EXPERIMENTAL NEUROLOGY 39, 372-380 (1973)

Lipid Composition of Muscles of Nearly

Homogeneous Fiber Type

W. FIEHN ANLI J. B. PETER i

Neuronmscular Discascs Research Group, UCLA School (,f Medicine,
Los AngPlcs, Californics 90024

Reccivcd November 1, 1972

The lipid concentrations in guinea pig and rabbit muscles composed pre-
dominantly or exclusively of one type of fiber were analyzed. Muscles com-
posed of slow-twitch-oxidative fibers contain more total lipid than do fast-
twitch-oxidative-glycolytic or fast-twitch-glycolytic fibers. This difference
reflects a two-fold concentration of triglycerides and cholesterol in the
neutral lipid fraction. In the guinea pig, phospholipids are found in decreasing
concentration in fast-twitch-oxidative-glycolytic, slow-twitch-oxidative and
fast-twitch-glycolytic muscles. In all cases phosphatidylcholine is the major
phospholipid followed by phosphatidylethanolamine. The large amount of
mitochondria and fragmented sarcoplasmic reticulum in fast-twitch-oxidative-
glycolytic fibers explains their high phospholipid concentration. When these
data are combined with known fiber composition of muscles previously
analyzed, major discrepancies among previous reports on lipid concentrations
in various muscles are explained.

INTRODUCTION
The quantity and type of lipids in mammalian skeletal muscle have been
assessedby a number of previous investigators (2, 8, 10, 12, 13). In no
case, however, have the analyses been made on muscles defined as to
physiological, histochemical, and biochemical characteristics of the popula-
tion of fibers composing the muscle.
Previous studies from our group have shown that certain skeletal muscles
of the guinea pig and rabbit are composed predominantly or exclusively
of one of three major fiber types of skeletal muscle which we have termed
fast-twitch-oxidative-glycolytic, fast-twitch-glycolytic and slow-twitch-oxi-
1 Supported by NIH grant NS07.587. The use of facilities of Professor J. F. Mead
is gratefully acknowledged as is the assistance of L. Rusdal, K. Stempel and L.
Meinburg. W. Fiehn is the Paul Cohen Postdoctoral Fellow of the Muscular
Dystrophy Associations of America.

372
Copyright 0 1973 by Academic Press, Inc.
All rights of reproduction in any form reserved.
 MUSCLE LIPID 373

dative (15). The fibers are classified as fast-twitch or slow-twitch according

to the relative speed of contraction of muscles which are nearly homo-
geneous in fiber population as determined histochemically (1). The speed
of the contraction-relaxation cycle correlates directly with the specific
activity of myosin ATPase isolated from the same muscles ( 1) and with
the amount and character of the sarcoplasmic reticulum (5). Fibers are
subclassified according to metabolic properties determined by histochemical
properties (1, 3, 15) and quantitative biochemical analysis (15).
In this study we have analyzed the lipid composition of guinea pig and
rabbit muscles composed predominantly or exclusively of one fiber type.
The differences detected are best understood when related to both the
physiological and biochemical properties of the muscles.

METHODS
Adult, male Hartley strain guinea pigs (about 450 g) and adult male
New Zealand white rabbits (3-4 kg) were fed Purina Lab Chow ad lib.
Following a blow to the neck and exsanguination the muscles were care-
fully freed of fascia, tendons and adipose tissue followed by weighing,
mincings and homogenization in a Polytron (Brinkmann; Westbury, New
York) with 20 volumes of chloroform :methanol (2 :l, v/v). After ex-
tracting overnight at 4 C the solutions were filtered through glasswool and
evaporated to dryness. The lipids then were taken up in 2 :l chloroform-
methanol, and water was added to get a biphasic system according to
Folch, Lees, and Sloane Stanley (7). The lower phase was evaporated
under a stream of nitrogen and the total lipids, as well as neutral and
phospholipids, were separated on a silica gel column followed by weighing
after 12 hr storage in vacua over concentrated H,SO+ The neutral lipids
(NL) were eluted from the column with ether: petrolether: acetic acid
= 25 : 75 : 1 and the phospholipids (PL) with methanol containing log,
chloroform.
Thin-layer chromatography and densitometry were performed according
to Fewster, Burns, and Mead (4) on precoated silica gel plates (Merck;
Darmstadt, Germany), either with the separated fractions of NL and PL
or with the total lipid extract. In the latter case the plate for PL cletermina-
tion was developed in the PL solvent (chloroform : methanol : ammonia :
water = 60 : 35 : 9 : 1) followed by chromatography in the NL solvent (chlo-
roform : petrolether : acetic acid = 25 : 75 : 1) to remove free fatty acids from
the phosphatidylethanolamine fraction. Similarly the NL plate was de-
veloped a second time in petrolether to separate small amounts of various
hydrocarbons from the cholesterol esters. On each plate four standard
mixtures were co-chromatographed in amounts chosen to be in the range
of the expected amount of lipid in the sample.
 TABLE 1
LIPID CONTENT OF DIFFERENT MUSCLES FROM GUINEA PIG AND RABBITS

