Decline with age of the respiratory chain activity in human skeletal muscle

BB ELSEVIER

Biochi~mic~a et BiophysicaA~ta

Biochimica et Biophysica Acta 1226 (1994) 73-82

Decline with age of the respiratory chain activity in human skeletal muscle D. Boffoli a, S.C. Scacco a, R. Vergari a, G. Solarino

b

G. Santacroce

b

S. Papa a,,

a Institute of Medical Biochemistry and Chemistry, University ofBari, Bari, Italy, b Orthopaedic Clinic II, University ofBari, Bari, Italy (Received 5 July 1993)

Abstract

Mitochondrial respiratory systems have been screened in 63 othopaedic patients of age ranging between 17 and 91 years. The results show a statistically significant definite decrease with ageing of mitochondrial respiratory activity with pyruvate plus malate, succinate and ascorbate plus TMPD. This pattern is associated with an equally significant decrease with age of the enzymatic activity of complex I, II and IV. No significant decrease with age is, on the contrary, observed in the mitochondrial content of cytochromes a + a3, and c + c x. Preliminary Western blot analysis indicates an altered polypeptide pattern in cytochrome c oxidase. This study provides evidence for a decline with age of mitochondrial respiratory activity in human skeletal muscle, affecting complex I, II and IV. Key words: Ageing; Mitochondrial respiration; Respiratory chain; Cytochrome c oxidase; NADH-ubiquinone oxidoreductase; Cytochrome

1. Introduction

Ageing in humans is associated with a decrease in the mass and functional capacity of skeletal muscle, brain and other tissues, in particular those having a high demand for oxidative phosphorylation [1,2]. Morphological [3-5] and biochemical observations [6-10] indicate a progressive decline with age of the mitochondrial respiratory system in human tissues. This, together with the finding of respiratory defects in neuromuscular disorders and other degenerative diseases [2], suggest that progressive decrease with age of mitochondrial respiration and oxidative phosphorylation is critical for development of structural and functional decline of neuromuscular, endocrine and other tissues which is associated with ageing [2,11].

* Corresponding author. Fax: 80-278-429. Abbreviations: UQH2-cyt.c reductase, ubiquinol-cytochrome c oxidoreductase; COX, cytochrome c oxidase; TMPD, N,N,N',N'tetramethyl-p-phenylenediamine; CCCP, carhonyl cyanide m-chlorophenylydrazone; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electophoresis. 0925-4439/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0925-4439(93)E0132-U

The mitochondrial respiratory chain is composed of three oligomeric redox enzymes: complex I (NADHubiquinone oxidoreductase), complex III (ubiquinol-cytochrome c oxidoreductase) and complex IV (cytochrome c oxidase) [12,13]. Electron flow in each of the three complexes is directly associated with generation of transmembrane electrochemical gradient of protons [12-14], which is in turn utilized by complex V (FoF 1 ATP synthase) to synthetize ATP from ADP and Pi [12-16]. Thirteen component polypeptides of the four complexes of oxidative phosphorylation are encoded by mitochondrial genes: seven subunits of complex I, one of complex III, three of complex IV and two of complex V [17]. The remaining subunits are encoded by nuclear genes [17]. The mitochondrial D N A (mtDNA) functions as template also for two rRNAs and all the tRNAs involved in mitochondrial protein synthesis [17]. The enzymes and protein factors for replication, transcription and translation in mitochondria are all encoded by the nucleus, mitochondrial biogenesis thus depending on the concerted action of two different genomes [17].

D. B(.~]~/bli et al. / Biochimica et Biophysica Acta 1220 (1994) 73 ,'~'2

74

immediately processed fl)r mitochondria isolation. The patients were free of overt symptoms of neuromuscolar disease.

There have been independent reports of increased levels in old people of a common deletion encompassing 4997 np [18-23] and other deletions [24,25] in m t D N A of skeletal muscle and other tissues. There is discrepancy with regard to the respiratory chain complexes affected and the extent of their functional impairment in ageing [6-10]. Evidence of age-related decrease in the content and activity of the FoF 1 ATP synthase in mitochondria from rat tissues has also been obtained [26]. In order to contribute towards clarifying these aspects and in order to obtain age-related control values in normal subjects with which to compare values from subjects with mitochondrial myopathies, we initiated a systematic study two years ago. The results of the analysis of 63 persons from the region of Apulia, in Italy, are described in this paper. They provide evidence for a decline with age of mitochondrial respiratory activity in human skeletal muscle, affecting complex I, II and IV.

