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480 P. J. LARGE, D. PEEL AN) J. R. QUAYLE 1961
Peel, D. & Quayle, J. R. (1961). Biochem. J. 81, 465. Quayle, J. R. (1961). Annu. Rev. Microbiol. (in the Press). Quayle, J. R. & Keech, D. B. (1959a). Biochem. J. 72,
Quayle, J. R. & Keech, D. B. (1959b). Biochem. J. 72,
Quayle, J. R. &^ Keech, D. B. (1960). Biochem. J. 75,
Rabinowitz, J. C. (1960). In The Enzymes, vol. 2, p. 185. Ed. by Boyer, P. D., Lardy, H. & Myrback, K. New York: Academic (^) Press Inc.
Sakami, W. (1955a). Handbook of Isotope Tracer Methods, p. 1. Cleveland, Ohio: Department of Biochemistry, Western Reserve University. Sakami, W. (1955b). Handbook of Isotope Tracer Methods, p. 5. Cleveland, Ohio: Department of Biochemistry, Western Reserve University. Trevelyan, W. E., Procter, D. P. & Harrison, J. S.^ (1950). Nature, Lond., 166, 444. van Niel, C. B. (1954). Annu. Rev. Microbiol. 8, 105. Vishniac, W., Horecker, B. L. & Ochoa, S. (1957). Advanc. Enzymol. 19, 1.
Bioch,em. J. (^) (1961) 81, 480
Composition of Phospholipids of Rabbit, Pigeon^ and^ Trout Muscle and Various Pig Tissues
BY G. M. GRAY* AND MARJORIE G. MACFARLANE
Lister Institute of Preventive Medicine, London, S.W. 1
(Received 17 April 1961)
Studies on the distribution and structure of the
phospholipids in certain mammalian tissues and
subcellular components have been reported pre-
viously (Gray & Macfarlane, 1958; Gray, 1960a, b;
Macfarlane, Gray & Wheeldon, 1960; Macfarlane,
1961a). The object of the present work was to
obtain a general picture of the pattern of fatty
components in phospholipids in other normal
tissues, and to examine further the distribution of
choline plasmalogen. This compound is less than
1 % of the total phospholipid in brain (Klenk,
Debuch & Daun, 1953; Webster, 1960) and a minor
component in liver and spleen; but it is a major
component (36 (^) %) in ram (^) semen and in ox and pig
heart (Lovern, Olley, Hartree & Mann, 1957;
Gray, 1960a; Klenk & (^) iDebuch, 1955; Marinetti &
Erbland, 1957; Gray & Macfarlane, 1958). It
appeared possible that choline plasmalogen is
associated directly with a contractile process,
though the small amount found in cod muscle
(Garcia, Lovem & Olley, 1956) indicated a con-
siderable species difference; Hartree & Mann
(1959), on the other hand, suggested that fatty
acids derived from plasmalogen participate in the
aerobic endogenous metabolism of sperm.
