294
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 5 5 0 2 1
A D I E T A R Y I N V E S T I G A T I O N ON T H E V A R I A T I O N S IN P H O S P H O L I P I D C H A R A C T E R I S T I C S OF R E D - C E L L M E M B R A N E S "
J. D E G I E R AND L. L. M. VAN D E E N E N
Department of Biochemistm,, Laboratory of Organic Chemistry, State University,
Utrecht (The Netherlands)
(Received D e c e m b e r 9th, 1963)
SUMMARY
Differences in phospholipid distribution and fatty acid composition between erythrocytes of different mammalian species, were investigated by dietary experiments
with rats and sheep. The nature of the ingested fats, and the non-participation of the
ruminal micro-flora, appeared not to affect the proportions of the major phospholipid
classes in the erythrocytes studied. The differences in the composition of fatty acid
constituents of red-cell phospholipids were shown to be attributable both to different
dietary habits, ruminal processes and species differences in the biosynthesis of fatty
acids.
INTRODUCTION
The integral part performed by phospholipids in biological membranes led several
investigators to suggest that the chemical composition of these lipids may participate
in determining the properties of the limiting cell membrane. This view has been
supported by studies from this laboratory, showing that coincidences exist between
differences in the permeability behaviour and the composition of the phospholipids
from erythrocytes of various mammalian species 1,2. A possible correlation between
lipid characteristics and permeability properties of erythrocytes may be complex,
since both differences in the proportions of various classes of phospholipids and
variations in the fatty acid constituents have been established. In addition red cells
from several species which differed both in lipid composition and permeability
characteristics have been found to exhibit a difference in the amount of so-termed
loosely-bound phospholipids a,4. It is difficult to judge whether the differences in the
polar or the apolar moiety of the phospholipid molecule, or both, are responsible for
the distinction in the surface properties of the erythrocytes concerned.
Perhaps valuable information on the problem outlined can be obtained by inducing a defined variation in the lipid composition of erythrocytes from one animal
species and to investigate whether such an alteration is accompanied by a change in
the permeability behaviour 5. Variations in the fatty acid constituents of red-cell lipids
* P a r t of this p a p e r was presented at the Deuel Conference on Lipids, S a n t a B a r b a r a ,
i 5 - i 8 F e b r u a r y , 1962.
Biockim. Biophys. Acta, 84 (I964) 294-3o4
D I E T A R Y E F F E C T S ON R E D CELL L I P I D S
295
can be brought about by dietary means, as has been demonstrated by feeding diets
deprived of essential fatty acids or rich in certain unsaturated fatty acids 5-~. However,
inadequate information is available on the possible dietary interference to the phospholipid distribution in the red-cell membrane.
The present paper is confined to a study of the influence of some diets on the redcell phospholipids, and their fatty acid pattern, from rat and sheep. These species of
animals were selected because their red cells differ extremely, both in the proportions
of major types of phospholipids and the fatty acid constituents as well as in permeability behaviour. This approach, which may form a basis for future investigations on
the role of the lipids in membraneous properties, furnished information on how far
dietary habits are involved in determining the differences in phospholipid characteristics between red cells of various mammals.
EXPERIMENTAL
Dietary experiments
Four groups of 6 male Wistar rats, 3 weeks old, were given ad libitum for 2o weeks
a regimen containing besides optimal amounts of proteins, vitamins and minerals the
following main sources of calories.
Group I:
72% calories as sucrose and 5% calories as sunflower oil.
Group I I : 72 % calories as hydrogenated arachis oil and 5 °/o calories as sunflower oil.
Group I I I : 77 °/o calories as sucrose.
Group IV: 77% calories as hydrogenated arachis oil.
After the given period the animals were killed and the blood obtained by heart
puncture was pooled for each group.
The experiments with sheep were started with two I-week old male lambs. The
animals were kept for I month on a cow's milk diet from which the fat was removed
and substituted by corn oil and coconut oil respectively to a concentration of 4%.
Blood samples were collected before and at the end of the dietary period, as well as
2 months after withdrawal of the diet during which period the animals were in the
meadow.
Lipid analyses
The erythrocytes were freed from serum by washing with saline and centrifuging.
