Glycerophosphate acylation by microsomes and mitochondria of normal and dystrophic human muscle

II3

Clrnrcu Chrmrcu Actu, 140 (1984) 113-124 Elsevier

CCA 02860

Glycerophosphate acylation by microsomes and mitochondria of normal and dystrophic human muscle D. Kunze a**, B. Riistow a and D. Olthoff ’ ” Institute for Pathological

cmd Cltnical Biochemistry and h Department of Anaesthesiology, Humboldt -lJnruersit~~, Berhn (GDR)

(Received

October

3rd. 1983; revision February

Chant6 Hosprtul,

10th. 1984)

Key words: Muscular dystroph.v; Inborn enzymtc defect; Glyceroliptd htosynthests; ltptds; Phospholiprds tn memhrurtes

G3P mcorporatron tnto

Summary The incorporation of [‘4C]glycerophosphate into phosphatidic acid, ditriacylglycerol and phosphatidylcholine by microsomes and acylglycerol, mitochondria prepared from normal and dystrophic human muscle incubated in vitro in the presence of 0.3 mmol/l CDP-choline was measured. In mitochondria only phosphatidic acid and diacylglycerol are labelled; the rate of incorporation into these two compounds showed no difference between dystrophic and normal mitochondria. In dystrophic microsomes the incorporation into phosphatidic acid was delayed and decreased. No incorporation of glycerol into diacylglycerol, phosphatidylcholine and triacylglycerol could be measured. Thus in dystrophic muscle microsomes only PA was labelled during an incubation of up to 45 min. In both types of microsomes the concentration of endogenous free fatty acids and diacylglycerol was nearly identical. The level of phosphatidylcholine was 186 and 79 nmol/mg microsomal protein in normal and dystrophic muscle microsomes, respectively. Whether the results could be explained as secondary changes was discussed. Despite some unsolved problems the conclusion was drawn that a better explanation

und Klinische * Correspondence should be addressed to Dr. D. Kunze. Institut fDr Pathologische Biochemie. Bereich Medizin (Charit&) der Humboldt-Universitit, Berlin, GDR. Ahbreoiafions: PL. phospholipids; PC, phosphatidylcholine; PL-ase A and D. phospholipase A and D. respectively; DG, 1,2-diacylglycerol (diglyceride); TG, triacylglycerol (triglyceride); PA. phosphatidic acid; LPA, lysophosphatidic acid; FA, fatty acid; G3P, sn-glycerol-3-phosphate; PA-P-ase, phosphatidic acid phosphatase (EC 3.1.3.4); PE. phosphatidylethanolamine.

0009-8981/84/$03.00

0 1984 Elsevier Science Publishers

B.V

114

of the results is to assume a primary defect involving microsomal-bound phatidic acid phosphohydrolase and possibly glycerol-P-acyltransferases.

phos-

Introduction

For 15 years variations of the PL-pattern in human dystrophic muscle have been known [l-4]. The decrease of PC and the increase of sphingomyelin have, however. produced little interest, but the few investigations carried out in the following years have confirmed these results [5-71. In human muscular dystrophy in the last few years the PL-changes have mainly been seen as a consequence of the infiltration of the dystrophic muscle with fat and connective tissue [8,9]. It seems to us that a decision as to whether the fatty degeneration of dystrophic muscle simulates or masks a changed PL-pattern in the dystrophic muscle cell itself will be impossible to make as long as only content is estimated considering the small amount of the bioptic specimen and the heterogenous nature of dystrophic muscle. The overall conclusion drawn by us has been that in lipids, results about content will always represent an uncertain basis. On the other hand we believe that the investigation of the metabolism in the field of lipids will give a more stable basis. Therefore both other authors [lo,11 a.o.] and our own team [7,12,13] began to research the PL-metabolism of dystrophic human muscle. We have concentrated our efforts on the anabolic site of PL-metabolism. We first studied the water-soluble pathway of PC biosynthesis, that is the formation of CDP-choline and its transfer to DC synthesizing PC [13]. We found normal activities of the enzymes which transferred choline. The other part of the de novo PC-synthesis - that is the acylation of G3P and the biosynthesis of DG - is common to both TG and PE. DG is the precursor of PC, PE and TG. Because PE and TG are not decreased in muscular dystrophy at first sight, an investigation of this route seemed to be valueless. However, investigations on the origin of the species pattern in glycerolipids, which is different in PC, PE and TG have led to consideration of different pools of DG (for example, in lung [14], in brain [15]). Experimental results have been presented which suggest that the glycerolipid species distribution is not only caused by the Lands De-Reacylase cycle but also by de novo synthesis. This argues against a unitary DG pool. Following this idea a defect in PC de novo synthesis seems to be possible without affecting the synthesis of other glycerolipids. The aim of our experiments has been to study the G3P acylation in microsomes and mitochondria of human dystrophic and normal muscle. In the same microsomal preparation the content of PC, DG and free FA has been estimated. Material

and methods

Material Normal muscle was obtained during routine surgery from the gluteus (application of an artificial hip, spinal anaesthesia. without muscle relaxants). Dystrophic muscle

