Phosphatides of human blood cells and their role in spherocytosis

STRUCTURE DES GLYC]~RIDES LYMPHATIQUES DU RAT x7 p. Is p. 19 F. 20 j . at G. ~2 B.

571

SAVARY ET P. DESNUELLE, Biochim. Biophys. Acta, 3 x (1959) 26. SAVARV, J. FLANZY ET P. DESNtI~LLE, Biochim. Biophys. Acta, 24 (t957) 414 • H. MATTSON ET E. S. LtJTTON, J. Biol. Chem., 233 (I958) 868. H, BRAGDON XX A. KARME.'~, J. Lipid Research, 1 (196o) 167. CLIgMENT ET J. CLgMENT, Bull. soc. chira, biol., 36 (I954) 1319BORGSTR6M, J. Biol. Chem., 214 (1955) 671.

Biochim. Biophys. Acta, 48 (1961) 562-571

P H O S P H A T I D E S OF HUMAN BLOOD CELLS AND T H E I R R O L E IN SPHEROCYTOSIS* M. K A T E S , A. C. A L L I S O N AND A . T . J A M E S

Division o[ Applied Biology, National Research Council, Ottawa (Canada), National Institute/or Medical Research, London (Great Britain) (Received O c t o b e r 7th, 196o)

SUMMARY

Spherocytes were found to contain a higher surface concentration of phosphatides than normal cells, but no limitation of overall phosphatide synthesis or turnover, and no significant variation from normal of fatty acid or long chain aldehyde composition was observed. However, the phosphatides of spherocytic cells were found to contain a significantly lower proportion of phosphatidyl ethanolamine and a correspondingly higher proportion of lysophosphatidyl ethanolamine. The latter component when added to normal serum was observed to produce sphering of normal red cells. It is concluded that the primary genetically controlled abnormality in hereditary spherocytosis probably lies in a partial block in the enzymic system for conversion of lysophosphatidyl ethanolamine to phosphatidyl ethanolamine. All other observed differences between normal and spherocytic cells could be secondary to such a defect.

INTRODUCTION

Patients with hereditary spherocytosis are distinguished by the spherical shape of their red cells, as compared with the normal bi-concave disc shape. Several metabolic differences between blood of patients with spherocytosis and normal blood have been described, e.g., differences in inorganic phosphate levels, sodium transport, and intermediary carbohydrate metabolism (see review by PRANKHERD1). It appears likely, however, that these differences are secondary to a defect in the red cell membrane which is manifested by the reduced surface area to volume ratio in spherocytes. Although the primary genetically controlled defect is not yet known, the striking abnormality in shape of the red cells in spherocytosis suggests that a component ot the cell membrane, possibly a phosphatide, may be involved. Abbreviation : EDTA, ethylenediamine tetraacetate. " T h i s p a p e r is issued as N.R.C. No. 6214.

Biochim. Biophys. Acta, 48 (196T) 571-58~

572

.~l. KATES, A. C. ALLISON, A. T. JAMES

For this reason we have investigated the phosphatide composition of spherocytes, as compared with normal cells, as well as the rate of incorporation of 3zp into phosphatides of normal and abnormal blood cells. A characteristic difference between normal and spherocytic blood lipids has been revealed, and this difference may possibly explain the development of spherocytosis in susceptible subjects. MATERIALS AND METHODS

Blood was obtained from two young adult female subjects with typical hereditary spherocytosis and family histories of the condition. One subject (patient II) had had a splenectomy.

Incubation with [~P T-orthophosphate and lipid extraction Normal or abnormal blood (20 ml) was incubated under sterile conditions with [3*Pjorthophosphate (2o/~C/ml) in acid citrate-dextrose and EDTA for 5 h at 37 °, as described previously2, a. Total cells were separated by centrifugation and washed with one-half volume of saline (0. 9 %). Total lipids in the centrifuged cells and in the plasma plus washing were extracted by the procedure of BLIG~I AND DYER4, modified as follows: To 1. 3 volumes of centrifuged cells, or I.O volume of plasma ( + washing) were added 3.75 volumes of methanol- chloroform (2 : I, v/v), and the mixture was shaken gently at room temperature for 1-2 h (or alternatively left overnight at room temperature); 1.25 volumes each of chloroform and water were added successively, and the mixture was shaken for a few minutes and centrifuged. The supernatant aqueousmethanol phase was removed quantitatively by pipette and discarded. The chloroform phase was decanted carefully from the insoluble residue and filtered through cotton wool; the insoluble residue was washed with several small portions of chloroform, and the washings were filtered through the cotton wool filter. The combined filtrates were evaporated to dryness in a rotary evaporator (below 35 °) and the lipid residue was dissolved in chloroform and made to volume (25 ml). Phosphorus analysis s (lipid-P), counting of 32P-activity, and paper chromatography were carried out with suitable aliquots of this solution.

