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					       The apparent transfer of fatty acid from
       phosphatidylcholine to phosphatidylethanolamine
       in human ervthrocvtes            J               J

                        STEPHEN B. SHOHET
                        Division of Hematology, The Children’s Hospital
                        Medical Center, and Department of Pediatrics,
                        Harvard Medical School, Boston, Massachusetts 02115

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ABSTRACT In previous studies an apparent transfer of                   active acylation of serum lysophosphatides with free fatty
14C-labeledfatty acid from phosphatidylcholine to phosphati-           acid within the membrane. Recent studies from our
dylethanolamine was obsxved in prelabeled human erythro-               laboratory (7) have indicated that the major outflow
cytes reincubated in fresh serum. These data could have been           pathway for passively incorporated PC is by exchange of
explained by direct fatty acid transfer from phosphatidyl-
                                                                       the entire PC molecule with preformed serum PC. O n
choline to phosphatidylethanolamine or by an apparent trans-
fer simulated by either demethylation of labeled phosphatidyl-         the other hand, most of the fatty acid actively incorporated
choline to phosphatidylethanolamine or base-exchange of                into PC by the acylation pathway is subsequently re-
phosphatidylcholine with ethanolamine.                                 turned to seruni as free fatty acid. I n the course of these
   To explore these possibilities, RBC containing phosphatidyl-        studies, when following the reincubation in fresh serum
choline doubly labeled with palmitic a~id-9,10-~H with   and           of cells briefly and actively prelabeled with fatty acids, it
cho1ine-l,2-l4C were prepared. Upon reincubation in fresh              was observed that the radioactivity in PC dropped while
serum, incorporation of aH (fatty acid) into phosphatidyleth-          that in PE rose before the return of labeled fatty acid to
anolamine was observed without incorporation of 14C(choline).          the serum. When these observations were combined with
In similar experiments in which RBC labeled with 3H-labeled            the further observations that the concentration of PC and
fatty acid alone were used, 14C-ethanolamineadded to the               PE did not change during the course of reincubation and
incubation was not incorporated into the isolated phosphatidyl-
                                                                       that these phosphatides accounted for the majority of the
ethanolamine which again showed incorporation of the fatty
                                                                       total label, it was hypothesized that fatty acid was trans-
  The data indicate that direct transfer of fatty acid from phos-      ferred from PC to PE before its final release back to the
phatidylcholine to phosphatidylethanolamine can occur in               serum (8). However, since we did not detect increased
human erythrocytes incubated in fresh serum.                           lysophosphatidylcholine during these incubations, two
                                                                       other possible explanations were considered to explain
                                                                       the observed changes. As shown in Fig. 1, the first of
SUPPLEMENTARY KEY WORDS                     palmitic acid
                                                                       these alternatives was that PC was converted into PE by
                                                                       demethylation of the choline moiety by a novel, as yet
                                                                       undemonstrated, reaction which would be the converse

PREVIOUS    STUDIES (1-6) have shown that renewal of
the erythrocyte membrane can occur by both the passive
                                                                       of the methylation reaction originally described by
                                                                       Bremer and Greenberg (9). T h e second of these alterna-
                                                                       tives was that PC was converted into PE by base-ex-
exchange of preformed serum phosphatides and the                       change in a type of reaction similar to the action of
   Part of the material presented here has previously been published   phospholipase D found in cabbage leaves by Yang, Freer,
in abstract form (Ref. 7).                                             and Benson (10). If either of these alternative pathways
  Abbreviations: PC, phosphatidylcholine ; PE, phosphatidyl-           had been operative, PC containing labeled fatty acid
ethanolamine; LPC, lysophosphatidylcholine; LPE, lysophos-
phatidylethanolamine; TLC, thin-layer chromatography.                  would have been converted into PE containing labeled

                                                                           JOURNAL LIPIDRESEARCH
                                                                                 OF            VOLUME 1971
                                                                                                    12,                         139
                            I                                                                           I
                          -C-    FA                                                                 -C-FA
                                 ~ F A                                       L P E (orPE)
                                                                                                -       I
                                                                                                    - C -FA
                            IS     fi          I       I       +   CH3
                                                                                                        I       O
                                                                                                                11       I     I   +I
                          - C-O-P-O-C-C-N:CH,                                                       - C-0-P-0-C-C-Nc
                            I          I       I       I           CHs                                      I   I    l         l
                                   -                                                                            -

