Vitamin E in young and old human red blood cells

84

~himwa

et ~ h y ~

A~a 860 (1986) 84-90

~

BBA 73220

VRamin E in young and old human red blood cells *

Graham W. Burton, Shee C. Cheng, Ann Webb and K.U. Ingold D i ~ o n ~ Chem~ry, National Research Coun~l ~ Canada, Ot~w~ O n ~

KIA OR6 (Canada)

(Recdved January 9th, 1986)

K~ w ~ :

~Tocop~rol; ~ m i n E; Amc~do~c add; A~ng; (Human e ~ t ~ o c ~

Young and ~d human ~ d ~ood cells contain about the same amou~ ~ ~ c o p h e r o i , a compound w~ch h ~ previou~y been shown to be the m~or ~ s ~ u ~ ch~n~rea~ng anfioxidant w ~ e ~ ~ such cells. ~ n c e ~ d ~ood cells ~ up ~ c& 20% ~ ~ d m a ~ ~om ~ r mem~ane ~ ~ e y age, ~ e ~tocopherol/ memb~n~li~d ~fio ~ l y f i ~ s w i ~ age ~ ~an declining ~ might h ~ e been expe~ed on ~ e ba~s ~ the ~ee ~ c ~ ~ e o ~ ~ a ~ n ~ The a - ~ c o p h ~ / ~ d o n ~ a~d m ~ e ~ ~fios ~ e ~ e ~ ~e ~de~ young wd ~ood cells < ~d ~ d ~ood cells < ~ m ~ wh~h ~ g u ~ ag~n~ ~ e ~gge~ed memb~ne s~l~g d~ ~ ~copherol/~do~c a~d m ~ e ~ c o m ~ e x ~ .

The red blood cell pro~des an e x c d ~ n t modal for ~udying the process of cellular a ~ n g because young and old cells have different denfities and so can be separated by careful centrifugation [1-15]. It has been found [10] that older red blood cells have increased hemoglobin and N a + concen~afions rdafive to younger cells but decreased K +, acety~holine esterase, fiatic a~d, membrane protein, Z ~ d i p h o s p h o ~ y c e r a t e , ~ u t a m i ~ o x ~ o a c e t i c ~ansaminase [1~, ATP, creatine [ & l ~ , and p h o ~ pholipid [2,3] concen~ations, together with a de-

* NRCC No. 25977. Data supNementa~ to tNs article are depofited wi~, and can be obtNned ~om E I ~ SNence Pubfishers BV/1000 BH Ams~rdam, The Netherlands. R ~ e n c e shoed be made m no.: BBA/DD/342/73220/860 (198~ 84-9~ The supp~mentary ~rmafion include: Thee ~b~s contaiNng the O-ac~ fat~ and compofifion of human red Nood cdN ~p~med by den~ff ~f~rence~ the Nasma lipid concen~afions and fatff and compofifion ~om dono~ of different age, and a comparison of heptaneqsopropanol and SDS 1 ~ e ~ r a ~ n s of young and Md ~d Mood cell~

creased concen~afion of fatty a ~ d s of CN or longer chNn ~ n g t h rdative to those C18 and shorter [3,6]. In additiom the acti~fies of catNase, Nucos~6-phosphate dehydrogenas~ Nutathione reductase, Nutathione p e r o ~ d a s e [15] and supero ~ d e dismutase [13] have been shown to decline in older cells. During the aNng process red blood cells decrease in size [5], losing about one-fi~h of ~hNr membrane (probably as cytoplasm-filed micro-ve~des [16,17]) but tittle or no hemo~obin; they Nso become osmotically more ~agile ~,8,18]. D e s p i ~ the weNth of information about the cellular ~ a n ~ o r m a t i o n s that occur with aNng, the actuN mechanism of the aNng process is still p o o ~ y understood. If the free radicN theory of aNng [19] has generN validity it should, as has sommimes been assumed [20], be spedfically applicable to red blood cells. We have pre~ouMy shown that ~ t a m i n E (mNnly a-tocopheroO is essentially the only lipid-soluble, chNn-breaking anfioxidant present in human plasma and red blood call ghost membranes [21,22]. One might therefore expect that the older red blood cells would be d e p e n d in ~ t a m i n E rdafive to the younger calls. SurprifinNy, no data have been