The lipids were extracted with chloroform-methanol ( I : I , v/v), according to the
TABLE
I
THE RELATIVE DISTRIBUTION OF MAJOR CLASSES OF PHOSPHOLIPIDS FROM RED CELLS
OF RATS FED ON SEVERAL DIETS
In sphingomyelins a trace of lysophosphatidyl ethanolamine may be present. In lecithins a small
a m o u n t o f l y s o l e c i t h i n is i n c l u d e d . E F A -- e s s e n t i a l f a t t y a c i d s .
Diet
Group I:
carbohydrate
plus EFA
(%)
Sphingomyelins
Lecithins
Cephalins
18 (15.3-21.3)
63 (62.9-62.1)
19 (21.8-16.6)
Group II:
hydrogenated
arachis oil
plus EFA (%)
carbohydrate
(%)
Group I I I :
22 (23.1-21.9)
61 (62.2-59.2)
17 (15.9-17.7)
22 (18.4-25.3)
58 (58.2-58.0)
20 (16.7-23.4)
Group IV:
hydrogenated
arachis oil
(%)
19 (17.8-19.7)
58 (57.3-59.2)
23 (23.0-23.0)
B i o c h i m . B i o p h y s . A c t a , 84 (1964) 294 304
296
j.
D E G I E R , L. L. M. VAN D E E N E N
method of REED et al. 1% The lipids extracted from the rat red cells were separated on
silica-gel-impregnated papers using di-isobutylketone-acetic acid-water (40:25:5,
v/v) as a developing solvent. The sheep lipids were separated on thin-layer plates of
silica gel with chloroform-methanol-water (65:35:4, v/v) as a developer. Phosphorus
analyses of the separated spots using the BARTLETT11 modification of the method of
Fiske and SubbaRow as described by BOTTCHERet al. 12 revealed the relative distribution of the major types of phospholipids. F a t t y acid analyses were carried out by
gas-liquid chromatography using polyethylene glycol adipate columns for the separation and argon ionization detection. Details of the methods have been described
recently 13.
RESULTS
The data on the distlibution of the major classes of phospholipids from the erythrocytes of rat and sheep fed on different diets are compiled in Tables I and II. Though
the chromatographic methods utilized have certain limits, the distinction in the
phospholipid composition between both animals is striking. Regardless of the diet
given, the phospholipid fraction of rat erythrocytes contained a high amount of
TABLE
THE RELATIVE
DISTRIBUTION
II
OF M A J O R C L A S S E S OF P H O S P H O L I P I D S
FROM RED CELLS
OF YOUNG SHEEP ON LIQUID DIETS PREVENTING THE INTERVENTION OF THE RUMEN
IN COMPARISON TO THAT OF NORMAL SHEEP
Lambs before the diets
Diet
Sphingomyelins*
Lecithin*
Cephalins
I (%)
I1 (%)
54
12
34
57
io
33
Lambs 1: month on diet
I (%)
I I (%)
(coconut
(corn
oil)
oil)
63
4
33
66
2
32
Data previously
reported on red
cells of mature
sheep (%)
61 - 6 5 . 5
o - 1. 5
34.5-37
" See T a b l e I .
lecithin, whereas in the sheep erythrocytes sphingomyelin was the prevailing cholinecontaining phospholipid.
Variations in the regimen of rats apparently did not significantly affect the
proportions of the major phospholipids of the erythrocytes. The results suggest a
small increase in the relative amount of cephalins (phosphatidyl ethanolamine) of the
erythrocytes from animals deficient in essential fatty acids, when compared with
their controls. However, the accuracy of the methods used does not allow a definite
conclusion, and further experiments are necessary to elucidate whether essential
fatty acids deficiency indeed does cause such an alteration.
The two lambs fed on a milk diet supplemented with coconut and corn oil did not
reveal any difference in the proportions of major phospholipids. Just as in the erythrocytes of mature sheep previously analysed a very low content of lecithin balanced
by a significant amount of sphingomyelin was detected in the red cells from both
animals. However, the 1-week old lambs before starting the dietary experiments apBiochim.
Biophys.
Acta,
84 (1964) 2 9 4 - 3 o 4
297
D I E T A R Y E F F E C T S ON R E D C E L L L I P I D S
TABLE
FATTY
ACID
COMPOSITION
UNDER
OF THE
SEVERAL
Abbreviation:
.
14:o
14:1
15:o
15 i s o 16:o
16 : I
17:o
0. 3
.
0. 3
17 iso-
18:o
18:1
18:2
18:3
20:O
20:3
20:4
LIPIDS
.