                          PHOSPHATIDYLCHOLINE                                                       PHOSPHATIDYL-
                                                                                                    ETHANOLAMINE(tFA) 4
                                                                                                    LYSOPHOSPHATIDY L
                            I                                                                           I
                          - C-    FA                                                                -C-FA
                            I                                            METHYLTRANSFERAS5            I
                          -C-FA                                              ACCEPTOR X             -C -FA
                            I          ::
                          - C-0-P-0-C-C-N
                                                   I       I   +e"-                                    I         I I
                                                                                                    - C -0-P-0-C-C-Ni

                            I          I           I       1                                            I        I       I     I


                          PHOSPHATIDYLCHOLINE                                                       PHOSPHATI DYL-
                                                                                                    ETHANOLAMINE t
                                                                                                    METHYL X    -

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                          - CI - FA                                                                 - C- FA
                            I                                             . .iOSPHOLIPASE   D         I
                          - C-FA                                           ETHANOLAMINE             - C-FA
                                                                                                      I     P
                                                                                                    - C-0-P-0-C-C-N;I        I I t ,

                                                                                                        I   I    I

                                           -                                                                -

                          PHOSPHATIDYL CHOLINE                                                      PHOSPHATIDYL-
                                                                                                    ETHANOLAMINE               +

                                 FIG.1. Possible pathways for the apparent conversion of PC to PE.

fatty acid. This would have speciously simulated a trans-volume of fresh blood-group-compatible human serum
fer of fatty acid from PC to PE.                         and with glucose (10 m),ATP (1 m),GOA (0.1 mM),
  The current experiments were designed to examine       penicillin (100 units/cc), and streptomycin (100 pg/cc).'
these three possible mechanisms for the apparent transferThe hematocrit was taken to 15% with Krebs-Henseleit
of PC fatty acid to PE during the incubation of human    buffer (pH 7.4) and the cells were reincubated with
erythrocytes in fresh serum. The results indicate that   gentle rocking agitation at 37°C. On occasion, com-
the first pathway considered is responsible for most of the
                                                         paratively large amounts of PC were added to the rein-
observed increase in radioactivity in PE.                cubation media as follows: a thin film of PC was de-
                                                         posited upon the wall of the reincubation vessel by evap-
                                                         oration of a chloroform-methanol solution of PC prior
                                                         to the addition of serum. The serum was then added, and
Erythrocytes with PC labeled with palmitic a ~ i d - ~ H the vessel was swirled and sonicated at 4°C for 5 min a t
primarily in the 2-position and with 14C in the choline  60 watt-sec. 94% of added PC was found to be in the
moiety were prepared by incubation of RBC with choline-  liquid phase following this procedure.
1,2-14C-labeled LPC, palmitic acid-9,103H, GOA, and          Following incubation, cell lipids were isolated (11) and
ATP as described by Mulder and van Deenen (3). The       separated by TLC (12). PC and PE were then isolated
LPC was prepared by the action of phospholipase A2       and their specific radioactivity determined by estimations
(Calbiochem, Los Angeles, Calif.) upon commercially      of phosphorus content (13) and counting in a system
supplied choline-labeled PC (Tracerlab, Waltham,         designed to separate 14C and 3H radioactivity (5).
Mass.). After incubation, the cells were repeatedly
washed with defatted albumin as previously described (5)    'Coenzyme A and ATP were not required cofactors in these
                                                         studies with fresh cells. These additions, however, reduced the
to remove the superficial neutral lipids. Erythrocytes   variability when similar studies were performed with older cells
thus prepared were then reincubated with an equal        and were used here to be consistent with such studies.

             OF     RESEARCH    12,
                           VOLUME 1971
Specific activities were calculated in terms of dpm/pmole                                        ACTIVITY PHOSPHATIDES
                                                                                   TABLE 2 SPECIFIC      OF         ACTIVELY
                                                                                  LABELED PALMITIC
                                                                                        WITH       AcID-~H REINCUBATED
                                                                                                          AND        IN SERUM
of fatty acid, assuming a ratio of fatty acid:phosphorus                                      WITH ETHANOLAMINE-I4C
of 2 in the isolated phosphatides. Identification of PC
and PE was as previously described (5) and the absence                          Dura-                 Expt 1*                             Expt 2 t
                                                                                tion of
of significant amounts of LPE in the PC spot was in-                            Rein-            aH             '4C                  'H              1 'C
dicated by a negative malachite green-staining reaction                         cuba-
                                                                                  tion    PC          PE         PE          PC             PE       PE
in this area of the TLC plates (14, 15).
                                                                                  hr                        dpmlpmole fatty acid X   10-2

