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A scientific research paper published in the Journal of Biological Chemistry in 1961. The authors, Gray and Macfarlane, studied the composition and structure of phospholipids in various tissues of rabbit, pigeon, and trout, as well as pig tissues. They used various methods to fractionate and analyze the phospholipids, including silicic acid column chromatography and gas-chromatography. The paper also discusses the distribution of phospholipids in different tissues and their fatty acid composition.
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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
component (36 (^) %) in ram (^) semen and in ox and pig
Gray, 1960a; Klenk & (^) iDebuch, 1955; Marinetti &
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-
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-
and re-extracted twice with 1 vol. of chloroform-methanol
three times with 0-2 vol. of salt solution (0.04M-MgCl2 or
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
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
5-
5- 3-
2- 2- 2-
Total cholesterol
(mg.)
18*
0- 009
0*
25- 27-
63-
Phospholipid*
(g.) (% of total lipid)
051 045
2- 2X
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.
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
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-
characterized by analysis for ethanolamine, serine, 31
aldehyde, inositol, alkali-stable P^ etc. and by
choline and choline plasmalogen, (5) sphingo- myelin. The methods of^ analysis for^ various^ groups
are, however, not so^ accurate^ that^ the^ presence of
tion in^ Tables^ 3-5.^ Comments^ on^ the^ individual tissues are made below. Rabbit myofibrils. Choline plasmalogen was
portion than in the whole muscle; about^50 % of^ the
About 43 % of the total was^ phospholipid, 2 % Bioch. 1961, 81.
Vol. 81
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
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-
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-
(^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-
The kephalin fraction contained about (^11) % of
of this, considerable decomposition occurred in this fraction on storage before saponification, and
The lecithin fraction contained less than 1 % of
from the silicic acid column, containing 19, 20 and
and a ratio N:P of 0-96-1-08: 1; these fractions
96 % of the P was labile to mild alkali and acid. On saponification by refluxing for 2 hr. in 0-5N-
glycerophosphate P by Burmaster's (1946) method. The three lecithin fractions were analysed separ- ately for fatty acids (Table 4); nearly 40 % of the
linoleic acid.
phospholipid conspicuous by its absence (Table 2). Compared with^ that in heart tissue the amount of
cardiolipin from this source because the small
the major aldehydogenic compound in all^ three tissues with serine plasmalogen (3%) and choline plasmalogen (3-1 %) present as^ minor components.
amounts (12-19 %). Phosphatidylinositol was
proportion (5 %) was^ greater than that of^ cardio- lipin, serine^ plasmalogen or^ choline^ plasmalogen.
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
corresponding kephalin and^ lecithin,^ with^ the exception of lung choline plasmalogen, which con- tained (^71) % of saturated acids and only (^24) % of a
The aldehydes (Table 6) from the choline plas- malogen and kephalin plasmalogen of lung and the 31-
Vol. 81 483
c
-L
0 0 0 t
CO
E (^) O
a)c^ aq(Cg. >-00.1^ (D a
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VDmL° C b s ACi CA
q*o^ t^0
(^64) "_
Cs^ CO^ D
A I_Se~4A
o (^) s 1 * C. (^) I4*
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6 6
O0 (^) O0O CO
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(^6 )
CO
O O O CO
10 O. CO 6 66 o coa _ O O
4 6 8666 i
10 0 CO o 45
4 O
o CO 10 6 Cb 6
coo xb a)
o6 Cb U
10 0 Co
es
-CO CO
6 C
0.1o^ s^ o
o CO;
10 - I) oC) E
r P t m
to oCt 00
_ oA
o 100
4 X _ COo
*~~~
_ COCOC. c6 I r c
co
CO
C)
CO t-r
X
do
00
16
CO 0-.
eq (^410)
CO 6
10 CO
P-
CO (^6) C
oo
.6.
00
COe Cl
EH
to CS O
(^10) 0.14 Et-
CO-
C 0 ( (M
00
6 10
_ 10 t 16 :
ell E
.1-
CO 0. U 16 COC;Q
1-
tD
t (^) 0.
Cq CD t*.
P-4 (^) '-ICO.d410 .1W10 w F-^ 1cOcOC-l -^ P-^004 00 0.10.10.1^ q^ ci
co~~~~~~~~~~~~~C Ca (^) ~~~~~~
.0: R^ §^ e^ woR^ $^ *m^ t^ $^9 i^ J^ {^5
es
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q Er- Er- P4 O E- , , E-4 (^) N PA PA Ei A p (^) E
(^484 )
a)
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a)
t:~ a)
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, H
G. M. GRAY AND (^) MARJORIE G. MACFARLANE
portion (80 (^) %) of palmitaldehyde. The spleen
peculiar in the amounts of branched (^) C,, and branched C15 (8 (^) %) compounds present.
to show the^ C1l and^ C18 saturated^ acids, the mono-
the C22 polyenoic acids in the kephalins were
unsaturated acids occur between the (^) kephalin
these series, were the antei8o (^) Cl5 and C17 acids, 12-
20 so
(^0 30) £ 0£ a~0££0££F
in the triglyceride, kephalin and lecithin fractions from
traces of trienoic C18 acid); (^) EO, polyenoic (^) CsO acids; *, poly-
from Bottcher, Boelsma-van Houte, Romeny-Wachter,
486 1961
The distribution of palmitaldehyde (^) (CL,$) and
branched-chain aldehydes were minor components
though small amounts of branched (^) C14 and (^) CH,
pig-kidney and trout-muscle kephalin plasmalogen also included a branched-chain C01 aldehyde which
ogens of ox tissues (Gray, 1960c) had a far greater
methylpentadecanal, but in pig spleen only (^3) % of
range of aldehydes (^) (C04-Cl) present is narrower than that of the fatty acids (^) (C,<,-C2). Presumably
afl-unsaturated ether^ linkage^ in^ the^ plasmalogen
Table 7. Amounts of branched-chain aldehydes, normal saturated odd-numbered carbon chain aldehydes and unsaturated aldehydes present in pk&smalogens from different tissues
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
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-