Group IV
Group I I I :
(hydrogenated
(carbohydrate) arachisoil)
(%)
(%)
.
.
o.2
o. 3
.
.
0.2
-22.5
2.9
0. 3
-lO.3
36.2
lO.9
o.2
.
o.I
.
.
.
o.I
o.I
30.2
2.6
o.I
o.I
lO.5
26.6
4.1
.
.
.
.
.
--
--
31.1
2.2
0. 4
18.9
2.4
o.I
--
--
13. 7
18. 5
8.9
22.4
13.8
2. 3
24.0
RATS
= essential fatty acids.
hydrogenated
arachis oil
plus EFA
( /o/
o)
7.2
OF
CONDITIONS
Gr oup I I :
carbohydrate
plus EFA
(%)
12:O
ERYTHROCYTE
DIETARY
EFA
Gr oup I :
Fatty acid
III
o.8
32.3
16.2
8.7
6.0
lO.4
peared to contain in their erythrocytes a somewhat higher relative amount of lecithin.
Perhaps this finding explains some of the differences reported in the literature with
respect to the lecithin content of sheep erythrocytes 2,14.
Whereas the phospholipid distribution in the red cell was not altered by variation of diet, a considerable number of transformations appeared to be possible in the
apolar moiety of red cell phospholipids. Table III, giving the results of the fatty acid
analyses on the total lipids of the erythrocytes from rat, demonstrates that the four
groups of animals exhibited a different fatty acid pattern. In Groups I and II, both
supplied with essential fatty acids but receiving carbohydrate and hydrogenated
arachis oil respectively as major caloric source, the former animals showed a significantly higher level of 16 :o (palmitic acid) in the erythrocyte lipids when compared
with the second group. Among other differences, the second group revealed a higher
content of polyunsaturated fatty acids namely 18:2 and 20:4 in the lipids studied.
Deprivation of essential fatty acids (Groups III and IV) gave, as expected, a decrease
of these fatty acids in the red cell lipids, which is compensated at least in part by an
increase of the level of an acid having the gas-chromatographic properties of eicosatrienoic acid (20:3). Furthermore an significant increase of 18:1 fatty acids (e.g. oleic
acid) can be noted in both groups when compared with their respective controls.
Though the trend in the shifts of the fatty acid pattern of the red-cell lipids, from both
groups of animals not receiving essential fatty acids, is the same, some quantitative
differences can be noted. The animals with a high fat intake (Group IV) balanced the
decrease in 20:4 (arachidonic acid) mainly by an increase in 18 :i fatty acid(s) and to
a less degree by an augmentation of eicosatrienoic acid, in contrast to the animals
receiving carbohydrate (Group III) for which the reverse is true. The physical condition of Group IV was worse than that of Group III.
Biochim.
Biophys.
Acta,
84 (1964) 2 9 4 - 3 o 4
298
j.
D E G I E R , L. L. M. VAN D E E N E N
The foregoing o b s e r v a t i o n s were m a d e on the t o t a l lipid fraction e x t r a c t e d from
the e r y t h r o c y t e s concerned. Since in the red-cell p h o s p h o l i p i d s function as t h e m a j o r
carriers for f a t t y acid components, the results o b t a i n e d can be a t t r i b u t e d to this class
of lipids. The question r e m a i n s which t y p e s or sub-classes of p h o s p h o l i p i d s are concerned in the d i e t a r y i n d u c e d changes in the f a t t y acid composition of the red cell.
F o r t h a t p u r p o s e analyses were m a d e of the f a t t y acids from t h e m a j o r t y p e s of
p h o s p h o l i p i d s s e p a r a t e d b y c h r o m a t o g r a p h y on s i l i c a - i m p r e g n a t e d paper. The m a n i p u 16 °
16'
18 °
18'
18'
20"
20 m'
TOTAL LIPID
CEPHALINS
LECITHINS
SPHINGOMYELt N5
Fig. i. Influence of essential fatty acids deficiency on the main fatty acids of total lipids, and
cephalin, lecithin and sphingomyelin fractions. The open bars give the fatty acid patterns for
rats fed on the sucrose diet supplemented with essential fatty acids (Group I); the shaded bars
show the patterns of the animals fed on the fat-free diet (Group III). The abbreviations indicate
the number of C-atoms and double bonds e.g. 18" stands for octadecadfenoic acid.
