Studies of selected enzymes of phospholipid metabolism in the dystrophic human muscle

211

Clinica Chimica Actu, 108 (1980) 211-218 0 Elsevier/North-Holland Biomedical Press

CCA 1551

STUDIES OF SELECTED ENZYMES OF PHOSPHOLIPID IN THE DYSTROPHIC HUMAN MUSCLE

METABOLISM

D. KUNZE *, B. Rk3TOW and D. OLTHOFF Department of Clinical Biochemistry and Department Humboldt-University, Berlin (Germany)

of Anaesthesiology,

CharitkHospital,

(Received March 8th, 1980)

summary Proceeding from deviations of the phospholipid pattern in the muscle in progressive muscular dystrophy, the activities of a number of enzymes of phospholipid metabolism in the normal and dystrophic human muscle were measured. Twelve cases of the Duchenne type and four of the Becker-Kiener type were studied. The activities of CDP-choline:diglyceride-P-cholinetransferase, of CDPcholine:ceramide-P-cholinetransferase and of phosphatidylcholine degradation were determined in normal and dystrophic human muscle. A significant difference in enzyme activities between normal and dystrophic muscle could not be established. The activities of a phosphatidylethanolaminemethyltransferase and a transfer of Pcholine directly from phosphatidylcholine to sphingomyelin could be excluded for normal human muscle. The significance of these results for the integration of phospholipid findings in ideas about pathogenesis of progressive muscular dystrophy is discussed.

Introduction Lipid and PL changes in the muscle in cases of human and animal dystrophies have been described by various authors [l-5]. The interpretation of these findings as a primary and direct consequence of the basic defect or as a secondary effect remained speculative. The relationship between PL and cell membrane supported the first of these possibilities [6]. Therefore, we decided to study the activities of a number of enzymes of lecithin metabolism (see Fig. 1). The following introductory remarks may be made. Firstly, the study of * To whom correspondence should be addressed. Abbreviations: PL, phospholipids; PCh. phosphatidylcholine; phosphatidylethanolamine.

Sph. sphiigomyelin;

DG, diglyceride; PE.

212 Cemmlde

Dlglycerlde

i Fig. 1. Diagram of the reactions

tested. -:

speculative

metabolic

step.

PL metabolism of muscle has so far not proved to be of theoretical or practical medical interest. Therefore, there is a lack .of available data for many of the enzymes of PL metabolism in muscle. Secondly, if a primary defect of PL metabolism in pseudohypertrophic muscular dystrophy of Duchenne (DMD) is assumed, it should involve a muscle-specific step in the metabolism. Such a step is not known. Thirdly, factors affecting possible pathogenesis finally have to be tested in biopsy samples. Muscle biopsies, when available, tend to be small and show fatty degeneration producing practical difficulties in biochemical work; these will be discussed below. Material and methods Material

Muscle biopsies from non-dystrophic subjects were taken during routine surgery and were mainly from the M. pectoralis major (ablatio mammae). Dystrophic muscle from 16 patients also was studied: 12 boys between 4 and 15 years of age suffering from DMD and four patients with the Becker-Kiener type. The diagnosis was confirmed clinically, histologically, and biochemic~ly. Samples were taken from the M. quadriceps femoris. Biopsy was performed under local anaesthesia by subcutaneous infiltration. Care was taken that there was no infiltration below the fascia. The material was investigated immediately after sampling.

Reagents [ “C-Me]-CDPcholine (specific activity 58 mCi/mmol) and [ “C-Me]S-adenosy~ethionine (55 mCi/mmo~) were supplied by the Radiochemical Centre, Amersham, U.K. and [3zP]o~hophosphate by Isocommerz GmbH, Rossendorf, G.D.R. The purity of all labelled compounds was tested by thin layer chromatography with subsequent autoradiography for S-adenosylmethionine by the method of Schlenk and DePalma [ 71. 32P-labelled PCh and Sph were prepared from rat liver after injection of 20 mCi [32P]orthophosphate. The specific activity of PCh was 0.41 mCi/mmol and of Sph, 0.05 mCi/mmol. Pure PE and PCh were prepared from rat liver. 1,2-DG was obtained from PCh by enzymatic hydrolysis with phospholip~e C (Boehringer, Mannheim, F.R.G.) and subsequent thin layer chromatographic purification. Deoxycholate, GDP-choline, ceramide and all other reagents were commercial products of analytical grade.