0005-2736/86/$03.50 © 1986 Else~er Science Publ~hers B.V. (Biomedic~ Diction)

85 reposed with regard to the rdafive concengations of ~tamin E in young and old red blood cells. The present study was undertaken to rectify this omis~on. M ~ e d ~ s and M~hods

Fresh wh~e blood was obtained by v e ~ p u n ~ ture from seven m~e ad~t v ~ u n m ~ aged 26-56 year~ after an overnight fast. Two of ~ e v~unteers are cu~enfly ta~ng ~ther ~ m ~ - ~ e r~ acetate ( ~ ; 400 I.U. once per week) or ( R R R ~ m c o p h ~ ac~am (GB; 100 I.U. once p~ w~ ~ a ~e~ ~ p p ~ m e m . The b~ od (100 ~ . ) was drawn ~ ~ua~& ste~e tubes coated ~ t h ~ e anticoag~am, ~ u m ~h~ene~ m i ~ ~ Within h~ f an hour of b~ng draw~ the ~ o o d was ~ m r i ~ g e d ~ o ~ y (1500 ~ m ) for 10 min to ~move the b ~ k of the ~ m ~ pl~d~ and white b~ od c~ s . ~ 1 ~ n ~ g ~ t~ns w~e done at 4°C (except where noted othe~ ~ s ~ ~ a S o r v ~ RC2-B ~ n t r i ~ eq~pped ~ t h an SS-34 roto~ 4.25 in. ~ u ~ ) ~ a s m ~ obt~ned from the crude p l a ~ a fraction by ~ n t r i ~ n g at 6000 ~ m for 10 min, was stored at 4°C for 1 day prior to e ~ n of the fi~d m~eri~. The crude red ~ o o d c ~ fraction was washed three times by • e ~tion ~ 5 mM ~ o ~ ~ f ~ s~ (pH 7.~ [ 2 3 , ~ ~ ~ ~w ~ g ~ (1500 ~ for 10 rain. After the finM wash ~ e red blood calls were packed by ~ n ~ n g at 10 000 ~ m for 10 m ~ and the ~ p e m ~ a m was ~moved. The packed c ~ s were then washed Mth 5 mM p h o ~ h ~ o b u f f ~ e d sMine c o n t ~ n g 5 mM ascorbate ~ H 7.~ and ~ n t r i ~ d at 10000 ~ m for 10 rain. After ~ e ~ p e m a m m had been remove& 20 ~ of the packed c ~ s were ~ d ~ t h 10 v~.% of the same b u f ~ This red b ~ o d call suspenfion Oem~ocfit ca. 90%) was then fa~n~ed by denfiff a ~ o r d ~ g to the procedure of M u s h y [9]. (Nora the a-tocopherol does not sur~ve t~s s~p if the ascorbate is not present.) The mp 10% and b ~ m m 10% (ca. 2 ml eac~ of the red ~ o o d c ~ s were h a ~ d after ~ m o v ~ of the ~ p ~ n ~ a m . Each fraction was ~ t e d to a known v~ume (5 ~ ; hem~ocfit ~ . 40-45%) ~ t h the ascorbate comMn~g phosphate-buffered s~. S a m ~ of each red blood c ~ suspenfion were