Preparation of human skeletal muscle mitochondria The muscle was homogenized in a medium containing: 100 mg wet weight tissue/ml, 250 mM sucrose, 2 mM EDTA, 10 mM Tris-HC1 and 50 U heparin/ml, final pH 7.4 [27]. Mitochondria were isolated according to Bookelman et al. [28]. Mitochondrial proteins were determined as in Ref. 29.

Polarographic measurement of oxygen consuinption Mitochondrial respiratory rates were measured at 37°C in a thermostatically controlled micro oxygen chamber (YS! 5356, Yellow Springs Incorporated, USA) equipped with a micro oxygen probe (YSI 5357) coated with a standard membrane (YSI 5775). 0.10-().25 mg proteins of freshly prepared mitochondria were incubated in 0.6 ml of a mixture containing 75 mM sucrose, 30 mM Tris-HC1, 50 mM KCI, 0.5 mM EDTA, 1 mM MgC12, 1 mM potassium phosphate buffer, final pH 7.4. Respiration was started by adding different respiratory substrates followed, in order, by the addition of A D P and CCCP.

2. Materials and methods

Collection of specimens Skeletal muscle biopsy samples (1-2 g) were obtained, with informed consent, from proximal vastus lateralis of patients, who underwent routine orthopaedic surgery under general anaesthesia, and were

Pyruvate÷Malate

Measurement of NADH-cytochrome c reductase activity Mitochondria were disrupted by exposure to ultrasound at 0°C in a mixture containing: 23 /xg / m l

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45

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Fig. 1. Polarographic traces of 0 2 consumption by respiring human muscle mitochondria. Mitochondria, 0.24 mg proteins, were suspended in 0.6 ml of the medium described under Section 2. Respiration was activated by adding respiratory substrates at the followingfinal concentrations: 20 mM pyruvate plus 10 mM malate, 20 mM succinate and 2 mM ascorbate plus 250/zM TMPD. Succinate oxidation was measured in the presence of 2/zg rotenone, TMPD plus ascorbate oxidation in the presence of 2/xg antimycin A. ADP: 0.3 mM; CCCP: 3/~M. The numbers on the traces indicate respiratory rates in ngatom oxygen min-1 mg protein-1. The respiratory rates were calculated by assuming the solubility of oxygen in the assay medium to be 398 ngatom oxygenper ml [39].

D. Boffoli et al. / Biochimica et Biophysica Acta 1226 (1994) 73-82

Measurement of cytochrome c reductase activity The ubiquinol-cytochrome c reductase activity was measured in frozen-thawed mitochondria (5 /~g proteins/ml) in a medium containing 50 mM potassium phosphate buffer (pH 7.4), 2 mM KCN and 10 /~M ferricytochrome c. The reaction was started by addition of 30/zM duroquinol. Enzymatic activity was measured at 37°C following the rate of reduction of ferricytochrome c.

mitochondrial proteins, 25 mM potassium phosphate buffer pH 7.4, 2.5 m g/ m l bovine serum albumin, 5 mM MgC12 and 10/xM cytochrome c [30]. The suspension of disrupted mitochondria was supplemented with 2 mM KCN and the reaction started by the addition of 25 /~M NADH. Reduction of ferricytochrome c was followed at 37°C at 550-540 nm, AemM = 19.1 [31] in a Johnson Foundation dual wavelength spectrophotometer. Activities were corrected for those determined in the presence of 2 /zg/ml rotenone.

Measurement of cytochrome c oxidase activity Cytochrome c oxidase activity was estimated on frozen and thawed mitochondria (5/xg proteins/ml) in 50 mM potassium phosphate buffer (pH 7.4) at 37°C. Reaction was started by the addition of 10/zM ferrocytochrome c [33].

