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Phospholipid Composition in Rabbit, Pigeon, Trout Muscle, and Pig Tissues, Lecture notes of Biochemistry

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.

What you will learn

  • What were the major differences in the fatty acid composition of phospholipids from different tissues?
  • What methods were used to fractionate and analyze the phospholipids in the study?
  • What was the distribution of phospholipids in different tissues according to the study?

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bg1
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,
623.
Quayle,
J.
R.
&
Keech,
D.
B.
(1959b).
Biochem.
J.
72,
631.
Quayle,
J.
R.
&
Keech,
D.
B.
(1960).
Biochem.
J.
75,
515.
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
out
as
described
by
Gray
(1960b).
*
Beit
Memorial
Fellow.
pf3
pf4
pf5
pf8
pf9

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Download Phospholipid Composition in Rabbit, Pigeon, Trout Muscle, and Pig Tissues and more Lecture notes Biochemistry in PDF only on Docsity!

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

c

-L

G. M. GRAY AND MARJORIE G. MACFARLANE

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, H

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