withdrawn and microhematocrit~ red cell coun~ (measured wilh a hemocytomete~, mean cell volum~ and mean cell hemoglobin concen~ations were all determined by standard methods [25]. The ~ v d of creafine was estimated according to the method of Griffiths [26] uNng 0.5 ml of each red blood cell suspension diluted to 1 ml with distil~d wate~ Lipid was extracted from 0.5 ml of each red blood cell suspen~on by a solvent mixture cf heptane-isopropanol (3 : 2, v/v) as described by Radin [27]. The heptane-isopropanol solvent was removed in vacuo and the ripid re, d u e was taken up in 2.0 ml of n-heptane. Vitamin E extraction was performed on 3 ml of each red blood cell suspenNon by our new sodium dodecyl sulphate (SDS) method [28]. Lipids and vitamin E were extracted f o m 1 ml of plasma u~ng 1 ml each of ethanol and n-heptane [22]. a-Tocopherol was measured as described earlier [28]. O-Acyl fatty adds and cholesterol were determined ~multaneou~y by capillary gas chromatography after thor conver~on to the corresponding methyl esters and the ~butyldimethyl~lyl ethe~ respectivdy. The fatty add methyl esters were obtNned in the usuN way [28] by sodium metho~de-cat~yzed ~anse~erification of dupficate 0.8 ml samp~s of the fipid di~olved in nheptane (co~esponding to 0.1 ml or ~ss packed red blood cell). Tripentadecanoin and triheptadecanoin (NuChek Prep, Elyfian, MN.) were added as i n ~ r n ~ standards to the n-heptane extracts and the n-heptane was removed by evaporation under a ~ream of ni~ogen prior to the addition of the sodium methoxide reagent. Silylation of cholesterol was carried out by evaporating down, under a ~ream of ni~ogen, a hexane solution of faay add methyl esters and cholesmrol obt~ned after the uansestefificafion s~p, adding 150 ~1 of ~ butyldimethylchlorofilane/imidazole reagent (Appried Sdenc~ State Colleg~ PA) to the refidue and heating the mixture at 80°C for 20 min [29]. Excess reagent was removed by adding chloroform and washing three times wilh wate~ The chloroform hyer was dried over sodium sulfat~ decanted, and the chloroform removed under a ~ream of nivogen. The refidue was taken up in 0.5 ml of n-heptane. Samp~s (1 ~ were introduced into a 50 m × 0.25 mm inner diameter fused

86

silva capilhry column cont~ning a bonded 75% cyanoprop~ methyl sil~one phase (CPS-1; 0.25 ~ film thickne~; Quadrex Corp., New Haven, CT) by means of a Vafian 1095/11095 on-column i~ector mounted on a Vafian modal 3700 gas chromatograpE The inje~or was programmed to rise from 180°C to 240°C at 60 Cdeg/min immediatdy a f a r injection. The oven ~mperature was programmed to hold at 60°C for 1 rain a~er i~e~ion, then rise to 235°C at 20 Cdeg/min and m ~ n t ~ n that ~mperature for a fu~her 10 min. The d e ~ o r was set at 300°C and the helium flow rate was 3 ml/rnin. Resd~ Despite the modification we made by harves~ ing the top 10% and bottom 10% (instead of 5% as sugges~d by Murphy [9D of the fractionated packed cells, distinct biochemicM differences exist between the two ~actions which indicate that the cells in the top ~action are younger than those in

the bottom one. Spedfic~ly, the creatine ~vds in the top fractions are confiderably higher than in the bottom fraction ~,12] (see Table I). There are ~so differences in other red call indices between the two fractions which are in good agreement with earlier repot s [3,10] *. Lipid an~ysis showed that the top ~actions cont~n more cholesterol and fatty adds than the bottom ones (Tab~ I). (The top ~acfions were ~so usually found to cont~n more arachidonic add, in agreement wilh pubhshed data [2,3,6]).

It has been p ~ n t e d out t h ~ n o r m ~ v ~ u ~ ( ~ e ~ , ReL 2 ~ for mean cell h e m o ~ o N n are 32-36 g/100 ml and for ~ e n u m b ~ of cells/ml of packed cells 0 ~ 8 - 1 2 ~ . 1 0 ~°. Some of our v~ues for ~ese quantities (and for O-ac~ fatW a d d / c e l l and c h ~ r ~ / c d D may therefore be in error caused in pa~, at least, by ~ f f i c d t i ~ m co~ecdy counting cells w i ~ a h e m o c ~ o m ~ . F o r ~ n a t d ~ ~ ~so n o , d , all our more impoaant condu~ons are based on r ~ N e ( l i ~ d / l i ~ d ; y o u n g / O d ) dma and s h o d d ~ e f o ~ be una~ ~cted by t h e e prob~ms.