115

(approximately 3 g) was obtained by biopsy from the quadriceps femoris from three males (aged 6, 6 and 8 years) suffering from Duchenne muscular dystrophy. The small patients were able to walk and the quadriceps had only a slightly reduced power. The biopsy was performed under local anaesthesia. Attention was paid to avoid subfascial infiltration with procaine. Quadriceps and gluteus are of a mixed composition and contain an equal amount of red and white fibers. The biopsy specimen was homogenised immediately after sampling in a Potter homogeniser in a medium containing Tris (10 mmol/l, pH 7.4). saccharose (0.25 mol/l), EDTA III (1 mmol/l). After centrifugation for 10 min at 800 x g the supernatant was centrifuged for 20 min at 20000 x g. The resulting pellet was taken as the mitochondrial fraction, The supernatant was centrifuged for at least 90 min at 100000 x g and the pellet taken without further purification as the microsomal fraction. The preparation was stored in a deep frozen state until use (3 weeks). Parallel to this a normal muscle sample was prepared in the same way, the final preparation of microsomes and mitochondria diluted to the same protein content as in dystrophic probes and also stored for nearly 3 weeks. The pooled mitochondria from dystrophic muscle had a total protein of 10 mg and the total protein of microsomes from dystrophic muscle was 3.38 mg. After first practicing the G3P incorporation with normal human muscle, the main experiment was done on a parallel basis with normal and dystrophic organelles in the same concentration. 0.4 mg microsomal protein was in every microsomal incubation; the mitochondrial tubes contained 0.73 mg protein. Reugents “C-U-labelled G3P (specific activity 6.33 GBq/mmol) was supplied by the Radiochemical Centre, Amersham UK; CDP-choline, palmitoylcarnitine, Florisil. different types of silicagel and all other biochemicals were commercial products of analytical grade; the solvents were redistilled before use. G3P incorporation The composition of the reaction mixture accords to that described by Fox and Zilversmit 1161 for liver microsomes. The incubation mixture contained HEPES buffer 50 mmol/l, pH 7.8; EDTA 0.1 mmol/l, cysteine 10 mmol/l, CoA 0.167 mmol/l, CDP-choline 0.333 mmol/l. KC1 150 mmol/l, MgClz 3 mmol/l, ATP 3.5 mmol/l, 14C-G3P 0.5 mmol/l, specific activity 2252 dpm/nmol. The final volume was 0.6 ml. This basic mixture was taken for the incubation of microsomes and mitochondria. In the cases quoted in the legends, palmitic acid (0.133 mmol/l) and albumin (1.33 mg/ml) or Dr.,-palmitoylcarnitine (0.25 mmol/l) were added. After a preincubation of 10 min the reaction was started by the addition of radioactive G3P and after a given time stopped by adding methanol/chloroform. Fractionation of lipids The lipids were extracted in accordance with 0.15 mol/I HCl instead of water.