METHODS
The estimation of (^) P, total (^) N, amino (^) N, choline, aldehyde, inositol, fatty acid ester (^) groups, alkali-labile P and phos- phomonoester were carried out as in (^) Gray & Macfarlane (1958). The method for the estimation of (^) ethanolamine and serine (^) (Axelrod, Reichental & (^) Brodie, 1953) was modified
slightly by substituting an acid hydrolysis and chloroform- methanol-extraction procedure used by Dr C. Long (per- sonal communication) for the usual alkaline hydrolysis. Chromatography of phospholipids on silicic acid-impreg- nated paper was done in diisobutyl ketone-acetic acid- water (40: 20: 3, by vol.) at 20 and of glycerides in light petroleum (b.p. 60-80')-dii8obutyl ketone (96: 6, v/v) at room temperature (Marinetti, Erbland & Kochen, 1957). Chromatography on Whatman no. 1 acid-washed paper in butan-l-ol-water-conc. NH3 (100: 15:^ 2, by vol.;^ Coulon-
Morelec, Faure & Mareehal, 1960) was also used^ (Rp cardio-
lipin 0-74; phosphatidic acid 0-48). Chromatography^ of water-soluble esters obtained^ by mild^ hydrolysis^ was^ done according to Dawson (1954). Myofibrils were prepared from rabbit muscle^ by the method of Perry & Grey (1956); the lipid was compared with that extracted from a portion of the whole muscle from the same animal. Muscle from rainbow trout and pigeons was extracted within 1 hr. after death, and pig tissue obtained from a slaughterhouse within 2 hr. The extraction of lipid from the tissues was based on the method of Folch, Lees & Sloane-Stanley (1957) and Gray (1960b). In^ general the^ tissue^ was^ blended^ mechani-
cally with^5 vol. of cold^ chloroform-methanol (1^ :1,^ v/v)
and re-extracted twice with 1 vol. of chloroform-methanol
(2: 1, v/v). The combined extracts were washed two or
three times with 0-2 vol. of salt solution (0.04M-MgCl2 or
0.05N-NaCI), and the^ chloroform^ solution^ was^ dried^ and
evaporated. The residue was dissolved in light petroleum and dialysed according to van Beers, de Iongh & Boldingh (1958) to separate most of the non-P lipid (neutral lipid fraction) from the phospholipid. The phospholipid was fractionated on silicic acid as in Gray (1960b) and neutral lipid by the system of Hirsch & Ahrens (1958). The isolation of the fatty^ acids^ from^ phos- phatidylethanolamine, phosphatidylserine and phospha- tidylcholine and of the fatty acids and aldehydes from the ethanolamine, serine and choline plasmalogen was carried
- (^) Beit Memorial Fellow. out as described by Gray (1960b).
COMPOSITION- OF TISSUE PHOSPHOLIPIDS^481
Table 1. Yield of total lipid and pho&pholipid from variou8 ti8eq8u Weights are calculated for 100 g. of fresh tissue.
Tissue
Rabbit Skeletal muscle 1 2 Pigeon Heart muscle Breast muscle Trout Muscle 1 2 Pig Spleen Lung Kidney Ram Semen
Total lipid
(g.)
5-
5- 3-
2- 2- 2-
Total cholesterol
(g.)
Lipid P
(mg.)
18*
0- 009
0*
25- 27-
63-
Phospholipid*
(g.) (% of total lipid)
051 045
2- 2X
- (^) Calculated from P content x 25.
The fatty acids as methyl esters and the aldehydes as dimethyl acetals were identified by gas chromatography. The analysis of aldehydes from the plasmalogens of dif- ferent tissues by gas chromatography described by Gray (1960c) made possible the identification of a number of hitherto unknown, naturally occuring aldehydes with branched carbon chains. Further work (Gray, 1961) has shown that these aldehydes belong to either the iso- or antei8o-series of compounds analogous to the branched- chain acids found in animal tissues, and can be positively identified by their gas-chromatographic behaviour on polar and non-polar stationary phases.
EXPERIMENTAL AND RESULTS
In the fractionation of phospholipid mixtures on silicic acid columns the volume of any one solvent mixture necessary to separate a component varies with the proportion of the component and the nature of the mixture. After passage of chloroform to elute the remaining non-P lipid, the stepwise increase in methanol concentration was not made at stages preset by the volume of solvent to be passed, but at stages determined for individual columns by the separation curve, based on analysis of eluent fractions for P and for amino N, fatty acid esters etc. as appropriate. In general, elution was begun with 2 % (v/v) methanol in chloroform, increased in steps of 2 % until the cardiolipin
fraction was eluted, and then in larger^ steps,^ e.g.