213

CDP-choline:diglyceride-P-cholinetransferase

(EC 2.7.8.2)

The composition of the reaction mixture was as described by Mudd et al. [S]. The homogenate was produced in the following buffer solution: 0.1 mol/l KCl, 0.05 mol/l Tris-HCl, pH 7.4, 0.005 mol/l MgS04 and 0.001 mol/l EDTA. In a total volume of 0.7 ml the mixture contained 0.5 ml homogenate, 0.5 mg DG and 200 nmol labelled CDPcholine with a radioactivity of 83 nCi. The incubation was performed for 20 min at 37°C. The sonication of the homogenate was carried out as described previously [ 71. Muscle slices were obtained and incubated as described in a previous paper [9]: with 3 ml incubation solution the concentration of GDP-choline in the medium was 600 nmol and the radioactivity level 200 nCi. CDP-choline:ceramide-P-cholinetransferase

(EC 2.7.8.3)

The assay mixture was that of Scribney [lo]. The homogenate had the same composition as in the preceding enzyme assay. The addition of ceramide was difficult, because it emulsified only in a 1 : 1 mixture with PE at ultrasonication in 0.02% deoxycholate (0.5 mg/reaction vessel). An alternative form of application was to dry the ceramide on the wall of the incubation vessel by blowing off the organic solvent. With either method, addition of ceramide only slightly stimulated Sph formation. It should be noted that normal muscle contains a considerable amount of ceramide [ 111. Measurement

of the transfer of P-choline

from PCh to ceramide

The reaction mixture contained [32P]PCh; the incubation time was extended to 90 min [ 12,131. Ceramide was added using both methods described above. Phosphatidylethanolaminemethyltransferase

(EC 2.1.1.17)

The assay conditions corresponded to the procedure of Schneider and Vance [ 141 including pretreatment with ultrasound and deoxycholate. Phospholipase

A (EC 3.1 .1.4) and degradation

of PCh

The intensity of PCh degradation was measured in two ways. Firstly, the measurement of CDPcholine:DG-P-cholinetransferase covered also the labelling of Lyso-PCh. Secondly, the degradation of endogenous PCh was measured with the “chase” technique [15]. To an incubation mixture containing 0.5 ml muscle homogenate, 0.0002 mol/l [ 14C-Me]CDPcholine with a specific activity of 0.41 mCi/mmol and 0.1 ml of Ca-acetate solution (0.5 mol/l) were added after 30 min incubation (labelling of PCh with [14C-Me]choline), mixed well and incubated for a further 60 min at 37°C. Under these conditions during the second stage of the reaction the Pcholine transfer to DG is inhibited and PLase A activated. The enzyme reactions, with PL as reaction products, were stopped by adding methanol/chloroform (1:2). Lipids were extracted by the usual Folch procedure. Proteins were determined by the method of Lowry et al. [ 161. PL were separated by 2-dimensional thin layer chromatography [ 171. Spots were localized by autoradiography and by staining with dichlorofluorescein. After delayering, radioactivity was measured by scintillation counting. Toluene was chosen as solvent for the lipid scintillator. Water soluble compounds were

214

counted in the dioxane scintillator. Mono- and dimethyl-PE by thin layer chromatography [ 181.

were also separated

Results Table I shows the activity of CDP-choline:DG-P-cholinetransferase in normal and dystrophic muscle. For the total homogenate no significant differences could be found. The liver enzyme is bound to the endoplasmic reticulum. Due to enzyme fixation to an intracellular structure, we determined the activities in three variants of tissue preparation: a total homogenate with partial destruction of structure, a homogenate with total destruction of structure by deoxycholate and ultrasonication [7] and one with well-preserved tissue structure (slice incubation). In the normal muscle, P-choline incorporation was greatest in the slice incubation, but compared with the total homogenate there was no significant difference. In variant II (Table I), it is possible that residues of deoxycholate in the homogenate could exert an inhibitory effect. For incorporation into dystrophic muscles by slice incubation only one sample could be used. Methods of determination were at first studied in rat muscles. Here the activity of CDPcholine:DG-P-cholinetransferase was about 7 times greater than in TABLE

I

ACTIVITY

OF

TROPHIC TREATMENT LINE

GDP-CHOLINE:

HUMAN

INTO

MUSCLE

WITH

DIGLYCERIDEI’-CHOLINE-TRANSFERASE IN

UNTREATED

ULTRASOUND

PHOSPHATIDYLCHOLINE

CHOLINE-LABELLED

CDP-CHOLINE

OVER

3 MIN

AFTER WAS

(II).