Chromatographic separation of lipid components A portion of the chloroform solution (total lipids) was evaporated to dryness in a stream of nitrogen and dissolved in isoamyl alcohol-benzene mixture (i : I, v/v) to a concentration of 0.5-0.7 mg P/ml, IO/~1 of this solution was applied as a spot to a strip of Whatman 3MM paper (4.5" × I8") impregnated with silicic acid according to MARINETTI et a/.6, 7. To separate phosphatides, chromatography was carried out at room temperature for 18 h in diisobutyl ketone-acetic acid-water ( 4 0 : 2 5 : 5 , v / v ) by tile procedure of MARINETTIet al. 8. The chromatogram was dried in the fume-hood for 30 rain. Radioactive spots were located by autoradiography, and total lipids were detected by staining the chromatogram with Rhodamine 6G6, 7. Amino-lipids were detected with 1 % ninhydrin in butanol; choline-containing lipids with phosphomolybdic acid-stannous chloride reagentS; and plasmalogens with the SchiffFeulgen reagent ~. To separate neutral lipids, io/~1 of the amyl alcohol-benzene solution was applied Biochim. t3iophys. Acta, 48 (1961) 571 -5.~-'

PHOSPHATIDES OF HUMAN BLOOD CELLS

573

to a strip of W h a t m a n No. I paper impregnated with silicic acid x° and chromatography was carried out in n-heptane--diisobutyl ketone (96:6, v/v) for 5.5 h, according to the procedure of MARINETTIet ~.11,12. The lipids were detected with Rhodamine 6G e, n.

Quantitative estimation of individual phosphatides Two methods were used, depending on the amount of material to be analyzed: I. For small samples (containing about 5-20 ~g P), the material was applied to a strip of silicic acid-impregnated paper (5-7/~g P per spot) in 1-3 spots, and the chromatograms were run as described above. The dried chromatogram was stained with Rhodamine 6G and the spot(s) corresponding to each component was cut out and ashed directly with 70 % perchloric acid plus one drop of 8 % ammonium molybdate. The digest was dilutcd with 8 ml of water and the blue colour developed according to ALLEN5. After 15 rain, the colour was extracted into 4.0 ml of isobutanol- diisobutyl ketone (I : I, v/v) ; 3.0 ml of the organic phase was then diluted with 0. 5 ml of 95 % ethanol and the absorbancy was measured at 680 m/~ (see ref. 13). Values were corrected for blank value of P in the paper. Silicic acid does not interfere in this method. 2. For larger samples and when the separated components are required for further study, the material was applied to several strips of paper (6 spots per strip, 5-7 ~g P per spot), the chromatograms were run, and the lipids in the outside channels were detected with Rhodamine 6G. The unstained portion of the chromatograms was then cut into strips corresponding to each of the separated components, which were then eluted by downward chromatography with chloroform-methanol-water (75:2512, v/v), followed by methanol. The combined eluates of each component were then evaporated to dryness, the residue dissolved in chloroform to a known volume, and suitable aliquots were analyzed for total phosphorus, either by the method of ALLEN5 or b y the modification described in method (I). Values obtained by methods (1) and (2) agree within a few percent.

3~P-specifie activities of individual components After determination of P in each of the separated components by method (I), all of the organic phase containing the reduced phosphomolybdate, or a suitable aliquot, was evaporated on an aluminum planchet, with the aid of a hair-drier, and counted with an end-window Geiger-Mtiller-tube. With method (2) the radioactivity m a y also be determined by evaporating a suitable aliquot of the chloroform solution of each eluted component on an aluminum planchct and counting as above.