10, 15, 20, 25, 35, 50 and^70 % (v/v)^ methanol^ in chloroform. The usual type of separation curve has been illustrated previously (e.g. Gray,^ 1960b). Eluent (^) fractions were pooled on the basis of the separation curve, supplemented^ by^ paper^ chro-
matography, and the larger fractions further
characterized by analysis for ethanolamine, serine, 31
aldehyde, inositol, alkali-stable P^ etc. and by
paper chromatography. The procedure permitted
in general the substantial separation of (1) cardio-
lipin, (2) phosphatidylinositol, (3) phosphatidyl-
serine or phosphatidylethanolamine, or^ both, and
corresponding plasmalogens, (4) phosphatidyl-
choline and choline plasmalogen, (5) sphingo- myelin. The methods of^ analysis for^ various^ groups
and structures present in^ a^ phospholipid molecule
are, however, not so^ accurate^ that^ the^ presence of
small amounts of^ other^ compounds of^ similar
general structure can^ be^ excluded.^ For^ example,
the analyses have^ not^ indicated^ the^ possible
presence in the kephalin and^ lecithin fractions^ of
the corresponding glycerol ether^ compound (Carter,
Smith & Jones, 1958), but^ during some^ recent
investigations on^ the^ hydrolysis of^ plasmalogens
(Pietruszko &^ Gray, 1960) evidence^ was^ obtained
that glycerol ethers^ may normally occur^ in^ small
amounts in these^ fractions.^ However, it^ is^ thought
that the^ computation from^ the^ analysis gives a^ fair
picture of^ the^ distribution^ of^ phosphorus amongst
the main components, and^ the^ separation curves^ and
detailed analyses for^ each^ tissue have^ been^ omitted.
The yield of total^ lipid and^ lipid P^ from^ various
tissues is^ given in^ Table^ 1; the^ composition of^ the
phospholipid in Table^ 2 and the^ fatty^ acid^ composi-
tion in^ Tables^ 3-5.^ Comments^ on^ the^ individual tissues are made below. Rabbit myofibrils. Choline plasmalogen was
present in the myofibrils in slightly higher pro-
portion than in the whole muscle; about^50 % of^ the
kephalin was^ plasmalogen^ in both^ whole muscle^ and
myofibrils.
Pigeon-breast muscle. The^ lipid contwt was^ high.
About 43 % of the total was^ phospholipid, 2 % Bioch. 1961, 81.
Vol. 81
COMPOSITION OF TISSUE PHOSPHOLIPIDS
Table 4. Fatty acid composition of phospholipids of (^) pigeon and trout muscle Values are expressed as percentage of total methyl esters. Pigeon-breast muscle
Fatty acid Myristic Palmitic Stearic Total normal saturated Palmitoleic Oleic Total monoenoic Linoleic Eicosatrienoic Arachidonic Eicosapentaenoic
Total C2, polyenoic
Docosapentaenoic Docosahexaenoic Total C,, polyenoic
Designation 14: 0 16 : 0 18 : 0
Kephalin
4-
54-
Lecithin Trace 24- 17- 42- 16: 1 0-2^ 1- 18: 1 4-2^ 22-
- 4-4 24- 18: 2 20: 3 20: 4 20: 5
Choline plasmalogen Trace 9-
Trout-muscle lecithin
I II III Mean* 0-4 2-6 1- 6-6 29-6 33-2 26- 5-5 3-0 1-7 2- 12-1 33-0 37-5 31-
5-5 1-6 7-9 2-0 -^ 0-
(^21692) 63-8 t25-6 (^) t12-0 2-4 2-9 (^109) 33-6 5-4 33-5 14-0 9-1 11-1 11- 2-0 24-4 3-9 2-
- 0-5 7-5 61-0 43- Nil 2-5 31-9 64-9 45-
- (^) Weighted mean. Includes any linolenic acid. Small amounts of branched (^) Cl,, branched C17 and C07 acids in some firactions have been omitted.
The kephalin fraction contained about (^11) % of
plasmalogen and was highly unsaturated (double
bonds/molecule of P, 5-6); possibly in consequence
of this, considerable decomposition occurred in this fraction on storage before saponification, and
analyses for fatty acids were therefore not done.