Guinea pig Rabbit

Semimembranosus
Muscle Red vastus White vastus Soleus accessorius Soleus

Predominant Fast-twitch- Fast-twitch- Slow-twitch- Fast-twitch- Slow-twitch-

fiber type oxidative- glycolytic oxidative glycolytic oxidative
glycolytic
(%I (78) (71) (100) (86) (96)

mg lipid/ y0 mg lipid/ y0 mg lipid/ y0 mg lipid/ y0 mg lipid/ yc 2

a
g muscle g muscle g muscle g muscle g muscle
z
Total 17.79 - 15.00 - 24.24 - 12.74 - 33.24 -
lipids kO.17 zko.77 f1.48 hO.75 f0.91

Phospho- 12.98 73.0 9.22 61.5 11.45 47.2 7.67 60.2 8.77 26.4
lipids f0.43 f0.40 ~0.76 f0.12 f0.58

Neutral 4.81 27.0 5.78 38.5 12.80 52.8 5.07 39.8 24.47 73.6
lipids ho.38 ho.38 f1.24 f0.49 zkO.76

Q Milligram lipid per gram muscle wet weight Z!Z standard error of mean, and percentage of total lipids. Number of preparations: guinea
pig red vastus (four), white vastus (four), soleus (five) ; rabbit muscles (four of each).
 MUSCLE LIPID 375

Guinea pig muscles used for lipid determination were the soleus (100%
slow-twitch-oxidative fibers) and the red portion (78% fast-twitch-oxida-
tive-glycolytic fibers) and white portions (71% fast-twitch-glycolytic
fibers) of the vastus lateralis. The rabbit soleus (96% slow-twitch-oxida-
tive) and semimembranosus accessorius (86% fast-twitch-glycolytic) were
also analyzed. Kerr’s nomenclature (11) ii used to define the rabbit
muscles used. No muscle composed predominantly of fast-twitch-oxidative-
glycolytic fibers was found in the rabbit hind limb.

RESULTS
The total lipids extracted ranged from 1.3-3.370 of the wet weight of the
muscles analyzed, In both rabbit and guinea pig the total lipids were higher
in slow-twitch-oxidative than in the other fibers (Table 1) . This difference
reflects the large amount of triglycerides in the neutral lipid fraction of
slow-twitch-oxidative muscles (Tables 1 and 2). In the guinea pig the
phospholipid concentration was highest in fast-twitch-oxidative-glycolytic
and lowest in fast-twitch-glycolytic muscle.
In both guinea pig and rabbit the cholesterol content per gram wet
weight is twice as high in the slow-twitch-oxidative as in the fast-twitch-
glycolytic ; likewise the cholesterol concentration of slow-twitch-oxidative
muscle of guinea pigs was two times that of the fast-twitch-oxidative-
glycolytic muscle (Table 2). The PL/cholesterol ratios are 14. 11.5 and
7 for fast-twitch-oxidative-glycolytic, fast-twitch-glycolytic, and slow-
twitch-oxidative muscle of the guinea pig and 9 and 5 for fast-twitch-
glycolytic and slow-twitch-oxidative muscles of the rabbit. The cholesterol
ester concentration was similar in fast-twitch-glycolytic and slow-twitch-
oxidative muscles of the rabbit and very low in the guinea pig (Table 2).
In all muscles phosphatidylcholine was the major phospholipid. com-
prising about 50% of the total phospholipids in all muscles studied: the
absolute amount was greatest in fast-twitch-oxidative-glycolytic and slow-
twitch-oxidative muscles (Table 3). Phosphatidylethanolamine, phos-
phatidylserine and sphingomyelin were present in decreasing percentage
and amount in all muscles analyzed. The amount of phosphatidylethanola-
mine is higher in fast-twitch-oxidative-glycolytic and slow-twitch-oxidative
muscles than in fast-twitch-glycolytic muscles as expected from their higher
content of mitochondria (6, 14, 15 ; cf. Discussion). In both rabbit and
guinea pig the range of phosphatidylethanolamine to phosphatidylcholine
ratios is small, varying from 0.5 (rabbit, fast-twitch-glycolytic) to 0.67
(guinea pig, fast-twitch-oxidative-glycolytic) .