                                                                                   0      19.0        1.3       0           19.4            1.5      0
                            RESULTS                                                4      16.7        1.9       0.04        16.3            1.9      0.02
                                                                                   8      14.2        3.9       0.02        14.0            3.0      0.03
The results of reincubation with erythrocytes in fresh                            16      11.8        4.2       0.02        12.0            4.4      0.03
serum following the labeling of erythrocyte PC as
described above are shown in Table 1. While there was a                           * Expt 1, 0.5 m ethanolamine-'*C (2 pCi/pmole) in reincuba-
                                                                               tion serum.
niinimal drop in 14C (i.e., choline) radioactivity, there                         t Expt 2, 5.0 m ethan~lamine-'~C pCi/pmole) in reincuba-
                                                                                                 M                (2
was a major drop in 3H (i.e., fatty acid) radioactivity.                       tion serum.
At the same time, there was an increase in 3H radio-
activity in PE and no rise in the negligible 14Cradio-                         shown in Table 2. I n this experiment, the erythrocyte
activity. The observed rise in PE radioactivity accounts                                                                        Here
                                                                               PC was prelabeled with only palmitic a ~ i d - ~ H . it is
for about one-third of the fall in PC radioactivity. If                        shown that increasing doses of radioactive ethanolamine
uniform mixing of the PC label within a single PC pool                         are not significantly incorporated into cellular phos-

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is assumed, this implies that this pathway may be respon-                      phatides.
sible for about one-third of the catabolism of PC fatty
acid. If on the order hand, and as suggested by pre-
vious data (8), only a fraction of total cell PC enters                        The data in Table 1 confirm previous observations that
into this reaction, this estimate would have to bemodified.                    turnover of PC fatty acid occurs when cells are rein-
Moderate levels of 3Hwere found in PE at the end of the                        cubated in fresh serum (7, 8, 16). A portion of this turn-
preincubation in these experiments since the 3H was in-                        over appears to involve the transfer of the fatty acid
corporated within the membrane with both the exo-                              from PC to PE and may be mediated by phospholipase
genously supplied LPC-14C and endogenous nonradioac-                           (17) or acyltransferase (18) activity in the serum, or by
tive LPE. Additionally, there was no major loss of PC                          phospholipase activity in the cells theniselves (19). The
specific activity when comparatively large concentrations                      predominant initial 3H in PC was in the 2-position even
of cold PC were added to the reincubation media.                               though palmitic acid was used for labeling since the LPC
   The results of adding relatively large amounts of 14C-                      used was prepared by phospholipase A2 hydrolysis.
labeled ethanolamine to a similar set of incubations are                      Therefore, in agreement with previous workers (1-4), it
                                                                               i reasonable to conclude that the subsequent independent
          TABLE 1 SPECIFIC
                        ACTIVITY PC
                               OF                     AND    PE IN            fatty acid turnover occurs primarily at the 2-position.
                     ERYTHROCYTES*                                             Our inability to find increased LPC or LPE in these ex-
                                                                              periments may be a result of rapid reacylation of the lyso-
tion of                                                                       compounds generated during these experiments. Alter-
 Rein-              3H                                      1c
                                                             4                natively, lyso-compounds may be eliminated by phos-
  tion     PC                     PE         PC                      PE       pholipase B activity or transmutation of 2-LPC to PC
  hr                     dpm/pmole fatty acid X 1 0 - 2 t
                                                                              and glycerophosphorylcholine (20). The latter process
   0                                        14.8
                                                                              has been shown to be insignificant for the incubation
          24.2    (23.8)$        1.6                    (14.3)       0.2
   4      22.3    (22.0)         2.7        14.4        (14.2)       0.2      conditions used here (3, 21). The former process is un-
   8      19.0    (18.7)         4.7        13.8        (13.1)       0.1      likely since the total amounts of phosphatides remained
  16      16.5    (16.1)         5.3        13.5        (13.0)       0.1
  32      13.9    (13.6)         5.5        12.9
                                                                              constant, although, admittedly, small differences in
                                                        (12.7)       0.1
                                                                              lysophosphatides might not be detected by the techniques
   * PC labeled with 14C in the choline moiety and aH in the 2-               used here.
acyl moiety was actively incorporated into RBC by incubating                      These data also indicate that the apparent conversion
cells with 1-acyl LPC (labeled with ~holine-l,2-'~C) FAJH.
These cells were then reincubated in fresh serum. (88% of 3H                  of PC to PE is confined to the transfer of fatty acid and is
in isolated PC could be removed with phospholipase Az.)                       not due to the conversion of the choline moiety to
   t No significant changes in lipid phosphorus in PE or PC were              ethanolamine by demethylation, since a rise in 14Cin the
detected during the course of the reincubation.
   3 Numbers in parentheses indicate SA found after reincubations             isolated PE would have been expected if this had oc-
with 10 m PC present in the reincubation serum.                               curred.