Fatty acid and long chain aldehyde analysis A sample of total lipids (containing 15o-2oo t~g P) was hydrolyzed with 4.5 ml of methanol-HC1 (approx. o. 7 N) under reflux for 2 h. The solution was made alkaline with o.5 ml of 8 N aqueous NaOH and refluxed again for I h. Total aldehydes (as dimethyl acetals) together with unsaponifiable material were extracted with two or three 5-ml portions of petroleum ether (b.p. 40-60°). F a t t y acids were extracted with petroleum ether after acidification of the methanol phase with 5 N sulfuric acid. The aldehyde dimethyl acetals were analyzed by gas-liquid chromatography on Apiezon L, at 197 °, as described by GRAY 14. The fatty acids were converted to methyl esters by treatment with diazomethane and analyzed by gas-liquid chromatography on Apiezon L, at 197 °, as described by JAMES 15. Biochim. Biophys. Acta, 48 (I96I) 57x-582

574

M. K A T E S ,

A.C.

A L L I S O N , A. T. J A M E S

Determination of sphering action Phosphatide components of normal and abnormal ceils were separated chromatc,graphically and eluted from the paper as described above. Each component was suspended in normal serum to give a final concentration of 2 to I2 times the normal concentration of the component (see Table VII). Normal red cells were added to the sera in silicone-treated tubes in the ratio 2 : 3 (v/v), and the suspensions were incubated at 37 ° with occasional agitation. At 2-h intervals, the cells were examined in fresh wet film preparations by phase-contrast microscopy, which shows clearly the overall shape of the cells. RESULTS

Fatty acid and aldehyde composition No significant difference in fatty acid composition of the total lipids was found between normal cells and spherocytes (Table I) ; the slight variations observed, e.g., in the percentages of palmitic and oleic acids are well within the normal range reported previously le. The major long chain aldehydes present in both normal and abnormal cell lipids were found to be palmitaldehyde and stearaldehyde (Table II). Spherocytes appeared to have a higher content of stearaldehyde than normal, and C19, Cz0 and Czl aldehydes were found in normal cells but not in spherocytes. However, these differences may not be significant since the normal variation in aldehyde composition is not known. These long chain aldehydes are probably present in "bound" form in both the phosphatide (plasmalogens) and non-phosphatide components (see below).

Phosphatides and neutral lipids The total phosphatide content (per milliliter of blood) was lower in spherocytic blood than in normal blood, but this was a result of the lower cell count and the lower TABLE FATTY

ACID COMPOSITION

OF TOTAL LIPIDS

I

OF N O R M A L

BLOOD CELLS AND

SPHIzROCYTES

C o m p o s i t i o n e x p r e s s e d a s p e r c e n t a g e s of t o t a l a r e a u n d e r t h e p e a k s o n t h e c h r o m a t o g r a m A b b r e v i a t i o n s : t r , less t h a n o. I Oo ; n d , n o t d e t e c t e d . Fatty acid

Lauric Myristic n-Cl~ Palmitoleic Palmitic Branched-Cry n-Ct~ l.inoleic Oleic Oleic isomer Stearic Arachidonic C2o t r i e n e n-Ct0 C22 u n s a t u r a t e d C2~ u n s a t u r a t e d

Normal cell

(a) (b)

0. 3 0.8 0.3 i. i 41 .o o.3 0.3 15.3 IS. 9 tr 7.9 7.9 i. 5 nd 2. 5 z.o

tracing,

Sphtrccytes I

o. i 0. 7 0.3 ~ .3 4o.5 o.3 0. 4 r 4.9 21.9 tr 7 .8 6.o i .7 nd 1.7 2. 3

II

o. x .o 0.3 i .o 38.9 o.2 0. 3 14.6 2t. 7 tr 9. I 6. 7 1.9 0.6 2. i 1.5

Biochim. Biophys. Acta, 48 ( I q 6 1 ) 571--58~

PHOSPHATIDES OF HUMAN BLOOD CELLS

575

TABLE II LONG

CHAIN

ALDEHYDE

NORMAL

COMPOSITION

BLOOD

CELLS

AND

OF

TOTAL

LIPIDS

OF

SPHEROCYTES

A b b r e v i a t i o n s : tr, less t h a n o. I ~o; nd, n o t d e t e c t e d ; - - , n o t d e t e r m i n e d .