DISCUSSION
The muscles chosen for study were previously characterized as to their
physiological, histochemical and biochemical characteristics ( 1, 3, 5, 14,
 TABLE 2
NEUTRAL LIPID COMPOSITION OF DIFFERENT MUSCLES FROM GUINEA PIG AND RABBIT'

Guinea pig Rabbit

Semimembranosus
Muscle Red vastus White vastus Soleus accessorius Soleus

Predominent Fast-twitch- Fast-twitch- Slow-twitch- Fast-twitch- Slow-twitch-

fiber type oxidative- glycolytic oxidative glycolytic oxidative w
CI
glycolytic
(%) (78) (71) (100) G36) (96) 2

mg lipid/ 7c mg lipid/ yc mg lipid/ yc mg lipid/ 7c mg lipid/ yc

z
g muscle g muscle g muscle g muscle g muscle
i2
Triglycerides 3.81 80.5 4.98 86.2 11.19 87.4 4.00 78.9 22.73 92.9
F3
f0.09 zto.33 f0.34 f0.01 +0.20 m

Cholesterol 0.93 19.3 0.80 13.8 1.60 12.5 0.87 17.2 1.69 6.9
f0.02 f0.05 f0.06 zkO.06 f0.22

Cholesterol Trace - Trace - Trace - 0.17 3.5 0.12 0.5

esters qJ.02 f0.02

a Values are milligrams of lipid per gram of muscle (wet weight) f standard error of mean, and percentage of total neutral lipids. Number
of samples as in Table 1.
 TABLE 3
MAJOR PHOSPHOLIPID CLASSES OF GUINEA PIG AND RABBIT MUSCLES"

Guinea pig Rabbit

Semimembranosus
Muscle Red vastus &‘hite vastus Soleus accessorius Soleus

Predominant Fast-twitch- Fast-twitch- Slow-twitch- Fast-twitch- Slow-twitch-

fiber type oxidative- glycolytic oxidative glycolytic oxidative
glycolytic
(7%) (78) (71) (100) (86) (96)

mg lipid/ Ye mg lipid/ % mg lipid/ % mg lipid/ ‘i; mg lipid/ r/;j

g muscle g muscle g muscle g muscle g muscle
Phosphatidyl- 6.18 47.6 4.64 50.3 5.69 49.7 4.02 52.4 4.58 52.2
choline f0.08 bO.28 ~1~0.62 ZkO.15 zko.22

Phosphatidyl 4.12 31.7 2.66 28.9 3.28 28.6 2.01 26.2 2.47 28.2
ethanolamine f0.20 f0.03 zto.05 z!zo.14 +0.22

Phosphatidyl- 2.68 20.6 1.94 21.0 2.36 20.6 1.64 21.4 1.70 19.4
serine plus ho.08 fO.ll zko.12 f0.12 fO.10
Sphingomyelin

Q Results are expressed as milligram lipid/gram wet weight muscle =t standard error of mean, and percentage of total phospholipid fraction _
Number of samples as in Table 1.
378 FIEHN AND PETER