TABLE I RED BLOOD CELL I N D I C E ~ ~ A C Y L FATTY ACID A N D CHOLESTEROL OF H U M A N RED BLOOD CELLS SEPARATED BY DENSITY DIFFERENCES Source

Age (y)

SC

26

PY

32

DG

37

GB c (100) JW

38 43

JV

52

KI c (400)

56

Creafine ( ~ g / m l packed cd~)

Mean cell hemo~obin (g/100 ml packed c d h )

N ~ of cells/ml of packed cells ( × 10-10)

O-Ac~ fauy add b (mol/cd~ ( × 1016)

Cho~s~r~ b (m~/cd~ ( ~ 1016)

young a

~d ~

young

~d

young

~d

young

~d

young

o~

10.46 10.35 6.89 6.78 8.30 8.24 4.88 5.01 9.94 9.09 8.61 8.66 8.43 9.42

6.51 4.59 4.41 4.78 4.36 3.21 3.82 3.47 2.14 3.00 2.38 2.62 2.17 2.99

40.7 41.8 34.2 33.9 33.8 33.7 31.4 31.1 36.8 37.0 30.1 29.8 41.5 41.7

49.4 49.6 41.3 41.0 40.6 41.2 36.6 36.8 46.2 46.4 34.7 34.3 51.7 51.9

1.13 1.30 1.25 1.16 1.31 1.33 1.18 1.28 1.08 1.25 1.28 1.26 1.27 1.20

1.36 1.45 1.30 1.28 1.34 1.36 1.41 1.44 1.25 1.33 1.39 1.37 1.37 1.38

6.01 +_0.21 7.89 _+0.48 4.98 _+0.07 3.14 d -+ 0.20 5.90 +-0.28 4.71 _+0.17 5.34 ~ +_0.21

5.33 _+0.45 7.25 _+0.35 4.74 _+0.16 2.79 d -+ 0.01 5.73 _+0.19 4.14 _+0.13 4.72 _+0.23

3.29 _+0.12 3.95 _+0.12 2.91 _+0.21 2.69 d _+0.06 3.23 _+0.08 2.38 +-0.10 2.80 _+0.14

2.99 _+0.19 3.33 _+0.38 2.89 +0.21 2.45 d _+0.34 2.50 _+0.03 2.21 +-0.06 2.41 _+0.13

a Young = top 10%; old = bottom 10%. b Composi~ quantity derived from the mean of two measurements for each vafiabl~ Propagated error is c~cula~d from the de~afion from the mean for each quantity. ~ Numbers in parentheses represent the amount (I.U3 of (RRR)-a-tocopher~ ac~ate (GB) or all-rac-~-tocopherol acetate (KI) taken once per week by the volunteer as a dietary supplement. d Lipid obt~ned by SDS m ~ h o & Not ~l of the Hpid is ex~acted by this procedure (see ~xt).

87 TABLE II

TABLE HI

~TOCOPHEROL CONCENTRA~ONS IN HUMAN RED BLOOD CELLS SEPARATED BY DEN~TY DIFFERENCES

CELL AND [ I ~ D PARAMETERS FOR HUMAN RED BLOOD CELLS OF INTERMEDIATE AGE Blood was donald by JV. I n ~ r m e ~ e age ~ood was obtained by harv~ting the midd~ ~ f i o n of blood separated by den~.

a-Tocopherol (a-T) concen~afions were me~u~d by HPLC ufing fluorescence detection. See ~so footnote b, Ta~e I. a ~ / l i ~ d is a mole mfo. ~ # d is ~e m ~ ~ m of 0 - ~ fat~ adds and cholesterol ~ee TaMe I). Source

SC PY DG GB a (10~ JW JV KI a (~

a-T (mol/ceH~ × 1019)