lations were carried out u n d e r conditions t h a t were shown w i t h s y n t h e t i c phospholipids not to affect t h e p o l y - u n s a t u r a t e d f a t t y acid c o n s t i t u e n t s 15. The results of a
t y p i c a l e x p e r i m e n t , concerning a c o m p a r i s o n of the e r y t h r o c y t e lipids b e t w e e n Groups
I a n d I I I are r e p r e s e n t e d in Fig. I. The differences e n c o u n t e r e d in the analyses of the
t o t a l lipids are m o s t clearly reflected b y the phosphoglycerides n a m e l y lecithin a n d
p h o s p h a t i d y l e t h a n o l a m i n e . S t r i k i n g is t h e occurrence of a high a m o u n t of eicosatrienoic in the e t h a n o l a m i n e - c o n t a i n i n g p h o s p h o l i p i d s of the e r y t h r o c y t e s of the
a n i m a l s from G r o u p I I I . The results clearly p o i n t to a significant i n t e r c h a n g e of t h e
2o :3 a n d 2o :4 f a t t y acids within this p h o s p h o l i p i d class.
The results of t h e f a t t y acid analyses of t h e lipids from sheep e r y t h r o c y t e s a r e
r e c o r d e d in Table IV. The f a t t y acid p a t t e r n of t h e t o t a l lipids from l a m b e r y t h r o c y t e s
Biochim. Bioph>,s. Acta, 84 (I964) 294-304
299
D I E T A R Y E F F E C T S ON R E D C E L L L I P I D S
TABLE
IV
F A T T Y ACID C O M P O S I T I O N OF T H E E R Y T H R O C Y T E S
PREVENTING
THE INTERVENTION
OF Y O U N G S H E E P
ON L I Q U I D D I E T S
OF T H E R U M E N , IN C O M P A R I S O N W I T H T H A T
OF N O R M A L M A T U R E S H E E P
L a m b s before the diets
F a t t y acid
I (%)
12 :o
i4;o
14:1
15:o
15 iso
16:o
16:1
17:o
17 iso
18:o
18 : i
18:2
18:3
20:0
20:3
20:4
0.6
1.8
0.2
I.I
0. 3
I5.O
4 .1
2.0
0.8
8.4
54.6
7 .6
+
+
I.O
2.5
IT (%)
I.O
1.8
0. 3
I.I
0.2
I6.1
4.9
1. 9
0.8
8.3
5°.6
7.4
+
+
1.2
4.3
L a m b s s, mont h on diet
I:
coconut
II:
corn oil
oil (%)
(%)
0. 4
2.2
+
1.8
o. 4
14. 3
2.6
i.i
0. 4
6.9
52.5
13.6
+
0.9
i.o
1.8
0. 4
i.i
0. 3
1. 3
0. 3
16.2
3.4
1. 7
+
5.4
32.8
33.0
+
1.5
1. 3
1. 3
L a m b s z months after
withdrawal of the diet
N o r m a l mature
sheep
I ( /o~
o)
0. 4
i.o
0. 5
i.o
0.2
13. 5
2.5
1. 3
0. 7
7.5
48.9
18. 4
I.I
0. 7
0. 7
1. 4
II (%)
0. 5
1.2
0. 5
1. 3
0. 3
16. 4
2.9
1. 3
0. 7
7 .6
45.2
17.9
I.I
0.8
o. 7
1.6
0.6
1.6
0. 3
1. 4
+
16.o
2.3
1. 7
0. 3
9.7
49.6
i i .5
0. 7
0.9
1.9
1. 4
turned out to be practically identical to that found for normal adult sheep. It may be
recalled that the fatty acid composition of these erythrocyte lipids is characterized by
a strikingly high content of octadecamonoenoic fatty acid(s) (18:1) and a low content
of poly-unsaturated fatty acids particularly of 20:4. In this respect a significant
difference exists between erythrocytes from sheep and rat, while in addition the latter
erythrocytes contain a higher content of palmitic acid (compare Table III). Several
investigations, recently reviewed by G A R T O N 16, showed that the fatty acid composition of the tissue lipids from ruminants reflect the microbial-effected changes of
ingested fatty acids occurring in the rumen. During passage through the rumen the
unsaturated fatty acids are partly hydrogenated and otherwise modified, resulting in
the formation of geometric and positional isomers. Apparently, the alterations brought
about in the rumen are also reflected by the fatty acids from the red-cell lipids. For
this reason induction of a higher content of poly-unsaturated fatty acids in the tissue
lipids needs the action of ruminal micro-organisms to be precluded. OGILVIE et al. 17
studied the effect of administration of poly-unsaturated fatty acids by duodenal
fistulas and demonstrated that in this way the content of these acids, normally
occurring in the depot fat of sheep in a very low concentration only, could be significantly enhanced. Another approach, applied in the present study, involves the use of