with the Bligh-Dyer procedure, washing The lipid extract was evaporated and

116

fractionated using two-dimensional thin-layer chromatography on Silicagel H/Florisil plates. PA prepared from boiled microsomes by PL-ase D hydrolysis was added as tracer to the lipid extract. Two-dimensional thin-layer chromatography was carried out as described by van Heusden and van den Bosch [17] using two solvent systems. In the first direction we used chloroform/ methanol/ ammonia/ water, (90 : 54 : 5.5 : 5.5 by vol.), and in the second direction chloroform/methanol/acetic acid/water (90 : 46 : 12 : 12, by vol.). After drying the plates were exposed to iodine vapour and the spots outlined. Spots relating to PA and PC are scraped off. Then the plate was auto-radiographed over 10 days to localise further labelled lipids. There was no other labelling to be observed, especially not in LPA. Nevertheless, the region of LPA also was scraped off. Silica gel scrapings were transferred to scintillation vials. Radioactivity was measured with a Packard liquid scintillation spectrometer (Model 3380) in a solution of POPOP and PPO in toluene (for neutral fats) and of POPOP, PPO and naphthaline in dioxane (for PL). The front area containing the neutral fats was delayered before fractionation in the two-dimensional chamber, eluted and the lipid extract separated by one-dimensional thin-layer chromatography on silica gel H in the system hexane/ether/acetic acid (70 : 30 : 4, by vol.). Analytical procedures Protein was determined by the Lowry method [18] with purified albumin as standard. PL was estimated as PO:- after digestion with perchloric acid [19], with modifications. We eluted the silica gel with an acidic solvent and digested with only 0.2 ml perchloric acid. With this miniaturisation we were able to estimate phosphate in the range of 10 nmol. DC was estimated by a HPLC using a Hewlett-Packard After 1084 B with a previously described method [20], with little modification. derivatisation with cY-naphthylisocyanate, the chromatography was performed on an RP 18 (5 I”) column with methanol/water as solvent system. The derivatives were detected with a fluorescence detector. The internal standard was 1.2-distearylglycerol. The sensitivity was in the range of 10 pmol. FA were estimated by GLC using a Varian 2100 equipped with FID. C,, fatty acid was used as internal standard. The fatty acids were estimated as their methylester and separated on a 10% EGSS-X on Gaschrom Q column. Results Table I shows the incorporation of [‘4C]glycerophosphate in glycerolipids by microsomes of human normal and dystrophic muscle. The method of incubation had already been practiced with many previous incubations with normal human muscle microsomes testing changing concentrations of the additions. The greatest dependency was registered in the concentration of G3P. We varied the concentration between 0.3 mmol/l and 3.3 mmol/l (with the same specific activity). The incorporation rate in PA increased from 452 pmol . mg-’ microsomal protein. min-’ to 893. For the experiment with dystrophic microsomes we chose a concentration of 0.5 mmol/l.

I

a, dpm. b. pmol/mg

microsomes

Normal

protein.

15 30 45

0

0 15 30 45

Min

131 116 80 127

120 101 125 9s

0 0 0

0 0 0

380 7609 6568 6456

326 1954 3085 3 822

a

a

of normal

PA b

in glyceroiipids

LPA

of [‘4C]glycerophosphate

see ‘Methods’)

Dystrophrc micro.wmes

incubation

Incorporation

TABLE

8 747 7487 7352

1970 3 340 4230

b

and dystrophic

101 175 137 167

113 682 1419 2612

a

DC

human

90 44 80

688 1580 3024

b

112 162 144 161

at different

135 642 1509 1992

a

PC

muscle microsomes

61 40 60

613 1663 2 247

b

229 343 567 756

200 156 144 130

a

TG

times of incubation

0 0 0

138 409 915

b

(conditions

of

118

The values for normal microsomes represent a large number of experiments (n = 8). Nevertheless, parallel with the dystrophic probe a normal sample of the same fresh weight was prepared, stored and incubated. The values listed in Table I for normal microsomes were obtained from this parallel sample. They corresponded to the other controls. In the dystrophic microsomes a delayed and decreased incorporation of G3P in PA was to be observed. The most surprising result was the lack of incorporation of G3P in DC, PC and also TG in dystrophic microsomes. We also listed the dpm, to show that the actual measured values lie in the same range as the blank values. By calculation of the rate of incorporation in DC. which may be theoretically expected following from the decreased PA labelling. we excluded a simulated defect at this point. The incorporation rate (pmol . mg ’ microsomal protein. mini ‘) calculated from the IO-min values is in the sequence LPA-PA-DC-PCTG for normal microsomes: not measurable -473-65-43-11 and for dystrophic microsomes: not measurable -132-4-3-0, respectively. In preparing the experiment we expected a generally decreased G3P acylation, mainly in PC. In this case the fundamental argument could have been that the level of substrate (fatty acids) would be too low. Therefore a parallel experiment with added fatty acids was carried out. The results are shown in Fig. 1. On a slightly decreased level there is a similar incorporation of G3P in the single glycerolipid fractions also. under these conditions. Table II gives a survey of G3P incorporation in LPA, PA, DC, PC and TG in normal and dystrophic human mitochondria under the conditions of added palmitic

pm0i my pr teln

jt !$I00 -

i

ip\\

\

Fig. 1. Incorporation of 14C-G3P in different human muscle microsomes dystrophic ( -) acid 0.133 mmol/l and albumin (1.33 g/l).