Measurement of succinate-cytochrome c reducatse activity The assay conditions were essentially those reported by King et al. [32]. Complex II was activated by preincubation of frozen and thawed mitochondria (5 /zg proteins/ml) for 30 min at 37°C in 50 mM potassium phosphate buffer (pH 7.4) in the presence of 20 mM succinate. The reduction of 9 /xM ferricytochrome c was followed at 37°C in the presence of 2 /~g/ml rotenone and 2 mM KCN.

Spectrophotometric determination of cytochrome content For evaluation of cytochromes, difference spectra of cytochromes were recorded in a Lambda 5 Perk•nElmer UV/Vis spectrophotometer, at room tempera-

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Fig. 2. Statistical analysis of the relationship between state 3 coupled respiratory activities of h u m a n muscle mitochondria and age in 63 subjects. The respiratory rates were m e a s u r e d as illustrated in Fig. 1. The statistical parameters of the plots are presented in Table 1. For experimental details see u n d e r Section 2 and legend to Fig 1.

D. Boftbli eta/./Biochimica et Biophysica Acta 1226 (1~)94) 73 -$2

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ture, after reduction of cytochromes with 20 mM succinate in the presence of 2 mM KCN, as reported by Bookelman et al. [34]. Both the reference and the sample cuvette (optical pathway 1.0 cm) were filled with a suspension containing 0.3-0.5 mg of mitochondrial proteins in 1.6 ml of 75 mM potassium phosphate buffer (pH 7.4), 187 mM KC1, 20 mM Tris-HC1, 4 mM EDTA, 5 mM MgC12. For calculation of the mitochondrial cytochrome content the following extinction coefficients were used: cytochromes a + a3, AEmM at 605630 n m = 14 [35], cytochrome b, AemM at 562-575 n m = 20 [36] and cytochromes c + c~ AemM at 550-540 nm = 19.1.

Biotechnology AB Multiphor II Novablol Electrophoresis System), using specific rabbit sera against cytochrome c oxidase purified from bovine heart [38]. Immunoreactive polypeptides were visualized using the immunoperoxidase method with 4-chloro-l-naphthol as substrate. Densitometry analysis was performed using a Camag TLC densitometer (Switzerhmd).

Chemicals Bovine serum albumin, horse heart cytochrome c (type VI), rotenone, antimycin A and CCCP were purchased from Sigma. Durohydroquinone (from K & K Laboratories) was dissolved in absolute ethanol and used within 4-5 h. T M P D was from BDH, England. N A D H and ADP were from Boehringer, Mannheim. All other reagents were of the purest grade commercially available.

Electrophoresis and immunoblotting Electrophoresis was performed as described by Schagger and Von Yagow [37]. Western blotting was performed using a semi-dry system (Pharmacia LKB

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AGE (Years) Fig. 3. Statistical analysis of relationship between state 3 uncoupled respiratory rates of human muscle mitochondria and age in 25-32 subjects. The respiratory rates are measured as illustrated in Fig. 1. Correlation factor and significance level were r = - 0.410, P < 0.05 for pyruvate plus malate; r = - 0.581, P < 0.01 for succinate and r = - 0.552, P < 0.01 for T M P D + ascorbate. For experimental details see unde r Section 2 and legend to Fig. 1.

D. Boffoli et al. / Biochimica et Biophysica Acta 1226 (1994) 73-82

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Fig. 4. Statistical analysis of relationship between state 3 coupled respiratory activity of human muscle homogenate and age (A). Correlation between cellular concentration of mitochondrial proteins and age (B). Muscle biopsy samples were homogenized in the medium described under Section 2. Respiration, supported by 2 mM ascorbate plus 250/xM TMPD, was measured polarographically at 37°C in the same reaction medium used for mitochondria. The concentration of mitochondrial proteins in the homogenate was estimated from the ratio between the specific respiratory rates of the homogenate and those of the mitochondrial fraction, both measured using TMPD plus ascorbate as respiratory substrate.

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Fig. 5. Statistical analysis of the activities of respiratory chain complexes in human muscle mitochondria as a function of age. The specific activities of redox complexes were measured as specified under Section 2. The correlation factors and significance levels obtained from the plots are reported in Table 2. For other experimental details see under Section 2.