The lecithin fraction contained less than 1 % of
choline plasmalogen and was very highly un-
saturated. Three successive fractions I, II and III
from the silicic acid column, containing 19, 20 and
60 % of the lecithin P, had a P content 3-8-4-0 %
and a ratio N:P of 0-96-1-08: 1; these fractions
gave a single spot on paper chromatography, and
96 % of the P was labile to mild alkali and acid. On saponification by refluxing for 2 hr. in 0-5N-
potassium hydroxide in 95 % ethanol, 93 % of the
P became water-soluble, all of which was present as
glycerophosphate P by Burmaster's (1946) method. The three lecithin fractions were analysed separ- ately for fatty acids (Table 4); nearly 40 % of the
acids were C,^ hexaenoic acids, and less than 2 %
linoleic acid.
Pig spleen, lung and kidney. Approximately
80 % of the total lipid in lung and kidney and 50 %
in spleen was phospholipid. The three tissues were
generally similar in^ composition^ with^ no^ particular
phospholipid conspicuous by its absence (Table 2). Compared with^ that in heart tissue the amount of
cardiolipin found was low, especially in spleen.
A fatty acid analysis was not carried out on the
cardiolipin from this source because the small
fraction obtained was contaminated with too
much phosphatidylethanolamine and phosphatidyl-
serine. The ratio of phosphatidylethanolamine to
phosphatidylserine varied from approximately 1:
in spleen to 3:2 in lung and^ 5:2^ in^ kidney. The
spleen both from pig and ox (Gray, 1960b) appears
to be one of the richest natural sources of^ phos-
phatidylserine. Ethanolamine^ plasmalogen was
the major aldehydogenic compound in all^ three tissues with serine plasmalogen (3%) and choline plasmalogen (3-1 %) present as^ minor components.
Lecithin was as usual the^ major tissue^ phospholipid
and sphingomyelin also occurred^ in^ substantial
amounts (12-19 %). Phosphatidylinositol was
present in^ minor^ quantities, though in^ lung^ its
proportion (5 %) was^ greater than that of^ cardio- lipin, serine^ plasmalogen or^ choline^ plasmalogen.
The fatty acid distribution in the lecithin,
kephalin, choline^ plasmalogen and^ kephalin plas-
malogen of^ the three^ tissues differed^ in^ a^ number of
ways (Table 5). Spleen had the^ highest degree of unsaturation (expressed as^ double^ bonds^ per molecule of (^) fatty acid) and (^) lung the lowest. A large proportion of the acids from^ the^ kephalin plasmalogen fractions^ were^ C20 and^ Cn polyenoic compounds, that^ in^ spleen kephalin plasmalogen being exceptionally high^ (75-6%^ of total^ acids). The (^) kephalin plasmalogen and choline plasmalogen
fatty acids were more unsaturated than those of the
corresponding kephalin and^ lecithin,^ with^ the exception of lung choline plasmalogen, which con- tained (^71) % of saturated acids and only (^24) % of a
monounsaturated acid.
The aldehydes (Table 6) from the choline plas- malogen and kephalin plasmalogen of lung and the 31-
Vol. 81 483
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G. M. GRAY AND MARJORIE G. MACFARLANE
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G. M. GRAY AND (^) MARJORIE G. MACFARLANE
choline plasmalogen of kidney had a high pro-
portion (80 (^) %) of palmitaldehyde. The spleen
aldehydes contained a higher proportion of un-
saturated aldehydes, mainly oleylaldehyde and
linoleylaldehyde but including some monounsatur-
ated heptadecanal, than lung or kidney. The kidney
aldehydes on the other hand had a higher pro-
portion of branched-chain compounds. The alde-
hydes from the kidney kephalin plasmalogen were
peculiar in the amounts of branched (^) C,, and branched C15 (8 (^) %) compounds present.
