. 1961, 7,327.

70. Mohler, D. N. Adenosine triphosphate metabolismin hereditary spherocytosis. J. clin. Invest. 1965,44, 1417.

71. Kennedy, E. P. Biosynthesis of complex lipids.Fed. Proc. 1961, 20, 934.

72. Weed, R. I., A. J. Bowdler, and C. F. Reed. Meta-bolic dependence of erythrocyte membrane struc-ture (abstract). Clin. Res. 1965, 13, 284.

73. Jacob, H. S. Membrane lipid depletion during cat-ion pumping: a mechanism for the genesis ofspheroidal red cells in hereditary spherocytosis(HS) (abstract). J. clin. Invest. 1966, 45, 1027.

185

Documents

Concomitant Alterations of Sodium Flux and Membrane ...€¦ · fore incubation. After removal of a sample of cells for counting of blood elements and hematocrit determination, the