78

D. Boffoli et al. / Biochimica et Biophysica Acta 1220 (19~)4) 7¢ ,'~'? Table I Correlation coefficient ( r ) a n d significance level ( / ' ) ~ 1 linear and exponential analysis of state III coupled respiratory activities with pyruvate plus malate, succinate plus rotenonc and asc~rbalc plu~ TMPD as substrates

3. Results

Respiratory activities The respiratory activity of mitochondria from muscle biopsies was assayed polarographically, within 1 h of their isolation, with pyruvate plus malate, succinate plus rotenone and ascorbate plus T M P D (added in separate samples). A D P and CCCP were added in sequence to the same sample so as to monitor sequentially state 4, state 3 coupled and state 3 uncoupled, respiratory rates and the respective respiratory control index in the same sample. A typical analysis of respiratory activity with the three substrates in mitochondria from a man of 62 years is illustrated in Fig. 1. The respiratory rates measured in state 3 (with A D P + Pi) with the three substrates, from 63 subjects ranging from 17 to 91 years, are plotted in Fig. 2 as a function of age. With all the three substrates, the measured values of respiratory rates were apparently quite scattered. Statistical analysis of the data, carried out by plotting the specific arithmetic values of respiratory rates as a function of age, showed, however, a significant negative correlation between the respiratory rates measured with all the three substrates and age. Comparison of the correlation coefficients (r) and significance levels (p) obtained from these plots with those derived from logarithmic plots (Table 1), shows

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that the linear analysis gives, in fact, the same satisfactory fit as the exponential analysis. Statistical analysis of the specific respiratory rates in the presence of the protonophoric uncoupler CCCP shows, in the uncoupled state, an equally significant linear decrease of the rates with respect to age with all the three substrates (Fig. 3). The specific respiratory activity, with ascorbate plus TMPD, measured in the homogenate (Fig. 4A), shows an age-dependence with a decline and a significance level identical to those found with the same substrate in mitochondria. The estimate of the cellular concentration of mitochondrial proteins, as obtained from the

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Fig. 6. Spectrophotometric difference spectrum of human skeletal muscle mitochondria. For experimental details see under Section 2. Mitochondria from muscle biopsy of a subject 17 years old were suspended in the medium described under Section 2 and difference spectrum obtained as reported.

D. Boffoli et al. / Biochimica et Biophysica Acta 1226 (1994) 73-82

Table 2 Correlation factor (r) and significance level (P) of linear analysis of the activities of the respiratory redox complexes

ratio b e t w e e n t h e specific r e s p i r a t o r y r a t e s o f t h e muscle h o m o g e n a t e a n d t h o s e o f t h e m i t o c h o n d r i a l fraction, d o e s n o t show, in fact, any significant c h a n g e with age in t h e c e l l u l a r c o n t e n t of m i t o c h o n d r i a l p r o t e i n s o f 31 m u s c l e b i o p s i e s f r o m subjects o f ages r a n g i n g f r o m 18 to 88 y e a r s (Fig. 4B).

Activity o f redox complexes and cytochrome contents Statistical analysis shows a significant n e g a t i v e corr e l a t i o n with a g e of t h e specific e l e c t r o n t r a n s f e r r a t e s f r o m f e r r o c y t o c h r o m e c to oxygen ( c o m p l e x IV), from N A D H to c y t o c h r o m e c ( c o m p l e x I plus c o m p l e x I I I ) a n d f r o m s u c c i n a t e to c y t o c h r o m e c ( c o m p l e x II plus c o m p l e x I I I ) (Fig. 5, T a b l e 2). T h e specific activity of c o m p l e x I I I ( e l e c t r o n t r a n s f e r f r o m quinol to cyt o c h r o m e c) d i d not, however, show significant dec r e a s e with age (Fig. 5, T a b l e 2). Thus, t h e r e is a significant d e c l i n e with a g e o f t h e activities of c o m p l e x I, c o m p l e x II ( s u c c i n a t e d e h y d r o g e n a s e ) a n d c o m p l e x IV, b u t n o t o f c o m p l e x III.

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For experimental details see under Section 2 and legend to Fig. 5.