new-born animals, where milk passes directly from tile oesophagus to the abomasum,
thus by-passing the rumen. This part of the stomach begins to function only when the
animal takes up rough food. The unwanted action of ruminal micro-organisms was
avoided by continuing the milk regimen of the lambs during the dietary period. The
animal supplied with corn oil additive to the fat-flee cow's milk revealed, after a
period of I month, a level of linoleic acid in the red-cell lipids which is nearly five times
as high as before the dietary period (Table IV). The augmentation of the linoleic acid
Biochim. Biophys. Acta, 84 ( I 9 6 ~ ) 2 9 4 - 3 o 4
300
j. DE GIER, L. L. M. VAN DEENEN
content is balanced b y a decrease in the content of mono-unsaturated f a t t y acids of
the C~s series. It is worth noting that the augmentation of linoleic acid was not
accompanied by an increase in the concentration of arachidonic acid in the red-cell
phospholipids. The results obtained on the animal fed with milk and coconut oil show
that under these conditions there was some rise in the amount of octadecadienoic
acids. On the other hand no incorporation of lauric acid, available in relatively high
amounts in the dietary coconut oil, into the erythrocyte lipids was observed. 2 months
after the regimen was stopped and the animals allowed to graze in the meadow, the
linoleic acid content was still significantly higher than in normal adult sheep (Table
IV). Though the present experimental results are too limited to allow any conclusion,
it m a y be suggested that the activity of the ruminal micro-flora to hydrogenate f a t t y
acids was not at top level yet in these experimental animals.
DISCUSSION
Whereas the normal erythrocyte contains a lipid composition that is characteristic for
each animal species, striking differences exist in this respect between different
mammalian species. In the sequence rat, man, rabbit, pig, ox and sheep a gradual
decrease of the ratio of lecithin to sphingomyelin has been observed, which turned out
to be accompanied by a comparable shift in the proportions of certain fatty acids e.g.
palmitic acid, oleic acid and arachidonic acid 1-a. These differences in both the phospholipid distribution and their f a t t y acid constituents raised m a n y questions 5. The
present dietary approach carried out on two animal species, which are most divergent
in the phospholipid characteristics of their red cells, appears to elucidate some of these
problems. The results obtained indicate that differences in the nature of the ingested
lipids, or the participation of ruminal processes, account at least to a significant part
for the variations in the f a t t y acid pattern of the red-cell lipids, but are not responsible
for the distinction in the proportions of various phospholipid classes. Consequently,
it can be concluded that the shifts in phospholipid distribution and f a t t y acid composition of the various erythrocytes are not directly related. This conclusion is in agreement with our earlier observation that the variations in the fatty acid patterns of
lipids from red cells of different animal species are localized predominantly in the
cephalin fraction 5.
As regards the distinction in the phospholipid distribution between the red cells
under discussion, no relation with dietary factors or the participation of ruminal
processes appears to exist. At present no reasonable explanation for the species
difference in the lecithin/sphingomyelin ratio, which is probably genetically controlled,
can be offered. Investigations on the haematopoietic bone marrow of sheep and ox
demonstrated no difference in the lecithin content, when compared with non-ruminants 18. A similar conclusion was reached b y THOMPSON AND HANAHAN 1~.