glycerolipids at different

by incubation of normal (incubation times with addition

- -_) and of palmitic

II

a, dpm. b, pmol/mg

protem

mitochondrru

Noi-maI

mirochondriu

Dystrophic

LPA

105

87

30

45

0 1986

1145

96 0

196 1158

95 0

0 0

1117 1524 204X

204

PA a

0

95 110

30 45

0

b

of normal

15

98 102

0

a

15

Min

Incorporation of [‘4C]gIycerophosphate in glycerolipids (0.133 mmol/l) and albumin (1.33 mg/ml) were added

TABLE

108X

577

585

555 803 1 I24

b

and dystrophic

175

210

178

130

112 180 332

101

a

DG

human

27

61

29

7 48 140

b

muscle mitochondria

1X6

204

125

126

9X 126 149

102

a

PC

at different

37

47

0

0 15 29

b

175

216

226

123

110 161 183

114

a

TG

timea of incubation:

57 32

63

0 29 42

b

palmitic

acid

120

TABLE

III

Effect of the addition of palmitic acid on the incorporation of ‘“C-G3P in phosphatidic and dystrophic human muscle mitochondria (pmol/mg mitochondrial protrm) Min incubation

15 30 45

Normal

mitochondria

Dystrophic

acid by normal

mitochondria

I

II

III

I

II

III

75 107

585 577 1088

16 34

386 719

555 803 1121

0 44

I. without addition of palmitic acid/albumin. II. addition of palmitic acid (0.133 mmol/l) and albumin (1.33 mg/ml). III. addition of Dt_-palmitoylcarnitine (0.25 mmol/l) instead of palmitic

acid/albumin

acid and albumin. Together with ATP and CoA (present in all incubations with mitochondria) a CoA-ester generating system is present. Although DG was not metabolised, we also measured the labelling in TG and PC. The conditions of incubation including the addition of CDP-choline were the same as for microsomes. There is no difference between normal and dystrophic mitochondria. The rate of incorporation calculated for 1 min from the IO-min values is in the sequence LPA-PA-DG for normal mitochondria: not measurable -39-3 (pmol . rng-’ microsomal protein. min-‘) and for dystrophic: not measurable -31-3, respectively. In mitochondria three variants of the incubation system, in relation to the palmitic acid added, were performed. Because mainly PA is labelled, the incorporation only in this compound was listed in Table III. Normal muscle mitochondria without added palmitic acid show a very low rate of G3P incorporation. By comparison, dystrophic mitochondria show under these conditions a six times higher labelling in PA. If palmitic acid/albumin is added, both types of mitochondria show the same incorporation. Palmitic acid added as carnitine ester shows a strict inhibition of the incorporation in both dystrophic and normal mitochondria. In this table the values for normal mitochondria also are similar to the controls (n = 7). Table IV shows the content of DG, PC and free FA in the same preparations of microsomes used for the incorporation studies. The analyses were carried out in single fractions containing 0.2 mg microsomal protein. In the case of normal microsomes they are in agreement with our other values (n = 6) and to those given in TABLE

IV

PC, DC and free FA in microsomes of normal and dystrophic human muscle: the microsomes aliquots from those taken for G3P acylation (Table I) (nmol/mg microsomal protein)