~-T/lipid ( × 104)

young

old

young

old

Z24±~12 3~±~ Z90±~03

2.96±~11 Z82±~14 3.35±0~3

Z4±~l Z 5 ±~ 2 3~±~1

3.5±0.2 3£±~3 4A±~l

~15±0D1 Z39±0D8 Z18 ±~09

~11±~10 Z38±~15 2.51±~11

5A±~2 2.6±~1 3.1±~2

1~8 ± ~

2.08± ~04

2A±~l

b

P~am~ Creati~ (~g/ml p a ~ c ~ M~n c ~ ~mo#oMn ~ u a f i o n ~ / 1 ~ ml p ~ d c ~ No. ~ m l p a ~ cel~(×10 10) O-Ac~ fat~ a~d (m~/c~0( × 10~6) C h ~ r M (m~/c~l~ × 10~6) a - T ~ o p ~ l (mol/cell)( × 1019) ~T~op~h~d ( × 104)

5~4 b 2~±~2 4D±~2

a

4.28, 4.45 30.5, 31.3 1.32, 1.34 4.18±0.35 a 2.40 ± 0.18 a 2.18±0.12 a 3.3 ±0.3 a

~ e ~ m n ~ e b in Ta~e I.

2~±~1

See footnote c ~ Table I. b See footnote d ~ TaMe I. a

T h e older cells a p p e a r to contMn as m u c h or e v e n m o r e a - t o c o p h e r o l / c d l than do the y o u n g e r cells (TabM II). This ~ m o r e a p p a r e n t if we confider the a - t o c o p h e r o l / l i p i d ratios in the old and y o u n g cells (Table ID. Cells f r o m the m i d d l e ~ c fion of o n e b l o o d sample (JV) are of i n ~ r m e d i a t e age to j u d g ~ for exampM, f r o m t h o r creatine M v d (TabM III) and the a - t o c o p h e r o l M v d in these

cells is Mso i n ~ r m e d i a t e b e t w e e n lhe vMues f o u n d for the y o u n g an d old cells. Thus, all our results i n d i c a ~ that in red b l o o d cells lhe a - t o c o p h e r o l / fipid ratio increases with i n c r e a f i n g cell age. T h e c o n c e n ~ a t i o n of a - t o c o p h e r o l in the red b l o o d cell m e m b r a n e is affected by the c o n c e n t r a tion in the p l a s m a an d by the c o n c e n t r a t i o n of p l a s m a fipids [28,30]. F o r this reason we det e r m i n e d the p l a s m a a - t o c o p h e r o l c o n c e n ~ a t i o n ~ h p i d c o n c e n t r a t i o n s an d c o m p o f i t i o n s of our donors. Partition c o e f f i d e n t s for a - t o c o p h e r o l between p l a s m a an d y o u n g red b l o o d cells and be-

TABLE W PARI~ON C O E F H ~ E N ~ OF ~TOCOPHEROL BETWEEN PLASMA AND YOUNG AND OLD RED BLOOD CELL~ S~ ~ m n m ~ a and b ~ TaMe I. R ~ ~e c M c ~ e d ~om male ~fios. ~ T ~ p ~ is a m~e ~fio. ~ d ~ ~e m d ~ ~ m ~ ~ ~ f f adds and c h ~ r ~ (see TaMe I). ( M ~ ~ f i ~ s of cholesterol and ~ ~ adds are ~ven ~ TaMe VII in the ~memary Ma~fiM.) RBC, r ~ M~d c~s. ~

(~ T / l i ~

~sm~

(~ T ~

~

(a~id)

( × 10 ~)

( ~ T / l i p i ~ yo~ngRsC

(~T/fi~a

SC PY DG GD(10~ a JW JV KI(40~ a

155±0A 1Z2 ± ~9 1%8 ± ~5 2&l ±~5 2~2±1~ 2Z4±1~ 15~ ± 1.1

6.5±0.3 ~9 ± ~5 5A ± ~2 4.5 ±0.2 7.8±~7 72±~7 6.3 ± 0.5

4A±~3 ~4 ± if4 ~5 ± ~2 &l ±~3 Z0±~8 5£±~5 52 ± 0A

Mean ± &E

185 ± 1~

6.1 ± 0.5

<9 ± ~5

a

S~ ~ m n ~ e c ~ Ta~e I.