THE

INCUBATION

ESTIMATED

protein muscle

0.292

muscle

(Duchenne)

0.265

muscle

(Becker-Kiener)

0.144

OF

AND

HOMOGENATE

INCORPORATION MUSCLE

OF SLICES

DYS-

AFTER

[14C-Me]CHOWITH

[ 14C.Me]-

III * mg-l

nmol

. h-1 * 0.154

0.15

+ 0.103

nmol

* h-1

protein

(n = 5) Dwtrophic

AND

II xx-1

(n = 9) Dystrophic

(I)

IN III

I nmd

Normal

IN NORMAL

HOMOGENATE

. mg-l

protein

f 0.129

0.411

(n = 8)

(n = 4)

0.157

0.170

(n = 2)

(n = 1)

. h-

*

+ 0.157

* 0.098

(n = 4)

TABLE

II

ACTIVITY

OF

DYSTROPHIC I-III:

THE

CDP-CHOLINE

HUMAN

see Table

I,

CERAMIDE-P-CHOLINE-TRANSFERASE

nmol . mg-l

protein.

muscle

Dystrophic

muscle

(Duchenne)

II

0.0239

+ 0.0148

0.084

* 0.035

muscle

(Becker-Kiener)

0.041 (n = 4)

* Significant

increase

@

<

0.01)

*

AND

f 0.038

III

0.067

_+ 0.049

0.011

0.130

*

0.048

(n = 2)

(n = 5) Dystrophic

NORMAL

h-l. I

Normal

IN

MUSCLE

(n = 1)

+ 0.073

215 TABLE III INCORPORATION OF RADIOACTIVE CHOLINE FROM [14C-Me1CDP-CHOLINE IN LYSOPHOSPHATIDYLCHOLINE BY MUSCLE IN VITRO (DEGRADATION OF ENDOGENOUSLY LABELLED PHOSPHATIDYLCHOLINE) I: muscle homogenate, II: homogenate after treatment with ultrasound, III: muscle slices.

mnol mg-’ protein. h-1

II nmol . mg-l protein. h-1

III nmol . mg-’ protein . h-1

0.058 * 0.0240 (n = 9)

0.0175 + 0.021 (n = 8)

0.079 c 0.045 (n = 4)

Dystrophlc muscle iDuchenne)

0.065 f; 0.03 (n = 4)

0.032 (n = 2)

0.048

Dystrophic muscle (Becker-Kiener)

0.0458 * 0.048 (n = 4)

I

Normal

muscle

human muscles (M. gastrocnemius of rat, 1.64 + 0.53 nmol - mg-l protein * h-l in the homogenate, n = 23); there were no significant differences between different muscles (M. tibialis anterior, M. quadriceps). The only literature data was the value for M. gastrocnemius of the mouse [ 51: 0.23 + 0.01 nmol - rngl protein * h-l. Table II shows the enzyme activities of CDPcholine:cerarnide-Pcholinetransferase for normal and dystrophic human muscles; one of these values differs significantly from the controls. As before, preliminary studies were made in rat muscles (M. gastrocnemius of the rat 0.203 + 0.08 nmol * mg-l protein * h-l in the homogenate, n = 15). With an incubation time of 20 mm, the addition of ceramide only slightly stimulated Sph-synthesis. In the incubation with labelled CDPcholine the labelling of lyso-PCh was also measured (Table III). Two enzymes are involved in this reaction: CDPcholine:DG-P-cholinetransferase (Table I), and the PL-ases Al and/or A2, which convert the endogenously labelled PCh into lyso-PCh. This latter is partly metabolised by reacylases or lysophospholipases. These complicated degradation processes are only summarily covered by the present test. The high standard deviation is an expression of the factors involved. Under these conditions the labelling of lysoPCh (as an expression of PL-ase A activity) in dystrophic muscle does not differ from that of normal muscle. To follow the degradation of endogenously labelled PCh, the “chase” technique was chosen (see Figs. 2a and b). During a 30-min initial incubation period, [‘4C-Me]choline-labelled PCh was formed. The degradation of this PCh was followed by two methods: addition of “cold” GDP-choline at loo-fold excess (which of course induces further PCh synthesis but due to decreased specific activity of the substrate the incorporation is “dumb”), or addition of Ca2+ to stop the synthesis and at the same time activate PL-ase A. In the alternative method the increase of labelled lyso-PCh and decrease of labelled PCh was taken as a measure of PL-ase A activity relative to the endogenous substrate in the sarcoplasmic reticulum. For normal muscles the values of 10 “chase” experiments were averaged and an activity of 0.015 nmol * mg-1 protein * h-l was obtained. For the study of dystrophic muscle two biopsy specimens were pooled (Fig.