DISCUSSION
It might be expected that differences in family,
species and diets would be reflected in variation in
the nature and proportion of the different phospho-
lipids and in their fatty acid composition. The
analyses reported here for various tissues indicate
little difference between beast, bird and fish in the
nature of the phospholipids in the same kind of
tissue, except for the virtual absence of choline
plasmalogen from trout muscle. Moreover there is
comparatively little difference in the fatty acid
composition of the same phospholipid from the
same tissue of different species in the proportions
of saturated and polyenoic acids: the most notable
exception to this is that cardiolipin from trout
muscle contained little linoleic acid and a high
proportion of saturated acids, and the lecithin
contained a high proportion of docosahexaenoic
acid.
The most^ substantial differences in fatty acid
composition in^ these tissues appear to lie between
the different kinds of^ phospholipid irrespective of
tissue or^ species. The results of^ the present and
previous work^ are^ presented graphically in^ Fig. 1
to show the^ C1l and^ C18 saturated^ acids, the mono-
and di-enoic acids, and the C20 and C22 polyenoic
acids in the triglycerides, lecithin and kephalin
fractions. The preponderance of stearic acid as the
saturated acid in kephalin and kephalin plasmal-
ogen and of palmitic acid in lecithin and choline
plasmalogen, noted^ previously (Gray, 1960b),
holds except for liver lecithin, which both in the
ox and the rat contains more stearic than palmitic
acid. The distribution patterns of other fatty acids
were far less predictable, though certain trends
were indicated; the proportions of C20 polyenoic
acids, especially arachidonic acid, and in general
the C22 polyenoic acids in the kephalins were
higher than in the corresponding lecithins and the
lecithins contained a higher proportion of linoleic
acid. Similar differences in the distribution of the
unsaturated acids occur between the (^) kephalin
plasmalogens and the choline plasmalogens.
The tissue triglycerides analysed, though few in
number, are remarkably similar to each other and
to those reported for normal aorta (Bottcher,
Boelsma-van Houte, Romeny-Wachter, Woodford
& van Gent, 1960) and differ substantially from the
kephalins in that palmitic acid is the preponderant
saturated acid, and from both kephalins and leci-
thins in the low content of linoleic and polyenoic
acid. It is clear that there is a considerable
selectivity towards the fatty components in these
compounds, as well as in cardiolipin and phos-
phatidylinositol, which should be borne in mind in
considering the general validity of routes for the
interconversion of tri- and di-glycerides and phos-
pholipids-for example, in the biosynthesis oflecithin
by transmethylation of phosphatidylethanolamine.
Saturated fatty acids with an odd number of
carbon atoms and branched-chain fatty acids with
both even and odd carbon numbers were minor
components of all the tissues. The most commonly
occurring, and very often the only members of
these series, were the antei8o (^) Cl5 and C17 acids, 12-
methyltetradecanoic and 14-methylhexadecanoic
acid. It seems possible that these branched fatty
acids are of bacterial origin, for it is now known
that they are the predominant or major kind of
20 so
coiicsoI X c.^ -, a.°ox^ o'o c.c.oxo cx^ to ,o
(^0 30) £ 0£ a~0££0££F
Fig. 1. Relative proportions of certain fatty acids occurring
in the triglyceride, kephalin and lecithin fractions from
different tissues. 0, Palmitic acid; *, stearic acid;
A, monoenoic C16 and C18 acids; *, dienoic C28 (including
traces of trienoic C18 acid); (^) EO, polyenoic (^) CsO acids; *, poly-
enoic C2, acids. Values for triglycerides of human aorta are
from Bottcher, Boelsma-van Houte, Romeny-Wachter,
Woodford &; van Gent (1960).