PC Free FA DC

Normal microsomes

Dystrophic microsomes

185.5 560 10.9

79.3 447 9.0

are

121

the literature [21,22]. The selection of the lipids estimated was based on the idea of controlling the conditions of G3P acylation. FA is the substrate and DG the final product of G3P acylation. Furthermore, it is generally accepted that the activity of membrane-bound enzymes is influenced by the lipid composition of the bilayer. PC is the main constituent of membrane lipids. Whereas the level of DG and FA are identical in dystrophic and normal microsomes, PC is decreased in dystrophic microsomes to nearly 2/3 of the control values. This corresponds to results given in the literature and mentioned in the ‘Introduction’ [1,5]. We have estimated PC only and listed the absolute value. It was impossible to detect the minor components of the PL pattern in the small specimen of dystrophic microsomes and also not the total PL. Therefore we are not able to give a total pattern of PL or a percentage of PC. Discussion G3P is the primary substance in the biosynthesis of glycerolipids [23,24]. G3P acylation takes place both in the endoplasmic reticulum and in the outer membranes of mitochondria (for references see [25]). The resulting PA is a key intermediate in the de novo synthesis of all glycerolipids. A specific PA-P-ase hydrolyses PA to DG [26]. Whereas in mitochondria the metabolic pathway ends at this point, in microsomes, 1.2-DG is the precursor in the biosynthesis of glycerophospholipids and triacylglycerol [27]. Most studies concerning de novo PL-biosynthesis were undertaken in the liver, brain and lung. In skeletal muscle, G3P acylation was studied in homogenate with labelled G3P [28] or in microsomes of rabbit muscle with radioactive fatty acid CoA-esters [29], measuring six times greater G3P acylation in neonatal muscle. The incorporation of [‘HIglycerol by incubation of dystrophic, normal and fetal muscle slices and in cell cultures of the same origin was measured [lo]. The incorporation was increased in dystrophic muscle, less in PL than in TG. For our study we used a microsomal system characterised by high flux through the entire de novo pathway from G3P to PC and TG. The microsomes were optimised for PC synthesis by addition of CDP-choline (current evidence suggests that the rate of PC biosynthesis is governed by the CDP-choline synthesis [30]), generating system containing palmitic acid. ATP Mg’+, KC1 and a palmitoyl-CoA and CoA [16]. Our main finding is the delayed incorporation of G3P in PA and the lack of further labelhng in all subsequent glycerolipids in microsomes of dystrophic muscle. It can be interpreted in two distinct ways. Firstly the results concerning G3P incorporation in dystrophic muscle microsomes can be seen as a secondary consequence of dystrophic degeneration. It is possible that the microsomal pellet contains a high percentage of adipose tissue microsomes with other enzymatic properties. However, in microsomes of adipose tissue an incorporation of G3P in the range of that found in muscle in our study has also been reported [31]. Furthermore, we obtained from 50 g adipose tissue (human) (wet weight) microsomes with a total protein content of 3.4 mg. From our small biopsy samples it would only be possible to isolate traces of microsomes of adipose

122

tissue. Therefore in our opinion. the idea of the influence of fatty degeneration can be rejected. Another possible interpretation of the lack of G3P incorporation as a secondary phenomenon is to take into account the changed properties of the dystrophic sarcoplasmic reticulum [1.5,8] resulting, in our opinion. from the abnormal PL pattern of the bilayer. The decreased PC level shown in Table IV confirms this statement. In every case the microenvironment of the G3P incorporating enzymes may be changed. It is possible that this varied activity occurs already under in vivo conditions as well as a result of increased sensitivity of the in vitro preparation. But we found a normal activity of the CDP-choline-DG-P-cholinetransferase in homogenate of dystrophic muscle [13]. This is a well-known membrane-bound enzyme. Therefore it is only for PA-P-ase of dystrophic microsomes that such sensitivity with regard to in vitro handling has to be assumed. On the other hand it is usual to estimate PA-P-ase activity with endogenous substrate obtained by PL-ase D treatment of microsomes (for rat liver microsomes see [17]). In our own studies with lung microsomes we used the back reaction of PC synthesis to obtain an increased level of endogenous DG. We found that the G3P incorporation under these conditions was not affected. In both cases the endogenous PL pattern was drastically shifted without influencing either the activity of PA-P-ase or G3P incorporation. The delayed and decreased incorporation of G3P in PA of dystrophic microsomes is the consequence of decreased activities of G3P acyltransferase or LPA acyltransferase, or both. Because the flux rate in LPA is very high we cannot decide which of the two acyltransferases is responsible. We cannot exclude the influence of the changed membrane structure. With regard to the normal level of fatty acids (Table IV) and the results obtained by incubation with added fatty acids, a lack of substrate cannot be the cause. Nevertheless the CoA ester of fatty acid is the direct substrate and the CoA-synthetase activity may be decreased. But this interpretation would be contrary to the results obtained with dystrophic mitochondria. To summarise, we did not find any convincing explanation on the basis of a secondary phenomenon. We cannot exclude the possibility that an altered membrane structure influences enzyme activity and this changed enzyme activity itself changes the structure of the membrane. However, we believe that the changed enzyme is the starting point of this vicious circle. As reported in many detailed studies, the relation between mitochondrial and microsomal G3P incorporation into PA in most organs is nearly 1 : 10 (see for example [25,26] a.o.). To our knowledge G3P acylation in muscle mitochondria has not been studied. We found in normal muscle mitochondria a strict dependency on the addition of palmitic acid/albumin. Dystrophic mitochondria independently of added palmitic acid/albumin show nearly the same incorporation of G3P in PA on the level which in normal mitochondria is only attained by adding palmitic acid/albumin. In both types of mitochondria the G3P incorporation was inhibited by the addition of palmitoylcarnitine. Secondly the results of the G3P incorporation of dystrophic microsomes can be interpreted as a sign of the primary defect. If we formally consider only the results of our incorporation study the situation is very clear: there is a defect of the enzyme PA-P-ase with an arrest of incorporation of G3P in all subsequent compounds. The main argument against this idea is the normal content of DG (Table IV). Further-