                                                                           SHOHET Phosphatide Fatty Acid Transfer in Eryihrocytes                       141
   In addition, the d a t a in T a b l e 1 confirm the previous               transport and incorporation into human erythrocytes zn
observations (8) that this turnover of PC fatty acid is                       vitro. J . Clin. Invest. 46: 1017-1027.
                                                                         5. Shohet, S. B., D. G. Nathan, and M. L. Karnovsky. 1968.
confined to a comparatively nonexchangeable pool of                           Stages in the incorporation of fatty acids into red blood
PC which is synthesized by the active incorporation of                        cells. J . Clin. Invest. 47: 1096-1108.
fatty acid rather than by the passive incorporation of                   6. Reed, C. F. 1968. Phospholipid exchange between plasma
preformed PC, since the addition of relatively large                          and erythrocytes in man and the dog. J . Clin. Invest. 47:
amounts of preformed PC to the serum, which should                             749-760.
                                                                         7. Shohet, S. B. 1969. Two pathways of catabolism of eryth-
influence the extent of the passive serum-cell exchange of                                                               ln
                                                                              rocyte phospholipid fatty acids. J . C i . Invest. 48: 77a.
preformed PC, did not significantly alter this independent                    (Abstr.)
PC fatty acid turnover.                                                  8. Shohet, S. B. 1970. The release of phospholipid fatty acid
   T h e d a t a in T a b l e 2 further support the concept of the            from human erythrccytes. J . Clin. Invest. 49: 1668-1678.
independent release of fatty acid from PC a n d its subse-               9. Bremer, J., and D. M. Greenberg. 1961. Methyl trzns-
                                                                              ferring enzyme system of microsomes in the biosynthesis of
quent transfer to, o r exchange with, PE. Here evidence
                                                                              lecithin (phosphatidylcholine). Biochim. Biophys. Acta. 46:
is presented against the possibility of significant conver-                   205-21 6.
sion of PC to PE by base-exchange under the influence of                10. Yang, S. F., S. Freer, and A. A. Benson. 1967. Transphos-
phospholipase D activity. If such a mechanism h a d been                      phatidylation by phospholipase D. J . Biol. Chem. 242: 477-
operative, some concentration-dependent incorporation                          484.
                                                                        11. Rose H. G., and M. Oklander. 1965. Improved procedure
of ethanolamine would have been expected.
                                                                              for the extraction of lipids from human erythrocytes. J .
   T a k e n as a whole, the d a t a support the concept of the               Lipid Res. 6: 428-431.
independent transfer of fatty acid on the 2-position of                 12. Skipski, V. P., R. F. Peterson, and M. Barclay. 1964.

                                                                                                                                                Downloaded from by guest, on July 26, 2011
PC to PE in red cells incubated in fresh serum. Reacyla-                      Quantitative analysis of phospholipids by thin-layer
tion apparently prevents the accumulation of lyso-                            chromatography. Biochem. J . 90: 374-378.
                                                                        13. LQwry,0. H., N. R. Roberts, K. Y. Leiner, M-L. Wu, and
compounds. U n d e r these conditions, conversion of PC to                    A. L. Farr. 1954. The quantitative histochemistry of brain.
PE by nieans of either demethylation o r base-exchange                        I. Chemical methods. J . Biol. Chem. 207: 1-17.
does not occur.                                                         14. Horhammer, L., H. Wagner, and G. Richter. 1959. Zur
This study was supported by USPHS HD 02777 and by a                           papierchromatographischen           Auftrennung von Phos-
grant from the Medical Foundation, Inc., of Boston.                           phatiden. Biochem. Z . 331: 155-161.
                                                                        15. Kaufmann, H. P., Z. Makus, and T. H. Khoe. 1961.
The author would like to thank Drs. David G. Nathan and                       Die Dunnschicht-Chromatographie auf dem Fettgebiet
Manfred L. Karnovsky of the Departments of Pediatrics and                     I11 : Uber die Sichtbarmachung der zu analysierenden
Biological Chemistry at the Harvard Medical School for help-                  Stoffe auf der Platte. Fette Seifen Anstrichm. 63: 689-691.
ful suggestions and interpretation during the execution of this         16. Fischer, H., and I. Haupt. 1961. Das cytolysierende
work.                                                                          Prinzip von Serumkomplement. Z . Naturforsch. 16B: 321-
Manuscript received 2 July 7970; accepted 26' October 7970.                   328.
                                                                        I / . Vogel, W. C., and E. L. Bierman. 1967. Post-heparin s x u m
                                                                        1 7

                                                                              lecithinase in man and its positional specificity. J . Lipid
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