486 1961
8CoOMPOSITION OF TISSUE PHOSPHOLIPIDS
fatty acid in certain bacteria such as Sarcina,
Bacillus subtilis and Micrococcus Iysodeikticus
(Akashi & Saito, 1960; Macfarlane, 1961 b). James,
Webb & Kellock (1961) note the occurrence of
heptadecanoic acid as well as 10-hydroxystearic
acid in the faecal lipids of subjects with steator-
rhoea and discuss the possibility of synthesis by
micro-organisms.
The distribution of palmitaldehyde (^) (CL,$) and
stearaldehyde (C18) in the plasmalogens of different
tissues was similar to that of palmitic acid and
stearic acid in the corresponding diacyl compounds
(Gray, 1960c). The main difference was that,
although there was^ more stearaldehyde in kephalin
plasmalogen than in choline plasmalogen, palmit-
aldehyde was a large and usually the major
normal saturated component in both. Saturated
aldehydes with an odd number of carbon atoms and
branched-chain aldehydes were minor components
of all the tissues examined in this recent work. The
C15 and C17 normal and branched-chain (both iso
and anteiso) compounds occurred most frequently
though small amounts of branched (^) C14 and (^) CH,
aldehydes were often present. The aldehydes from
pig-kidney and trout-muscle kephalin plasmalogen also included a branched-chain C01 aldehyde which
was probably 16-methyloctadecanal.
In general aldehydes isolated from the plasmal-
ogens of ox tissues (Gray, 1960c) had a far greater
proportion of branched-chain compounds than
those isolated from pig tissues (Table 7). In ox-
spleen choline plasmalogen half of the total alde-
hydes were branched compounds, mainly 13-
methylpentadecanal, but in pig spleen only (^3) % of
the total aldehydes were branched compounds.
The proportion of aldehydes with odd-numbered
carbon chains was small (2-11 %) varying only
slightly from tisue to tissue and species to species.
The proportions of unsaturated aldehydes varied
more in pig tissues than in ox tissue.
The data so far obtained on the fatty acid and
aldehyde composition of tisue phospholipids of
different species suggests further limitations (Gray,
1960b) to the enzyme systems which are involved in
the biosynthesis and metabolism of the plasnal-
ogens. In all the sources so far examined the
range of aldehydes (^) (C04-Cl) present is narrower than that of the fatty acids (^) (C,<,-C2). Presumably
the enzyme responsible for producing the typical
afl-unsaturated ether^ linkage^ in^ the^ plasmalogen
molecule will not in general act on^ compounds with
a carbon chain longer than 18 carbon^ atoms^ in the
ac-position of^ the glycerol moiety. Of^ course^ it^ is
also possible that the high degree of^ unsaturation
normally present in^ the C20 and^ C22 carbon chains
and not the chain^ length prevents the-^ enzyme
from functioning.
SUMMARY
1. The lipid was extracted from pig spleen, lung
and kidney, pigeon, trout and rabbit muscle, and
the phospholipid fractionated quantitatively on
silicic acid columns.
2. The distribution of the lipid phosphorus
amongst cardiolipin, phosphatidylinositol, eth-
anolamine-, serine- and choline-containing diacyl
Table 7. Amounts of branched-chain aldehydes, normal saturated odd-numbered carbon chain aldehydes and unsaturated aldehydes present in pk&smalogens from different tissues
Values for each class of aldehyde are expressed as a percentage of the total aldehydes.
Choline plasmalogen Ox (^) spleen Ox heart Ox liver Pig heart Pig spleen Pig lung Pig kidney Pigeon muscle Ram semen Kephalin plasmalogen Ox spleen Ox heart Ox liver Pig heart Pig spleen Pig lung Pig kidney Pigeon muscle Trout muscle
Normal saturated
'- - - ranclhed- odd-numbered
chain carbon chain
50- 15- 56- 2- 2- 2- 6- 0-
9-
2- 3- 23* 1*
8-
6-
2-
1-
8*
11-
Unsaturated
1- 1-
4* 15- 6- 2*
2-
8- 6- 5* 10- 20-
5-
Vol. 81 487