Cytochrome contents Fig. 6 shows a d i f f e r e n c e a b s o r p t i o n s p e c t r u m of i s o l a t e d m i t o c h o n d r i a ( s u c c i n a t e + K C N r e d u c e d minus oxidized) from which t h e c h a r a c t e r i s t i c p e a k s of c y t o c h r o m e s a + a3, b a n d c plus Cl, a r e i d e n t i f i e d a n d t h e i r specific c o n c e n t r a t i o n e s t i m a t e d . Statistical analysis d o e s n o t show any significant c h a n g e with age in t h e m i t o c h o n d r i a l c o n t e n t o f c y t o c h r o m e s aa 3 a n d c plus c~ (Fig. 7). T h e r e is, on t h e o t h e r h a n d , a small b u t

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Fig. 7. Statistical analysis of cytochrome contents of human skeletal muscle mitochondria as a function of age in 35-39 subjects. Cytochrome

contents were obtained from difference spectra as described under Section 2 and in the legend to Fig. 6. The mitochondrial content of cytochrome bs62 shows a significant decline with age: r = -0.371 P < 0.05. The content of the other cytochromes did not show significant changes with age. For experimental details see under Section 2.

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D. Boffofi et aL /Biochimica et Biophysica Acta 1220 (1904) 73 &?

significant decrease with age in the mitochondrial content of cytochrome b (r = -0.371, P < 0.05) (Fig. 7).

Polypeptide pattern of complex IV In order to examine whether the decrease in the catalytic activity of cytochrome oxidase could derive from an altered polypeptide composition, preliminary Western blot semiquantitative analysis of the relative content of complex IV subunits was carried out. Immunoblots of human muscle mitochondria with polyclonal antibodies raised against cytochrome c oxidase isolated from bovine heart showed good immunological reactivity of subunits If, IV, V, VII and VIII (Fig. 8). The immunoblot shows in the old subjects, as compared with the young one, an apparent decrease in the content of subunits II, IV, V, VII and VIII. In the sample of the old subject there was the appearance of

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VIII Fig. 8. Immunoblot analysis of subunits of cytochrome c oxidase. Mitochondria from muscle biopsies of two subjects, 31 and 87 years old respectively, were applied on the gel-slabs in the a m o u n t s showed in the Figure. Lane 1, muscle mitochondria from a subject of 87 years; lane 2, muscle mitochondria from a subject of 31 years; lane 3, cytochrome c oxidase isolated from beef heart.

immunoreactive material above subunit IV and subunit VII exhibited decreased electrophoretic ]nobility. These patterns, which were observed in other samples from old subjects, showed a significant alteration in the polypeptide composition of cytochrome oxidase in old with respect to young subjects, which was associated with a marked decrease of the turnover of the oxidasc (Fig. 9). The occurrence of subunit isoforms in human cytochrome c oxidase [40-42] has been reported, whose expression seems to be tissue-specific and age-dependent.

4. Discussion

The present study shows a statistically significant, linear decline with age of the mitochondrial respiratory activity in skeletal muscle (vastus lateralis) of orthopaedic patients. The large number of patients examined allowed us to evaluate the effect that other factors like sex and physical activity could have on the respiratory activity besides ageing. Young subjects are more likely to undertake regular exercise than elderly subjects. A training effect seems, however, to be excluded by the fact that no significant difference was observed in respiratory activity (with pyruvate plus malate, succihate or ascorbate plus T M P D ) between female and male subjects at the various ages examined. Furthermore no significant difference was observed in respiratory activity between orthopaedic patients affected by chronic diseases, which were hospitalized for several weeks, and patients who underwent emergency surgery within hours or days after traumatic lesions. Practically the same depression of specific respiratory rates is observed in ageing when pyruvate plus malate, succinate plus rotenone or ascorbate plus T M P D are used as respiratory substrates in isolated muscle mitochondria. It can be noted that the same decline observed in the respiratory activity of isolated mitochondria was also found in the total muscle homogenate. Furthermore there was no significant change with age in the mitochondrial content of cytochromes aa 3 and c plus c]. These observations, while excluding the possibility that the observed age-related decrease of respiratory rates results from contamination of mitochondria from elderly subjects with non-mitochondrial proteins, show that the changes described are primarily due to a progressive decrease with age of the turnover of respiratory enzymes. The same extent of decline of respiratory rates was observed in the phosphorylating state 3 (plus ADP and Pi) and in the presence of a protonophoric uncoupler. This is, in fact, in agreement with the observation that the activity of the FoF ~ ATP synthase is in excess with respect to that of the respiratory chain [43]. Direct analysis would, however, be desirable to verify whether