Ald¢hyale carbon chain

RettzCion at 197 ° on Apttzon L, relative to methyl myristcae

Normal cells

1.27 i .63 1.95 z. 15 2.44 2.55 2.98 3.74 3.85 4.47 5.83 6.o 7 6.75 8.75 13.I I9.7

tr 0.8 0.6 tr nd 0. 4 z4.2 1.7 7.5 I. 3 6.o 2.8 42.5 2.9 3.I 5.¢)

n-Cx4 Branched-C15 n-Cx5 H i g h l y branched-C16 Branched-C16 C16-monoene n-Cxe H i g h l y b r a n c h e d Cxv B r a n c h e d Cx7

n-Cl7 ('is-monoene lsomeric-Cls-monoene ~/-C18

U n k n o w n Ct9 U n k n o w n C20 U n k n o w n C21

Spheroc ytes 1

I1

tr 0.8 0.6 0.5 nd nd 27.7 1.7 tr 1.3 7.6 7.2 52.6 nd nd nd

tr 0.8 0.6 tr 1.7 0. 7

27.2 i.8 x.6 1.4 5.5 5.2 53.5 -----

TABLE Ill PHOSPHATIDE

Source o/lipids

f cells Normal / plasma S cells Spherocvtic 1 ). p l a s m a k' cells Spherocytic II t plasma

CONTENT

OF

NORMAL

BLOOD

CELLS

AND

Phosphatidc content* #mole/ml blood

Cell count, Cells/ml b l o o d

Cellular content of phosp~ffides, pmole/cell

T.I 5

5.2. IO9

2.2. )o -I°

2.13

1.o 3 1.38

0-95 i .33

--

4.7" lO9

Surface area Surface concentration of cells, of phospluaidts, pt/cell /*moles/it ~

I63

1.35" IO ' t z

13o

I. 7. l o -12

I3O

1.5s. l ° - t ~

--

2.2- IO- l °

--

4.8" lO9 --

SPHEROCYTES

--

2.0. Io -1° --

" E q u i v a l e n t t o / ~ m o l e s of l i p i d p h o s p h o r u s / m l of blood.

plasma phosphatide content oi spherocytic blood (Table III). When the values were calculated on a cellular basis, spheroeytes were found to have almost the same content of phosphatides as normal cells. Furthermore, since spherocytes have a smaller surface area than normal, it follows that the surface concentration of phosphatides is actually greater in spherocytes than in normal cells (Table III). This result is not in agreement with the older evidence of ERICKSOI~ et al. 1~ that phosphatide surface concentration in normal and spherocytic cells is the same. Chromatography of the total lipids on silicic acid-impregnated paper showed that, qualitatively, the phosphatide and neutral lipid composition of spherocytes and spherocytic plasma was the same as that of normal cells and normal plasma, respectively (Figs. I and 2). The probable identity of the neutral lipid (Fig. 2) and phosphatide (Table IV) components was established on the basis of their relative R$ values and staining behaviour towards specific reagents (see refs. 6, II, 12). Some Biochim. Biophys. Acta, 48 (I96I) 571-582

576

KATES,

M.

A. C. ALLISON,

A. T. JAMES

comment is however necessary concerning the identity of two of the phosphatide components (spots I and 2, Fig. I). Spot I, from both cells and plasma (Table IV), gave a weak but definitelypositive reaction with phosphomolybdic acid ("choline" reagent) and presumably contains lysolecithin. However, the fact that part of this spot showed a blue fluorescence when stained with Rhodamine 6G indicates the presence also of an acidic phosphatide, most likely phosphatidyl inositol (see ref. 18). Lysolecithin and phosphatidyl inositol have previously been reported present in human plasma 1~,1s-~°. Spot 2 (Table IV), from both cells and plasma, gave a strong reaction with the "choline" reagent and presumably consists mostly of sphingomyelin. However, this spot always gave a weak but positive reaction with ninhydrin and with the FeulgenSchiff stain (see ref. 18). When spot 2 was subjected to mild alkaline hydrolysis followed by mild acid hydrolysis .1 and then chromatographed on silicic acid-impregnated paper, it gave only one spot which stained positive with the "choline" reagent but was no longer ninhydrin positive. Furthermore, the aqueous phase of the hydrolysate contained a phosphate ester identified as glycerylphosphoryl ethanolamine *" (RF in butanol-acetic acid-water, o.19). It may thus be concluded that spot 2 is a mixture of sphingomyelin and lysophosphatidyl ethanolamine, the latter consisting of both ester and plasmalogen forms. It is also noteworthy that the lecithin (spot 3) and phosphatidyl cthanolamine (spot 5) components also occur in both ester and plasmalogen forms. The phosphatidyl

_,t,_j t.j cu, k.j t,J 0.9

-

o,,i

;,

6

,

,

;

6

0.8

0.7

0.6 .J 0.5

0.4

,o00, ,oo b: ,o00, ~')0

,:"" ,

G I

o "", "-'I,, ,"-'I 5

Y

"'

!

t)i

4

*

or')('; : , ',..; i!"