In agreement with previous investigations it can be stated that the influence of
dietary lipids is on the f a t t y acid composition, but the effects are complex. With
regard to the saturated-fatty-acid constituents, the experiments performed on rats
(supplied with essential fatty acids) indicated that b y a high caloric intake of hydrogenated arachis oil the palmitic acid content could be lowered when compared with
animals receiving carbohydrates. Feeding of fat containing a high amount of lauric
acid did not bring about any alteration in the concentration of this fatty acid in the
Biochim. Biophys. dcta, 84 (1964) 294-3o4
301
DIETARY EFFECTS ON RED CELL LIPIDS
red-cell lipids from the sheep. A similar observation was previously made in our
laboratory on rabbits ~0. This phenomenon endorses our observations on the selectivity
of the incorporation of fatty acids into red-cell phospholipids in vitro 2°. Notable shifts
are brought about by changes in regimen in the unsaturated fatty-acid constituents
of the cell. Deprivation of essential fatty acids induces a reduction of 18:2 and 20:4
fatty acids and a significant rise in the monoenoic fatty acids (18:1) and eicosatrienoic
acid (20:3) of the rat erythrocyte. However, when a diet low in essential fatty acids
but rich in hydrogenated fat, was given, the elevation of the 20:3 acids appeared to
t6 °
16 ~
17 e
18 °
18'"
18'
18 w
20 a
2 0 mn
NORMAL RAT
~n _ Oln
n
0
RAT ON
HYDROGENATED
FAT
n
n
[7
n
_
_
NORMAL SHEEP
Fig. 2. F a t t y acid p a t t e r n s of red-cell lipids from n o r m a l rats, n o r m a l sheep a n d from r a t s fed
on a h y d r o g e n a t e d fat diet (Group IV). A b b r e v i a t i o n s as in Fig. i.
be limited. An increase of monoenoic fatty acids in red-cell lipids in response to
essential fatty acids deficient diets has been observed also by WATSON9 on rats and by
GREENBERG AND IVfOON7 on monkeys. An elevation of the oleic acid content of lipids
from human erythrocytes, as reported by FARQUHAR AND AHRENS21, can be caused
also by feeding triolein; confirmatory results have been obtained recently in a joint
study with Dr. THOMASSON. A decrease of the oleic acid content of red-cell lipids
can also brought about by dietary means. A regimen with corn oil as major lipid
source caused an increase of the linoleic acid content of red-cell (phospho)lipids with a
concomitant lowering mainly of the oleic acid level. Such effects for instance have been
observed on rats by WITTING et al. 6, on rabbits in this laboratory s,2° and on human
erythrocytes by FARQUHAR AND AHRENS21. The present study demonstrated that it is
possible to cause such an interchange of oleic acid and linoleic acid in the red cell of a
ruminant, at least when the hydrogenating activity of the ruminal content is precluded. However, some species differences are to be noted. As demonstrated in a
convincing study by 1V[OHRHAUERAND HOLMANs both the concentrations of linoleate
and arachidonate in rat erythrocytes are directly related to the concentration of
linoleate in the diet. However, a comparison of our results obtained on rat and sheep,
Biochim. Biophys. Acta, 84 (1964) 294-304
302
j. DE G I E R , L. L. M. VAN D E E N E N
suggests that the ability of the rat to convert linoleic acid into arachidonic acid is
missing or less active in the sheep.
That different dietary habits, and alterations in ingested lipids by ruminal
processes, m a y explain part of the distinction in f a t t y acid composition of red-cell
lipids from various mammalian species is illustrated by the results in Fig. 2. The
change in the f a t t y acid pattern of lipids from rat erythrocytes brought about by
feeding a diet of saturated fats free of essential f a t t y acids results in an increase of
I 8 : I and a decrease of 16:o and 2o :4 f a t t y acids, thereby making this pattern nearly
identical to that of normal sheep. Apparently the ruminal processes, known to cause a
partial hydrogenation and isomerization of ingested poly-unsaturated f a t t y acids16,17
16°
16'
17°
18°
18~
_
Ilnn
18"
18I"
20 °
20 °l 20 TM
NORMAL RAT
Hn
LAMB ON
LIQUID CORNOI L
DIET
....
Fin
~
[7
FI_
~,-,
NORMAL SHEEP
Fig. 3. F a t t y acid p a t t e r n s of red-cell lipids from n o r m a l rat. normal sheep, and of a lamb fed
on a liquid corn oil diet. A b b r e v i a t i o n s as in Fig. I.
are reflected by the f a t t y acid composition of the erythrocyte phospholipids of the
ruminant and can be mimicked by giving a suitable diet to a non-ruminant. This view
was endorsed by the dietary experiment carried out with a lamb to which corn oil was
supplied under such conditions that the ruminal micro-flora was not yet developed.