123

more it is impossible that a cell can survive with a total defect at such an important metabolic point. But it may be that the defect is not total. PA-P-ase is a very heterogenous enzyme ([26,32] a.o.). It is present in cytosol and in a membrane-bound form in the endoplasmic reticulum and in the mitochondria. It is also possible that muscle with such a defect develops a compensatory mechanism. for example the uptake of mitochondrial DG. Whatever the unknown metabolic process is which may compensate, a decreased level of PC cannot be prevented. It is not our intention to speculate further in this direction. However, to summarise all our arguments, we believe that the thesis of a primary defect of microsomal PA-P-ase is quite probable and provides the best explanation for our results. Our future experimental activities will be directed towards finding musclespecific properties in the de novo PL-biosynthesis and to characterise the PA-P-ase in muscular dystrophy. Acknowledgement

We thank

Dr. Hansgen

for assistance

in obtaining

muscle biopsies.

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124 15 Binaglia L, Roberti R, Vecchini A, Porcellati G. Evidence for a compartmentation of brain microsomal diacylglycerol. J Lipid Res 1982; 23: 9555962. 16 Fox PL. Zilversmit DB. High de nova synthesis of glycerolipids compared to deacylation-reacylation in rat liver microsomes. Biochim Biophys Acta 1982; 712: 605-615. 17 Van Heusden GPH, Van den Bosch H. The influence of exogenous and of membrane-bound phosphatidate concentration on the activity of CTP-phosphatidate cytidyltransferase and phosphatidate phosphohydrolase. Eur J B&hem 1978; 84: 405-412. 18 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 2655275.. 19 Hallermayer G. Neupert W. Lipid composition of mitochondrial outer and inner membranes of Neurospora crassa.Hoppe Seyler Z Physiol Chem 1974; 355: 279-288. 20 Kruger J. Rabe H, Reichmann G, Riistow B. Separation and determination of diacylglycerols as their naphthylurethanes by high performance liquid chromatography. J Chromatogr, in press. 21 Takagi A. Lipid composition of sarcoplasmic reticulum of human skeletal muscle. Biochim Biophys Acta 1971; 248: 12-20. 22 Marai L, Kuksis A. Comparative study of molecular species of glycerolipids of sarcotubular membranes of skeletal muscle of rabbit, rat, chicken and man. Can J Biochem 1973; 51: 1365-1379. 23 Kornberg A, Pricer WE. Enzymatic esterification of o-Glycerophosphate by long chain fatty acids. J Biol Chem 1953; 204: 345-353. 24 Kennedy EP. Biosynthesis of complex lipids. Fed Proc 1961: 20: 934-940. 25 Carroll MA, Morris PE. Grospean CD, Anzalone T, Haldar D. Further distinguishing properties of mitochondrial and microsomal glycerophosphate acyltransferase and the transmembrane location of the mitochondrial enzyme. Arch Biochem Biophys 1982; 214: 17-25. 26 Hiibscher G. Glyceride metabolism. In: Wakil SJ, ed. Lipid metabolism. London: Academic Press, 1970: 280-352. 27 Beall RM. Coleman RA Enzymes of glycerolipid synthesis in eukaryotes. Ann Rev Biochem 1980; 49: 4599487. 28 Daae INW. The acylation of glycerol-3-phosphate in different rat organs and in the liver of different species (including man). Biochim Biophys Acta 1973; 306: 1866193. 29 Smith PB, Reitz RC, Kelley D. Acyl-CoA synthese and acyltransferase activity in developing skeletal muscle membranes. Biochim Biophys Acta 1982; 713: 128-135. 30 Vance DE, Choy PC. How is phosphatidylcholine biosynthesis regulated? TIBS 1979; 145-148. 31 Steffen DC, Plinney G, Brown LJ, Mersmann HJ. Ontogeny of glycerolipid biosynthetic enzymes in swine liver and adipose tissue. J Lipid Res 1979; 20: 2466253. 32 Lamb PG, Fallon JH. An enzymatic explanation for dietary induced alteration in hepatic glycerolipid metabolism. Biochim Biophys Acta 1974: 348: 166-178.