3

,000,

G

0©0,

oli f J ,,,!', ,-

0.3

~,

"7 ~

0.2

"" 0.1

Y

o

o

TOTAL

TOTAL

o

I II NOm~AL sp~¢mocvTic CELLS

NORMAL SPHi[ ItOCy T LC

PLASMA

Fig. z. Tracing of chromatogram of phosphatides from total cells and plasma of normal and spherocytic blood; chromatograms were stained with Rhodamine 6G. Abbreviations: Y, yellow; O, orange; B, blue; G, grey. Dashed lines indicate minor components.

%,/

"d

NORU&L S~K[nOGYTI¢ CELLS

I MORWAL

'1I

SPNEROGTTI¢

PLASMA

Fig. 2. Tracing of chromatogram ()f neutral lipids from total cells and plasma of normal and spherocytic blood (chromatograms stained with Rhodamine 6G; Y, yellow; (), orange; G, grey). Tentative identification of components: phosphatides, spot o, monoglycerides, spot z, diglycerides, spot 2, cholesterol, spot 3, triglycerides, spot 4, cholesterol esters, spot 5. Minor components, dashed lines.

Biochim. Biophys. .4cta, 48 (196tl 571 5,q2

PHOSPHATIDES

TABLE CHARACTERIZATION

577

OF HUMAN BLOOD CELLS IV

OF PItOSPHATIDE COMPONENTS OF NORMAL AND SPHEROCYTIC BLOOD

A b b r e v i a t i o n s : v w , v e ~ " w e a k ; w, w e a k ; m , m o d e r a t e ; s, s t r o n g ; - - , n e g a t i v e ; ( - - ) , w e a k l y positive but variable. Results apply to both normal and abnormal blood which gave qualitatively t h e s a m e c h r o m a t o g r a m s (see Fig. i). Spot No.

Average RF valve

Rhadamin¢ 6G stain

Ninhydrin stain

"Choline" Plasma2ogen stain" stain

Probableid~gity of components

T o t a l blood cells

x

0.40

G r e y - b l u e (w)

2

0.46

Y e l l o w (s)

3 4 5 ¢) 7

0.55 0.60 0.70 0.90 0.95

Y e l l o w (s) B l u e (w) Y e l l o w (s) B l u e (w) Y e l l o w (s)

--

wv

--

w

s

w

s

s

-vx~. s . . --

. .

. . --. . ---

s s

Lysolecithin + Phosphatidyl inositol f Sphingom)elin + lysophos~. phatidyl ethanolamine Lecithin Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidic acid Neutral lipids

~(

Plasma

x

o.4i

Y e l l o w - b l u e (m)

--

w

z

0.48

Y e l l o w (s)

vw

s

3 4 5 0 7

0.56 0.60 o.70 0.90 0.95

Y e l l o w (s) B l u e (vw) Y e l l o w (w) B l u e (w) Y e l l o w (s)

-¢± ) vw ---

s -----

" After first staining chromatogram

Lysolecithin + phosphatidyl inositol Sphingomyelin + lysophosvw { phatidyl ethanolamine s Lecithin -Phosphatidyl serine (+) Phosphatidyl ethanolamine -Phosphatidic acid s Neutral lipids --

with ninhydrin.

TABLE PHOSPHATIDE

{

COMPOSITION

V

OF NORMAL

AND

ABNORMAL BLOOD

C o m p o s i t i o n e x p r e s s e d a s p e r c e n t a g e of t o t a l p h o s p h o r u s r e c o v e r e d f r o m t h e c h r o m a t o g r a m s ( b l a n k c o r r e c t e d ) . V a l u e s for p h o s p h a t i d y l s e r i n e w e r e n o t d e t e r m i n e d b e c a u s e of o v e r l a p p i n g w i t h t h e l e c i t h i n s p o t (see F i g . i.) Total cells .',pot No.

I 3 4 5 6

Phosplw,lid¢ component

Lysolecithin + phosphatidyl inositol Sphingomyelin + lysophosphatidyl ethanolamine Lecithin Phosphatidyl serine Phosphatidyl ethanolamine Phosphatidic acid

Plasma

Sp~wvoeyles

Nor~ 1

II

3.3

3. t

3.8

22.9 45 .2 . 27.9 0. 7

32.5 45.5

32.9 4 z.1 . x9.5 I. 7

.