216

(a) Fig. 2. Labelling of PCh and lyso-PCh dystrophic muscle (b). The “chase” choline (see text). A PCh: degradation PCh dunng the same time.

(b) m the homogenate of normal muscle (a) and of 2 pooled biopsies of technique was started after 30 min pre-labelling with [ 14~Me]of labelled PCh for SO min. A lyso-PCH: production of labelled lyso-

2b). Here no PL-ase A activity could be demonstrated. With [ 32P]PCh the direct transfer of phosphorylcholine to ceramide accompanied by Sph formation was studied [ 12,131. Sph labelling could not be found either in rat liver or muscle, nor in normal human muscle, so study of the dystrophic muscle was not pursued. Methods of PCh formation by PE methylation were first of all tested in rat liver homogenate. Here the values given in the literature for liver homogenate [ 191 and liver microsomes [ 19,201 were reproduced. Methyl transfer could not be established either in rat or in human muscle (activity < 0.01 nmol . mg-l protein - h-1. Discussion This paper has been based on the idea that PL anomalies are an expression and direct consequence of the basic biochemical defect in dystrophic muscle. As the primary defect presumably concerns a protein as an immediate gene product, an enzyme defect in the biosynthesis of PCh in muscle was the basic suggestion of the present paper. Speculation about the consequences of such a defect and the possibility of overcoming it allowing cell survival could be presented for all metabolic reactions studied here, but will be avoided. Nevertheless, the reactions studied have been selected carefully. PCh was the focus of the reactions studied (see Fig. 1). This was because of PL findings in dystrophic muscle in DMD (for review see [6]), viz. PCh is decreased by about l/3; Sph is increased; PCh exhibits a changed fatty acid pattern with a decreased linoleic acid but increased oleic acid content. As the contents of Sph and triglycerides in dystrophic muscle are increased, the conclusion is justified that glycerophosphate acylation and DG supply on the one hand and choline activation (synthesis of CDP-choline) on the other, are intact. This accounts for our emphasis on the study of CDPcholine:DG-P-

217

cholinetransferase. Thus the enzyme activity was measured in different tissue preparations. This was based on the idea that, due to homogenisation and structural destruction, a loss of activity could occur which, at the higher enzyme activities of normal muscle would naturally be greater, and possibly then might approximate the values seen in dystrophic muscle. Using incubation of tissue slices the greatest activity is found, but its difference from dystrophic muscle was insufficient to regard it as evidence of the basic defect. This statement is also true for enzyme activity of the muscle of the Becker-Kiener type. In assessing the results, allowance must be made for the fact that lipid anomalies also affect the sarcoplasmic reticulum, as a biological membrane [21] which can secondarily alter the activity. P-choline transfer to ceramide (CDPcholine:ceramide-P-choline transferase) is increased in dystrophic muscle and shows no defect. The results of our studies of PCh degradation in rat muscle have been given in detail in a separate publication [ 151. In the study of dystrophic muscle, we were influenced by the following considerations: exogenous supply of substrate when using detergents, due to cellular heterogeneity of muscles, mainly involves intercellular connective tissue, which is drastically increased in dystrophic muscle. When measuring PL-ase A in the acid pH range, one is confined to the lysosomal enzyme, enhancing the emphasis on the interstitial connective tissue of the muscle. The acid hydrolase activity increases in the late stages of all muscular diseases [ 221. As the interstitial component cannot be eliminated when studying biopsy specimens, the total activity of PL-ase A in the homogenate is of less interest than investigation of PCh degradation in vivo. In our experiments, making all possible allowances for these factors, we measured the degradation of endogenously labelled PCh. As there is no optimal substrate concentration, enzyme activity cannot be validly given, but a measure of PCh degradation under the conditions described can be used to compare normal and dystrophic muscle. From our results we conclude that PCh degradation in DMD is not greatly altered. In muscle homogenate from dystrophic mouse, a significantly increased PL-ase A activity was found with an exogenous substrate supply at pH 5: normal 0.36; dystrophic 1.74 nmol - mg-1 protein - h-l [5]. The normal value given by the authors is the only reference to PCh degradation in muscle so far. In interpreting the values in mouse dystrophy, the heterogeneity of the muscle tissue has to be allowed for as mentioned above. PE methylation during PCh metabolism is located in the liver [ 23,241, in the lung [ 251 and,other organs. As expected no methyltransferase activity could be found in muscle. Although one group of authors described Pcholine transfer from PCh to Sph [ 12,131, this could not be confirmed in rat liver or in rat or human muscle. Studies of PL metabolism of muscle have so far been largely neglected. We have sought to remedy this omission, as part of our search for an enzyme defect in DMD, but unfortunately the results were negative. Nevertheless, since the search for the molecular defect in this mysterious hereditary disease must continue, we feel that our results are not without value.