p~ g~

88 TABLE V M O L A R RATIOS OF A R A C H I D O N I C A C I D IN RED BLOOD CELLS (RBC) A N D PLASMA A N D OF a - T O C O P H E R O L TO A R A C H I D O N I C A C I D IN H U M A N PLASMA A N D Y O U N G A N D O LD RBC ~ Source

20 : 4 ( R B C ) / 2 0 : 4 ~ l a s m ~

a - T o c o p h e r o l / a r a c h i d o n ~ a o d ( × 10 a )

young ~ RBC

~d ~ RBC

~ma

young RBC ~

~ d RBC b

SC PY DG

2.8 ± 0.1 Z2 ± ~1 Z6 ± ~ 2

Z6 ± ~1 Z3 ± ~1 2.3 ± 0.2

38 ± 2 23 ± 1 ~ ±3

2~ ± ~2 2.1 ± 0.2 3.5 ± ~1

3 2 ± 0.3 2.7 ± 0.2 ~9 ± ~3

GB ¢ (10~ JW JV

2.1 ± 0.1 ~8 ± ~2 Z8 ± ~1

2.2 ± 0.1 4 ~ ± 0.2 2.5 ± 0.1

47 ± 2 88 ± 8 50 ± 4

53 ± 0 ~ a Z5 ± ~ 2 Z3 ± 0 2

6~ ± ~2 a 3.1 ± ~ 2 3.5 ± ~ 2

KI ~ (400)

Z5 ± ~1

2.2 ± 0.1

30 ± 3

1.9 ± ~1

2~ ± ~2

~ b ¢ a

See See See See

footnote footnote fo otno~ footnote

b a c d

in in in in

Tab~ Tab~ Table Table

I. I. I. 1.

tween plasma and old red blood cells are Nven in Table IV. In Table V the molar ratios of a-tocopherol to arachidonic add in plasma and red blood cells are ~ven, These data are r~evant to the suggestion that a-tocopherol slabfl~es membranes by complex formation with arachidonic add moieties [31-34], vide inffa. Although we reposed earlier that the SDS method yidded recoveries of fipid ~om red blood cells that were equN to or better than those obtNned by ufing ~ther the Folch or Bligh-Dyer methods [28] we have fince discovered that the heptan~isopropanol method I27] gives 1.5-2.4 times better yidds of O-acyl hpid. Howevec it should be noted that the recovery of choles~rol by this method was no be~er than the recovery by the SDS method. Moreovec the recovery of atocopherol by the heptan~opropanol method was ex~emdy poor; so pooc in fact, that this method should not be employed for vitamin E anNyses. D~cus~on

The aging of red blood cells does not ~ad to any net depletion of thNr a-tocopherol content. On the contrary, the membrane fipids in the older calls are significantly enriched in vitamin E.

As was mentioned ~ the I n ~ o d u ~ red blood c d h ~se about 20% of thdr membrane fipid during thdr fi~ span. In ~ o ~ u ~ have shown that mi~o-vefid~ are r d e a ~ d by the a~ng red blood cells [16] and that t~s process can be induced by endogenous ATP deletion [17], or by incubation with fiposom~ of phosphatid~ch~ lines cont~ning p ~ y u n s ~ u r ~ e d fatff ac~ c h i n s [35]. In the latter cas< mi~ovefide rdease co~d be in~bi~d by flee r a d ~ scavenge~ and by the i n c o r p o r ~ n of a-tocophe~ol into lhe liposomes [35], w~ch c e ~ n l y suggests that h p ~ pero~dation is involved in the membrane v ~ a t i o n process. It t h ~ o r e appe~s mascnab~ to assume that fi~d p e r o ~ d ~ n precedes vefic~ation and actually occurs during the ~ vivo a~ng of red blood cells. If thh assumpt~n is correct then the a-tocopher~ th~ is o ~ z e d by lhe fipid p ~ o x ~ ra~c~s may be partiaHy or completdy 'regenerated' by w ~ - s ~ u b l e redudng agents that are present m the plasma (e.g., a ~ o r b ~ , as has been shown to occur in modal sysmms [36-40]. Any o~dized a - t o c o p h ~ that escapes regeneration must be replaced from the plasma lipoprotein p o ~ of a - t o c o p h ~ . It has been suggested [31-3~ that a-~copher~ exerts i~ membrane-s~bilizing effect ~a complex formation with ar~c~don~ m ~ e f i ~ of membrane p h o s p h o l i p ~ with the m ~ h ~ 'brancheg on the