. x8.3 0. 5

Sphcrocytes

Nomud I

II

xo.o

lO. 5

to.z

23.6 61.2

20.2 65. 5

23.5 59.6

3.6 0. 3

5.3 1. 4

.

. 4.9 0. 4

B i o c h i m . B i o p h y s . A c t a , 48 (T961) 571 -582

57 8

M. KATES, A, C. ALLISON, A. T. JAMES

serine component (spot 4) was always detectable with Rhodamine 6G on chromatograms of the cell lipids but was not always seen on chromatograms of plasma lipids; also the ninhydrin stain of spot 4 was very weak and was often obscured in the cell lipids by the strong stain given by phosphatidyl ethanolamine (spot 5).

Quantitative composition of phosphatides When the phosphatide composition was determined quantitatively, it was found that normal and spherocytic plasma had the same composition, but spherocytes contained a considerably higher concentration of spot 2 and a correspondingly lower concentration of phospbatidyl ethanolamine (spot 5), than normal cells (Table V). Since spot 2 contained sphingomyelin and lysophosphatidyl ethanolamine, which were not separable by the chromatographic system used here, it was difficult to establish whether the observed increase in spot 2 corresponded to an increase in either one or in both of these components. However, the close structural relationship between lysophosphatidyl ethanolamine, together with the fact that the increase in spot 2 was exactly balanced by the decrease in phosphatidyl ethanolamine strongly suggested that lysophosphatidyl ethanolamine was the component involved in this change. The quantitative composition of plasma phosphatides found here (Table V) is very similar to that reported previously by other workers is, ~9.

Incorporation of 32p into phosphatides 3*P-incorporation into total phosphatides was greater in abnormal than in normal cells (Table VI, compare normal cells and spherocytes I). This is probably due to the fact that spherocytic blood has a smaller phosphatide pool (Table III) and a lfigher concentration of leucocytes, which are responsible for most of the synthesis of phosphatides by blood cells in vitro ~, 24. The specific activities of individual phosphatide components of both normal cells and spherocytes were in the order phosphatidic T A B L E VI 32P-SPECIFIC

ACTIVITIES

OF

PFIOSPHATIDES

FROM

NORMAL

AND

ABNORMAL BLOOD

A b b r e v i a t i o n s : LysoPC, l y s o l e c i t h i n ; P I , p h o s p h a t i d y l i n o s i t o l ; L y s o P E , l y s o p h o s p h a t i d y l e t h a n o l a m i n e ; Sphing, s p h i n g o m y e l i n : PC, l e c i t h i n ; P E , p h o s p h a t i d y l e t h a n o l a m i n e ; PA, phosp h a t i d i c acid. Specific activity, counts/rnotl,ug P* Source ~f lipids j*

Total phosphatides

LysoPC -PI

LysoPE ~Sphing

I'C

I'E

PA

cells plasma cells Spherocytic I plasma

4.8 x. r b.9 z.6

o.5 4.7

o. l

7.2

o.i --

2.8 o.9 4.2 1.2

o. 4 -0.8 -

5. I 4.9 ----

S p h e r o c y t i c I I { cells plasma

17. t 14.o

IO.8 33

5.0 2.8

i 2.2 6.2

7-4 7-3

Normal

I43 I44

" A c t i v i t i e s are corrected for d e c a y ; t h e " d a s h " i n d i c a t e s v a l u e s too low for a c c u r a t e m e a s u r e m ent. "" R e s u l t s for n o r m a l and s p h e r o c y t i c blood from p a t i e n t [ are c o m p a r a b l e , t h e i n c u b a t i o n w i t h s ~ P - p h o s p h a t e h a v i n g been done u n d e r i d e n t i c a l c o n d i t i o n s a n d a t t h e s a m e t i me . Blood f r om p a t i e n t i I was i n c u b a t e d in t h e s a m e w a y e x c e p t t h a t t he acid c i t r a t e - - d e x t r o s e a d d i t i v e was a d j u s t e d to pH 7.5, a n d E D T A was o m i t t e d ; t h e s e c h a n g e s are c ons i de re d r e s p o n s i b l e for t h e h i g h e r degree of 32P-incorporation.