15). This information together with our recent detailed characterization

of the lipid content of sarcolemma, fragmented sarcoplasmic reticulum and
mitochondria isolated from rat skeletal muscle (6) allows some reasonable
explanations for the different lipid content of the various types of fibers
which comprise these muscles and for discrepancies in the literature on
lipid content of various muscles.
The triglyceride lipid may be an energy storage form on which the slow-
twitch-oxidative fiber greatly relies because of its poor glycolytic systems.
Recent studies show that skeletal muscle not only oxidizes free fatty acids
extracted from blood but also utilizes its own esterified fatty acids as an
energy source (2). On the other hand our data show that the triglyceride
content of muscle does not necessarily parallel mitochondrial content as
manifest by the similar triglyceride concentrations in the mitochondria-rich
fast-twitch-oxidative-glycolytic muscle and in mitochondria-poor fast-
twitch-glycolytic muscle (14, 1.5; Table 2). In addition the slow-twitch-
oxidative fiber of the guinea pig contains twice as much triglyceride as the
fast-twitch-oxidative-glycolytic fiber but has only 78% as much succinate
dehydrogenase activity and 37% as much cytochrome a (14, 15).
Part of the triglyceride might be of interstitial rather than intrafiber
origin and this interstitial lipid could account for the high triglyceride
concentration of muscles composed of slow-twitch-oxidative fibers. This
could explain the discrepancy between the results of our chemical analyses
and histochemical straining for lipids which suggest that those fibers which
contain the most mitochondria also contain the most neutral lipid (9).
Regardless of the precise location of the excess triglyceride it is clear that
muscles composed of slow-twitch-oxidative fibers contain much more of it
than do fast-twitch-oxidative-glycolytic or fast-twitch-glycolytic muscles.
The same is true of cholesterol.
Previous studies (13) showed a slight but significantly higher concen-
tration of phospholipid in slow-twitch muscle (soleus) of the mouse over
that in fast-twitch mouse muscle (extensor digitorum longus). Since the
mouse EDL is known to be composed predominantly of fast-twitch-
glycolytic fibers and the soleus of slow-twitch-oxidative fibers (V. R.
Edgerton, personal communication), these data (13) reflect the small but
significant differences between the phospholipid concentration of slow-
twitch-oxidative and fast-twitch-glycolytic fibers shown in Table 1. On
the other hand, Frtiberg (8) found much more phospholipid in the red
part than in the white part of the rat gastrocnemius. Since the red part of
the rat gastrocnemius is composed predominantly of fast-twitch-oxidative-
glycolytic fibers and the white part of fast-twitch-glycolytic fibers (1) these
larger differences in phospholipid concentration are expected (cf. Table 1) .
This explanation of these seeming discrepancies does not imply that sub-
stantial differences in the type and quantity of lipids should not be expected
 MUSCLE LIPID 379

in muscles of different species composed of what seems to be the same type

of fiber. Indeed, Masoro, Rowe11and McDonald (12) found very much
more phospholipid in the soleus than in the gastrocnemius of monkeys.
Their data, however, are impossible to compare critically with ours or
that of the literature since data on the fiber type composition of the monkey
gastrocnemius are not available.
The important point is that the physiology and histochemistry of grossly
red or white muscle must be defined if the studies are to be interpreted
critically. This is illustrated by a recent paper describing a much higher
phospholipid concentration in red than white skeletal muscle of the guinea
pig which had almost identical contents of triglycerides (10). Our data
(Table 1) show that these authors were actually studying fast-twitch-
oxidative-glycolytic and fast-twitch-glycolytic muscle and not slow-twitch-
oxidative and fast-twitch-glycolytic muscle as might have been assumed
from the invalid but widespread generalization that white muscles are fast
and red muscles are slow (cf. References 1, 13. 15 for discussion).
Phosphatidylcholine and phosphatidylethanolamine account for the higher
phospholipid concentration in fast-twitch-oxidative-glycolytic and slow-
twitch-oxidative compared with fast-twitch-glycolytic fibers (Tables 1 and
3). These phospholipids also contribute more than 7570 of the phospholip-
ids of skeletal muscle membranes including sarcolemma, sarcoplasmic
reticulum and mitochondria (6). The higher concentration of phosphati-
dylcholine and phosphatidylethanolamine in fast-twitch-oxidative-glycolytic
and slow-twitch-oxidative than in fast-twitch-glycolytic muscle apparently
reflects a larger total amount of mitochondria plus sarcoplasmic reticulum
in these types of fibers.
Another striking difference between the types of skeletal muscle is the
higher concentration of cholesterol in slow-twitch compared to fast-twitch
muscles (Table 2). This tendency is also apparent when cholesterol is
related to total phospholipid concentration. In rat muscle the sarcolemma
accounts for most of the membrane-bound cholesterol (6) but we cannot
say if the lipid composition of sarcolemma from fast-twitch muscles differs
from that of slow-twitch muscles.
These data re-emphasize the importance of defining the nature of the
muscle being studied if results are to be critically interpreted and compared
with those of others. In addition they provide important clues to possible
differences in lipid storage and metabolism of various types of skeletal
muscle fibers.

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380 FIEHN AND PETER

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