218

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Kunze, D., Olthoff, D. and Schellnack. K. (1968) Acta Biol. Med. Germ. 21, 669 Kunze, D. and Olthoff, D. (1970) Clin. Chim. Acta 29, 455 Hughes, B.P. (1972) J. Neurol. Neurosurg. Psychiat. 35. 658 Hughes, B.P. (1973) in Basic Research in Myology (Kakulas. B.A., ed.), Excerpta Medica, Amsterdam Kwok. C.T. and Austin, L. (1978) Biochem. J. 176.15 Kunze, D. (1977) in Pathogenesis of Human Muscular Dystrophies. (Rowland. L.P., ed.). Excerpta Medica. Amsterdam Schlenk, F. and DePalma, R.E. (1957) J. Biol. Chem. 229.1051 Mudd, J.B., Van Golde. L.M.G. and Van Deenen, L.L.M. (1969) Biochim. Biophys. Acta 176, 547 Kunze, D. and Qlthoff, D. (1969) Acta Biol. Med. Germ. 23, 19 Scribney, M. (1968) Arch. Biochem. Biophys. 126,954 Iwamori, M.. Costello, C. and Moser, H.W. (1979) J. Lipid Res. 20, 86 Marggraf. W.-D. and F.A. Anderer (1974) Hoppe-Seyler Z. Physiol. Chem. 355, 803 Diringer, H. and Koch, M.A. (1973) Hoppe-Seyler Z. Physiol. Chem. 354, 1661 Schneider, W.J. and Vance, D.E. (1978) Eur. J. Biochem: 85,181 Riistow, B. and Kunze, D., in preparation Lowry, O.H.. Rosebrough, N.J.. Farr, A.L. and Randall, R.J. (1961) J. Biol. Chem. 193, 265 Rouser, G., Fleischer, S. and Yamamoto. A. (1970) Lipids 5, 494 Poorthuis, B.J.H.M.. Yasaki, P.J. and Hostetler. K.Y. (1976) J. Lipid Res. 17, 433 Schneider, W.J. and Vance, D.E. (1979) J. Biol. Chem. 254, 3886 Tanaka, Y.. Doi, 0. and Akamatu, Y. (1979) Biochem. Biophys. Res. Comm. 87.1109 Takagi, A. (1971) Neurology (Minneap.) 21.457 Kar, N.C. and Pearson, C.M. (1977) in Pathogenesis of Human Muscular Dystrophies. (Rowland, L.P., ed.), Excerpta Medica, Amsterdam-Oxford Bremer. J. and Greenberg, D.M. (1960) Biochim. Biophys. Acta 37. 173 Hirate. F., Viveros, O.H.. Diliberto. E.J. and Axelrod, J. (1978) Proc. Natl. Acad. Sci. U.S.A. 75. 1718 Rooney, S.A. and Motoyama, E.K. (1976) Clin. Chim. Acta 69, 525