81

D. Boffoli et al. / Biochimica et Biophysica Acta 1226 (1994) 73-82

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Q 17-31 Years (n=5) R 71-87 Years (n=5) Fig. 9. Mean values of the relative abundance of subunits and catalytic activity of cytochrome c oxidase in two groups of young and old subjects. For experimental details see u n d e r Section 2 and legends to Figs. 1 and 8.

in human mitochondria there occurs with ageing also a decline of the activity of substrate transport systems [44] and of FoF 1 A T P synthase as observed in mitochondria from ageing rat tissues [26]. A primary defect in the respiratory chain activity is directly supported by the present observation of a statistically significant decline with age in the specific activities of complex I, complex II and complex IV. Complex III does not show, on the other hand, significant decline of activity with age, although the content of cytochrome b seems to decrease with age. This aspect deserves further attention. The age-related decline in the specific respiratory activity of mitochondria results in a corresponding decrease in the overall respiratory activity of the tissue as shown by the observation that the muscle homogenate shows pratically the same quantitative decline in the respiratory rate measured with ascorbate plus T M P D as that recorded for isolated mitochondria. No significant change in the cellular content of mitochondrial proteins is, in fact, observed in the course of ageing. The statistical analysis of the mitochondrial cytochromes shows that there is no significant change with age in the content of cytochromes aa 3 and c + c 1 but only a small decrease in the content of cytochrome b. The lack of decrease with age, in the content of cytochromes aa 3 indicates that the age-related decrease in the activity of cytochrome c oxidase (complex IV) is not associated with decrease in the content of hemes a and a 3 nor in the level of subunit I of this complex which holds the hemes. Age-related exponential accumulation of the common 4997 np deletion has been found in the m t D N A

of human skeletal muscle [18-23]. This is apparently accompanied by accumulation of less common deletions of m t D N A [24,25,45] as well as of the A-to-G transition mutation in the t R N A gene of mtDNA, characteristic of the M E R F syndrome (myoclonic epilepsy with ragged red fibers) [46]. Although the maximal level of the 4997 np deletion observed in the oldest persons examined is small ( < 0.1% of the total mtDNA), it has been proposed that this can reflect accumulation with age of numerous species of mutated DNA, particularly in slowly turning-over tissues like skeletal muscle, so that in aged tissues the mutated D N A may represent a significant fraction of the total m t D N A and may be responsable for phenotypic deficiencies of the respiratory chain (ref. 23; see, however, ref. 9). It can, however, be noted that the genes encoding for subunit II of cytochrome c oxidase and cytochrome b of complex III, two subunits whose level seems to decrease with age, are located in the mitochondrial genome, outside of the commonly deleted segments [2]. The best statistical fit for the age-related decline of the activities of the respiratory chain is given by a linear analysis thus exhibiting a behaviour different from that observed for the age-related accumulation of mutated mtDNA, whose best statistical fit is given by an exponential curve [2,22]. Besides mutations of mtDNA, other factors might be involved in the age-dependent decline of mitochondrial respiratory enzymes. These could be represented by mutations in nuclear genes encoding for proteins involved in mitochondrial biogenensis [2,17] or direct oxygen radical damage of protein components [47] a n d / o r particular phospho-

S2

D. Bqffoli et al. / Biochimica et Biophysica Acta 1226 (1994) 73 ,~'2

lipids specifically associated with respiratory enzymes [48].

5. Acknowledgements

We thank Prof. Gabriella Serio for helpful discussions and advice regarding statistical analysis. This research was supported by funds of C.N.R., target Project on 'Utilization sanitary data' (Rome, Italy) No. 91.04203.ST75.

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