As illustrated in Fig. 3, under these conditions there is a significant lowering of the
level of 18 : I f a t t y acids in the red-cell lipids. However, this approach does not furnish
a f a t t y acid pattern of the sheep erythrocyte, thereby indicating that an over-simplification has to be avoided. It can be seen that the sheep subjected to this experimental
treatment reveals a significantly elevated content of linoleic acid (I8:2), but there is
no increase in the amount of arachidonic acid in the red-cell lipids. The inability of the
sheep to convert the dietary linoleic acid to araehidonic acid apparently prevents the
f a t t y acid pattern of the red-cell lipids of the experimental animal from becoming
more like that of the red-cell lipids from normal rat. Thus it can be inferred that at
least three factors are involved in bringing about the distinction between the f a t t y acid
Biochim. Biophys. Acla, 84 (1964) 294-3o4
DIETARY EFFECTS ON RED CELL L I P I D S m
303
patterns of the phospholipids from red cells of various species, namely : (a) differences
in dietary habits, (b) the action of the rumen, and (c) species differences in the ability
to biosynthesize f a t t y acids. Though some of the variations in the fatty acid composition of the phospholipid fraction of various red cells m a y perhaps be attributed to
the specific proportions of several sub-classes of phospholipids, the present experiments strongly indicate that it is an over-simplication to connect a f a t t y acid pattern
of total phospholipids with the distribution of various types of phospholipids.
As outlined above, the differences in lipid characteristics of erythrocytes from
various species coincide in a striking manner with differences in permeability behaviour of the red cells concerned. Since the dietary influences are restricted to the
composition of the f a t t y acid constituents, possibilities arise to study within certain
limits some of the implications of f a t t y acid structure for the properties of the membranes concerned. Such studies are in progress in our laboratory. Obviously other
approaches are required to investigate the implications of differences in the phospholipid distribution for the permeability of the cell boundary.
The mechanism b y which the dietary-induced alterations in the red-cell fatty
acid constituents are brought about must now be considered. Though it cannot be
denied that during long-term dietary experiments such alterations will proceed even
in the early stages of blood-cell production in the bone marrow, a considerable effect
appears to occur in the circulating red cell as well. We have shown that in the rabbit
the dietary-induced augmentation of the linoleic acid content on a given regimen
reached its optimum after about io days~, ~°. In man the f a t t y acid pattern of red-cell
lipids is altered in a far shorter period than expected if this process was due to maturation of the red cell in the bone marrow 21. Also in our experiments on sheep the
augmentation of the linoleic acid content reached its highest level in a relatively short
time. Actually it has been shown in our laboratory that red cells in vitro have the
ability to incorporate fatty acids into their phosphoglyceridesS, 2°, and similar observations have been made by OLIVEIRA AND VAUGHAN22. These independent studies
revealed a selectivity with respect to the nature of the fatty acid incorporated, while
in addition it was found that the red cell is able to convert lysolecithin into lecithin.
Further studies in our laboratory, to be published soon, have elucidated the relationship between both phenomena.
As in our study on rabbits 2°, no significant uptake of the ingested lauric acid was
observed in sheep red cells. These findings are in keeping with the results obtained on
the incorporation of labelled f a t t y acids in vitro 2°. In the experiments both on sheep
and rat the dietary variations produced an interchange of several types of unsaturated
f a t t y acids e.g. the augmentation of linoleic acid was balanced by a decrease of oleic
acid content o~ the phospholipids from sheep red cells. This process resembles the
results of the studies in vitro which revealed that the incorporation of unsaturated
f a t t y acids, being of a higher rate than that of the saturated fatty acids, is positionally
specific 2°. Further, erythrocytes of ruminants have been found to be active in this
respect and to be able to convert lysolecithin and lysophosphatidyl ethanolamine
into the corresponding diacyl compounds.
The results of both the dietary approach and the isotopic studies are in good
agreement, and show that a renewal of the f a t t y acid constituents of the phosphoglycerides is a dynamic process of the cell membrane in circulating red cells.
Biochim. Biophys. Acta, 84 (1964) 294-3o4
304
J.
D E G I E R , L. L. M. VAN D E E N E N
ACKNOWLEDGEMENTS
The authors wish to thank Dr. J. THOMASSON (Unilever Research Laboratory, Vlaardingen) for the supply of the various groups of rats, and for much helpful discussion.
The dietary experiments on sheep were made possible by the kind cooperation of
Professor Dr. C. ROMYN and Dr. G. H. HUISMAN (Laboratory for Veterinary Physiology, State University, Utrecht).
This study was supported in part by a Research Grant (GM IO 198 ) of the
United States Public Health Service.
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