Caracterização de miopatias mitocondriais através da avaliação das atividades enzimáticas envolvidas no metabolismo energético

Pedroso, Fábio Cesar; Campello, Annibal P.; Werneck, Lineu Cesar; Klüppel, Maria Lúcia W.

doi:10.1590/S0004-282X1997000200013

Resumos

Foi determinada a atividade das enzimas NADH desidiogenase, NADH citocromo e redutase, succinato desidiogenase, succinato citocromo e redutase, citocromo e oxidase e citrato sintase em mitocôndrias de músculo esquelético humano normal e doente (suspeito de miopatia mitocondrial). O grupo controle foi constituído de 13 indivíduos normais e que não faziam uso contínuo de fárrnacos. O grupo doente era constituído de 10 pacientes cujo diagnóstico anatomopatológico indicava suspeita de miopatia mitocondrial. Observou-se redução na atividade das enzimas em todos os pacientes: 7 com anormalidades em todas as enzimas ensaiadas; 2 com deficiências em todas as enzimas exceto na citocromo e oxidase; e 1 paciente com disfunção apenas na atividade da succinato desidiogenase e succinato citocromo e redutase. Este perfil possibilitou caracterizar múltiplas deficiências ou deficiência combinada da cadeia respiratória, além da disfunção na citrato sintase em 9 pacientes. Um dos casos constituiu exceção, sendo a deficiência enzimática restrita ao complexo II. Foi possível concluir que a metodologia usada é adequada e facilmente aplicável aos objetivos clínicos. Os resultados obtidos possibilitam a caracterização dos complexos enzimáticos mitocondriais deficientes, mostrando que tais enfermidades são originadas de disfunção no metabolismo energético.

mitocôndria; músculo esquelético humano; miopatia mitocondrial; NADH desidiogenase; NADH citocromo e redutase; succinato desidrogenase; succinato citocromo e redutase; citocromo e oxidase; citrato sintase

The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome e oxidase and citrate synthase in normal and sick human skeletal muscle mitochondria were determined. A control group was formed by 13 normal people and without using continuous medication. The patient group was formed by 10 people whose pathological diagnosis indicated suspicion of mitochondrial myopathy. A decrease in the activity of the enzymes in all patient was observed: 7 with abnormality in all the tested enzymes; 2 with deficiencies in all the enzymes except cytochrome e oxidase; and 1 with dysfunction only in the activities of succinate dehydrogenase and succinate cytochrome e reductase. The results indicate multiple or combined deficiencies in the respiratory chain, besides dysfunction of citrate synthase in 9 patients. In one exceptional case, the enzymatic deficiency was restricted to complex II. It is possible to conclude that the methodology used herein is adequate and easily applicable to clinical objectives, and that the results obtained allow characterization of the deficient mitochondrial enzymatic complexes, thus showing that the origin of the diseases is an energetic metabolic dysfunction.

mitochondria human skeletal muscle; mitochondrial myopathy; NADH cytochrome e reductase; NADH dehydrogenase; succinate dehydrogenase; succinate cytochrome e reductase; cytochrome c oxidase citrate synthase

Caracterização de miopatias mitocondriais através da avaliação das atividades enzimáticas envolvidas no metabolismo energético

Characterization of mitochondrial myopathies through the evaluation of the enzymatic activities involved in the energetic metabolism

Fábio Cesar Pedroso^I; Annibal P. Campello^I; Lineu Cesar Werneck^II; Maria Lúcia W. Klüppel^I

^IDepartamento de Bioquímica Universidade Federal do Paraná (UFPR)

^IIClínica Médica Universidade Federal do Paraná (UFPR)

RESUMO

Foi determinada a atividade das enzimas NADH desidiogenase, NADH citocromo e redutase, succinato desidiogenase, succinato citocromo e redutase, citocromo e oxidase e citrato sintase em mitocôndrias de músculo esquelético humano normal e doente (suspeito de miopatia mitocondrial). O grupo controle foi constituído de 13 indivíduos normais e que não faziam uso contínuo de fárrnacos. O grupo doente era constituído de 10 pacientes cujo diagnóstico anatomopatológico indicava suspeita de miopatia mitocondrial. Observou-se redução na atividade das enzimas em todos os pacientes: 7 com anormalidades em todas as enzimas ensaiadas; 2 com deficiências em todas as enzimas exceto na citocromo e oxidase; e 1 paciente com disfunção apenas na atividade da succinato desidiogenase e succinato citocromo e redutase. Este perfil possibilitou caracterizar múltiplas deficiências ou deficiência combinada da cadeia respiratória, além da disfunção na citrato sintase em 9 pacientes. Um dos casos constituiu exceção, sendo a deficiência enzimática restrita ao complexo II. Foi possível concluir que a metodologia usada é adequada e facilmente aplicável aos objetivos clínicos. Os resultados obtidos possibilitam a caracterização dos complexos enzimáticos mitocondriais deficientes, mostrando que tais enfermidades são originadas de disfunção no metabolismo energético.