Biochim. Biophys. Acta, 48 (I96I) 57t- 58-'

PHOSPHATIDES

579

OF HUMAN BLOOD CELLS

acid > lecithin > lysolecithin + inositol phosphatide > phosphatidyl ethanolamine > lysophosphatidyl ethanolamine + sphingomyelin (Table VI). These results appear to exclude any defect of overall synthesis or turnover of cell phosphatides in hereditary spherocytosis. Unfortunately, specific activity data on the individual lysophosphatidyl ethanolamine component could not be obtained because of the inability ot the present chromatographic system to separate this component from sphingomyelin. The specific activity of phosphatidyl ethanolamine would appear to be relatively slightly higher in spherocytes than in normal cells. Although this result would be consistent with the smaller pool size of phosphatidyl ethanolamine in sphcrocytes, it should be noted that the observed difference may be within the experimental error in determining the low 32P-activity of this component. The raP-specific activity of plasma total phosphatides was also higher in spherocytic blood than in normal blood. This is a reflection of the higher specific activity of spherocyte phosphatides, since the activity of plasma phosphatides results from exchange with raP-labelled phosphatides synthesized in the cellsL Comparison of the specific activity of each component in the plasma with that of the corresponding component of the cells (Table VI) shows that the rate of equilibration of cellular and plasma phosphatides is different for each component, being highest for phosphatidic acid and phosphatidyl ethanolamine, the minor phosphatide components of plasma. It should also be noted that the lysolecithin + phosphatidyl inositol component has a higher specific activity in the plasma than in the cells, and indeed has a much higher specific activity than the major plasma component lecithin. The only explanation offered for this anomaly is that the phosphatidyl inositol component in the cells diffuses at a much greater rate than the lysolecithin component, the former having a much higher specific activity than the latter. However, further study will be necessary to resolve this question. TABLE

VII

SPHERING ACTION OF PHOSPHATIDE COMPONENTS FROM NORMAL CELLS AND SPHEROCYTES

PhosphaIid¢ comptmtnt

Excess concemrcaton" of component lrom Normal c e l l s

Sphering action a/tee z h by component ]tom

Spherocytes Normal cells Spherocytes

,ug P / m l serum

Lysolecithin + phosphatidyl inositol S p h i n g o m y e l i n -- l y s o p h o s phatidyl ethanolamine Lecithin Phosphatidyl serine Phosphatidyl ethanolamine

16 (1.4) 68 66 15 65

(2.5) (0.9) (3) (ti)

33 (2.9)

+

--

97 (3.6) 65 (o.9) Not tested 44 (8)

+ ---

--Not tested ---

--

P u r e substrates

Dimyristoyl lecithin (synthetic) Monopalmitoyl lecithin ** S p h i n g o m y e l i n " *"

65 (0.9) 72 (I.O) .58 (2.1)

+

* E a c h v a l u e in b r a c k e t s is t h e r a t i o of t h e e x c e s s c o n c e n t r a t i o n t o t h e c o n c e n t r a t i o n of t h e c o m p o n e n t in n o r m a l p l a s m a (see T a b l e V). ** G i f t f r o m Dr. D. J. HANAHAN, U n i v e r s i t y of W a s h i n g t o n , S e a t t l e (U.S.A.). "*" G i f t f r o m Dr. N. FISHER, B r i t i s h B a k i n g I n d u s t r i e s R e s e a r c h A s s o c i a t i o n , C h o r l e y w o o d , Herts. B i o c h i m . 13iophys. A c t a , 48 ( I 9 6 I ) 57x--582

580

M. KATES, A. C. ALLISON, A. T. JAMES

Sphering action of phosphatide components Each of the phosphatide components from both normal cells and spherocytes were tested for their ability to bring about sphering of normal red cells when added to normal serum in the concentrations given in Table VII. The only components which produced sphering were the lysolecithin + phosphatidyl inositol and the lysophosphatidyl ethanolamine q- sphingomyelin components. Since a sample of pure lysolecithin alone produced sphering, while a sample of pure sphingomyelin had no effect (Table VII), it may be concluded that both the lysolecithin and the lysophosphatidyl ethanolamine components are capable of inducing sphering of red cells. It should be noted that at the concentrations and under the conditions used, neither lyso-compound caused any hemolysis of the red cells. DISCUSSION