89 phytyl fide c h i n (or on the chroman ring [41]) fitting into the c # - d o u b ~ bond 'pockets' of the polyunsaturated fatty adds. One might therefore expect that fipids rich in arachidonic a d d r e , d u e s would ~ s o be rich in a-tocopherol. HoweveL this expe~afion ~ c e n ~ n l y not realized for the fipids in blood ( T a b ~ V). Thus, the red blood cell membrane fipids c o n t ~ n much less a-tocopherol than do the plasma fipids [22] (see ~ s o Tables II and IV), but they c o n t ~ n much mcre arachidonate ( T a b ~ V). While it might be suggested that the fipoprotein and membrane en~ronments are too different for this result to argue a g ~ n ~ complex formation, the same could not be s ~ d so readily about red blood cell membranes. N e v e n h d e s ~ lhe young ce~s have a lower ratio of a-tocopherol to arachidonate than the old cells ( T a b ~ V). We would therefore argue that a-tocopherol does not stabilize membranes by forming complexes w h ~ h e r static ~ 1 ~ 3 ~ cr dynamic [33] - with polyunsaturated fatty adds. (No relationship implying stabH~ation is apparent with 18 : 2 or 22 : 6 d t h e r 3 Our arguments are s u p p o s e d by the f a ~ that the old cells c o n t ~ n a higher concen~ation of a-tocopherol ffdafive to fipid) than the young cells but, n e v e ~ h d e ~ , are osmotic~ly more ~ a ~ than the young c d ~ [ L I ~ . Our resul~ ~ s o tend to argue a g ~ n ~ the existence of spedfic binding rites for a-tocopherol within the fipid portion of red blood cell membranes. If such rites exi~ed one might have expec~d to find a higher concentration of atocopherol in red blood call membrane fipids than in the p h s m a fipids, rather than the reverse. On the other hand, the exi~ence of such f i ~ particularly if they are at least partly p r o t d n in nature [42], could expl~n why a - t o c o p h e r d ~ not lost as the cell ages and sheds membrane m~rovefides. It r e m ~ n s to be seen if these microvesides c o n t ~ n ~ t a m i n E. None of our resul~ show any ~end with the age of the donor (26-56 year~. It ~ known that the red blood cells of a ~ n g rodents have a s h o a e r mean h ~ g f i ~ than those of younger a n i m u s together with reduced ~ v d s of free thiol, ~ u t a thione r e d u ~ a s ~ ~utathione peroxidas~ supe~ oxide dismutase, cat~ase, and ~ u c o s ~ 6 - p h o ~ p h a m dehydrogenase [13,15,43]. The red blood cell rqembranes of the older a n i m u s would therefore

appear to be more susceptible to pero~dative damage than those of the younger a n i m ~ g It has been found that in humans a-tocopherol concen~ations decrease with age of the su~ect in platel~s but not in plasma [44], which means that the ~ a-tocopherol)pl~sma/(a-tocopherol)pl~tel~t] ratio increases with age. Based on our own, very ~ m i ~ d , study the ~a-tocopherol)p~a~ma/(atocopheroD~d blo,,~,] ratio is not age dependenL FinallL we note that the paradox that ¼tamin E increases with age is not without a paralld. Spedficall~ Kunert and Edever [45] have very recently r e p o s e d that the content of Otamin E in beech ~aves and in fir needles increases with ag~

We thank the N a t i o n ~ Foundation for Cancer Research and the A ~ o d a f i o n for I n ~ r n a t i o n ~ Cancer Research for p a r t i ~ support of this ~esearch. We ~ s o thank the Red Cross (Ottawa) for taking the blood samples.

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