Palavras-chave: mitocôndria, músculo esquelético humano, miopatia mitocondrial, NADH desidiogenase, NADH citocromo e redutase, succinato desidrogenase. succinato citocromo e redutase, citocromo e oxidase, citrato sintase.

ABSTRACT

The activities of the enzymes NADH dehydrogenase, NADH cytochrome e reductase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome e oxidase and citrate synthase in normal and sick human skeletal muscle mitochondria were determined. A control group was formed by 13 normal people and without using continuous medication. The patient group was formed by 10 people whose pathological diagnosis indicated suspicion of mitochondrial myopathy. A decrease in the activity of the enzymes in all patient was observed: 7 with abnormality in all the tested enzymes; 2 with deficiencies in all the enzymes except cytochrome e oxidase; and 1 with dysfunction only in the activities of succinate dehydrogenase and succinate cytochrome e reductase. The results indicate multiple or combined deficiencies in the respiratory chain, besides dysfunction of citrate synthase in 9 patients. In one exceptional case, the enzymatic deficiency was restricted to complex II. It is possible to conclude that the methodology used herein is adequate and easily applicable to clinical objectives, and that the results obtained allow characterization of the deficient mitochondrial enzymatic complexes, thus showing that the origin of the diseases is an energetic metabolic dysfunction.

Key words: mitochondria human skeletal muscle, mitochondrial myopathy, NADH cytochrome e reductase, NADH dehydrogenase, succinate dehydrogenase, succinate cytochrome e reductase, cytochrome c oxidase citrate synthase.

Texto completo disponível apenas em PDF.

Full text available only in PDF format.

Aceite: 15-janeiro-I997.

Dr. Annibal P. Campello - Departamento de Bioquímica, UFPR - Caixa Postal 19046 - 81531-990 Curitiba PR - Brasil.

Estudo realizado com auxílio do Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Proc. nº 52.1478/96,30.0738/ 87).