The results described show that spherocytes do not differ significantly from normal cells with respect to total phosphatide content (moles/cell), overall phosphatide synthesis (or turnover), or fatty acid and aldehyde composition, nor does the lipid composition of the plasma of spherocytic blood differ from that of normal plasma. The only significant and consistent difference which could be demonstrated was the lower concentration of phosphatidyl ethanolamine and what is presumed to be the correspondingly higher concentration of lysophosphatidyl ethanolamine in spherocytes. Two interpretations of this finding can be offered. According to the first, spherocytic blood is presumed to contain an enzyme, phosphatidase A, which would hydrolyze phosphatidyl ethanolamine to produce lysophosphatidyl ethanolamine. However, since this enzyme acts also on lecithin to form lysolecithin~, this interpretation could not possibly account for the fact that in spherocytes it is the phosphatidyl ethanolamine concentration which is lower than normal, whereas the lecithin and lysolecithin concentrations are the same as in normal cells. Furthermore, the presence of this enzyme in blood would be extremely dangerous, if not fatal, since the enzyme is known to hydrolyze the lipids of red cells with resultant hemolysis. The second interpretation, which seems much more likely, is that patients with hereditary spherocytosis possess a defect or block in the biosynthesis of phosphatidyl ethanolamine. In attempting to locate the site of this defect, the following biosynthetic pathway for phosphatidyl ethanolamine (diester and plasmalogen types) is proposed : OOh/ H ~CO I'--OCH,CH aN H 2

H zC()H I HCOH

.... ~

H 2COR a-monoglyceride

HCOH

OO•/ H ~COP ..OCH 2CH 2NH 2 ~

H sCOR Lysophosphatidyl ethanoloamine (ester and plasmalogen types)

HCOCR' H 2CO R P h o s p h a t i d y l ethanolamine (ester and plasmalogen types)

O I. [R = -C-(CHs)nCHs(ester) ; or -C=CH(CHz) aCHs(enol ether) R ' = h y d r o c a r b o n chain]

Biochim. Biophys. Acta, 48 (I961) 57x-582

PHOSPHATIDES OF HUMAN BLOOD CELLS

58I

If the defect were in the last step, namely, the acylation of lysophosphatidyl ethanolamine (ester and enol ether type), the overall effect would be a decrease in phosphatidyl ethanolamine and an accumulation of the lyso-compound, precisely as observed in spherocytic blood cells. Some support for the above biosynthetic route is offered by the recent work of LANDS~ who demonstrated the existence of an enzyme system in rat liver which catalyzes the acylation of lysolecithin to lecithin. It is clear that the proposed metabolic block cannot be complete, since subjects with hereditary spherocytosis, being heterozygous, would have one normal gene as well as one abnormal gene at the locus concerned. The experiments on the sphering action of phosphatides added to normal serum suggest that lysolecithin and lysophosphatidyl ethanolamine are the only blood phosphatide components capable of inducing significant sphering of cells at near physiological concentrations. This result is consistent with the well known surface activity of these lysocompounds and their~bility to produce hemolysis in higher concentrations. The fact that lysolecithin is present at the same low concentration in normal and abnormal blood, and that this concentration in normal cells obviously does not produce sphering, would exclude the possibility that this compound plays a role in spherocytosis, as was once thought ~. The evidence would thus appear to favour lysophosphatidyl ethanolamine as the component directly responsible for the manifestation of the abnormal spherical shape of the red cells in hereditary spherocytosis. The question arises whether the metabolic differences between spherocytes and normal cells, summarized by PRANKERD t, are primary or whether they follow from the defect of synthesis described above. The latter interpretation is most likely since alterations in sodium and phosphate ion transport, for example, could well result from an abnormal membrane containing a higher proportion of a surface active component such as lysophosphatidyl ethanolamine. If the abnormality in phosphatide composition were secondary and resulted from a primary metabolic disturbance in, for example, phosphate ion transport, it would be remarkable if only a single pair of components were affected, all other phosphatides being present in identical proportions. It is therefore concluded that the most likely primary genetical defect in hereditary spherocytosis is in the final stage of synthesis of phosphatidyl ethanolamine, resulting in an accumulation of lysophosphatidyl ethanolamine which produces the characteristic sphering of the red cells.

ACKNOWLEDGEMENTS

The authors are indebted to Dr. T. NORRIS, Archway Hospital, for access to cases under his care, and to Dr. T. FREEMAN for assistance in collecting material. The technical assistance of Mr. A. BROWNSTONEis acknowledged. One of the authors (M. K.) is grateful to the National Institute for Medical Research for providing facilities for carrying out this work. This study was carried out under a work transfer of one of theauthors (M.K.) from the Division of Applied Biology, National Research Council, Ottawa to the National Institute for Medical Research, Mill Hill, London.

Biochim. Biophys..4eta, 48 (1961] 571-582

582

M, KATES, A. C. ALLISONp A. T. JAMES REFERENCES

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Biochim. Biophys. Acta, 48 (1961) 571-582