1. Behbehani AW, Goebel H, Osse G, Gabriel M, Langenbeck U, Berden J, Berger R, Schutgens RBH. Mitochondrial myopathy with lactic acidosis and deficient activity of muscle succinate cytochrome-c-oxidorediictasc. Eur J Pedinlr. 1984;143:67-71.
2. Bindoff LA, Desnuelle C, Birch-Machin MA, Pellissier JF, Serratrice G, Dravet C, Bureau M, Howell N, Turnbull DM. Multiple delects of the mitochondrial respiratory chain in a mitochondrial encephalopathy (MFRRF): a clinical, biochemical, and molecular study. J Neurol Sci. 1991:102:17-24.
3. Capaldi RA. Mitochondrial myopathies and respiratory chain proteins. TIBS 1988; 13:144-148.
4. Cooper JM, Hayes DJ, Chaliss RAJ, Morgan-hughes JA, Clarck JB. Treatment of experimental NADH ubiquinone reductase deficiency with menadione. Brain 1992,115:991-1000.
5. Di Mauro S. Bonilla F, Zeviani M, Nakagawa M, De Vivo D. Mitochondrial myopathies. Ann Neurol. 1985:17:521-538.
6. Hall RE, Henrikson KG, Lewis SF, Haller RG, Kennaway NG. Mitochondrial myopathy with succinate dehydrogenase and aconilase deficiency: abnormalities of several iron-sulfur proteins. J Clin Invest, 1993;92:2660-2666.
7. Ichiki T. Tanaka M, Nishirimi M, Suziki H, Ozawa T, Kobayashi M, Wada Y. Deficiency of subunils of complex I and mitochondrial cncephalomyopathy. Ann Neurol 1988;23:287-294.
8. Jung K, Reinholdt C, Scholz D. Inhibited efficiency of kidney mitochondria isolated from rats treated with ciclosporin A. Nephron 1987:45:43-45.
9. Kim SJ, Lee KO, Takamiya S, Capaldi RA. Mitochondrial myopathy involving ubiquiiiol-cytochrome e oxidoreductase (complex III) identified by immunoelectron microscopy. Biochim Biophys Acta 1987;92:270-276.
10. Lee CP. Biochemical studies of isolated mitochondria from normal and diseased tissues. Biochim Biophys Acta 1995:1271:21-28.
11. Lowry OH, Rosebrough NJ, Rut AC, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-275.
12. Luft R, Ikkos D, Palmieri G, Ernester L, Afzelius B. A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondria! respiratory control: a correlated clinical, biochemical, and morphological study. J Clin Invest 1962:41:1776-1804.
13. Mason TL, Poyton RO, Wharton DC, Shatz G. Cytochrome c oxidase from bakers yeast. J Biol Chem 1973;248:1346-1354.
14. Riggs JE, Schochet SS Jr., Fakadej AV, Papadimitriou A, Di Mauro S, Crosby TW, Gutmann L, Moxley III RT. Mitochondrial encephalomyopathy with decreased succinate-cytochrome c reductase activity. Neurology 1984;34:48-53.
15. Rivner MH, Shamsnia M, Hommes FA. Kearns-Sayre syndrome and complex II deficiency. Neurology 1989;39:693-696.
16. Schapira AHV, Cooper JM, Morgan-Hughes JA, Landon DN, Clark JB. Mitochondrial myopathy with a defect of mitochondrial-protein transport. N Engl J Med 1990;323:37-42.
17. Scholte HR, Agstcribbe E, Busch HFM, Hoogenraad TU, Jennekens FGI, Van Linge B, Luyt-Houwen IEM, Ross JD, Riters MHJ, Vcrduin MHM. Oxidative phosphorylation in human muscle in patients with ocular myopathy and after general anaesthesia. Biochim Biophys Acta 1990; 1018:211-216.
18. Singer TP, Ramsay RR, Ackrell BAC. Deficiencies of NADH and succinate dehydrogenases in degenerative diseases and myopathies. Biochim Biophys Acta 1995; 1271:211 -219.
19. Singer TP. Determination of the activity of succinate, NADH, choline and a-glycerophosphate dehydrogenase. Methods Biochem Anal 1974;22:125-175.
20. Somlo M. Induction des lactico-citochrome e redutases (D- et L-) de la levure aerobie par des lactates (D- et L-). Biochim Biophys Acta 1965;97:183-201.
21. Srere PA. Citrate synthase. Methods Enzymol 1969; 13:3-11.
22. Yonetani T, Ray GS. Kinetics of the aerobic oxidation of ferrocytochrome e by cytochrome e oxidase. J Biol Chem 1965;240:3392-3398.
23. Yorifuji S, Ogasahara S, Takahashi M, Tarui S. Decreased activities in mitochondrial inner membrane electron transport system in muscle from patients with Kearns-Sayre syndrome. J Neurol 1985;71:65-75.
24. Zheng X, Shoffner JM, Voljavec AS, Wallace DC. Evaluation of procedures for assaying oxidative phosphorylation enzyme activities in mitochondrial myopathy muscle biopsies. Biochim Biophys Acta, 1990.1019:1-10.

Datas de Publicação

Publicação nesta coleção
10 Nov 2010
Data do Fascículo
Jun 1997

Histórico

Recebido
15 Jan 1997

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. Behbehani AW, Goebel H, Osse G, Gabriel M, Langenbeck U, Berden J, Berger R, Schutgens RBH. Mitochondrial myopathy with lactic acidosis and deficient activity of muscle succinate cytochrome-c-oxidorediictasc. Eur J Pedinlr. 1984;143:67-71.

[2] 2. Bindoff LA, Desnuelle C, Birch-Machin MA, Pellissier JF, Serratrice G, Dravet C, Bureau M, Howell N, Turnbull DM. Multiple delects of the mitochondrial respiratory chain in a mitochondrial encephalopathy (MFRRF): a clinical, biochemical, and molecular study. J Neurol Sci. 1991:102:17-24.

[3] 3. Capaldi RA. Mitochondrial myopathies and respiratory chain proteins. TIBS 1988; 13:144-148.

[4] 4. Cooper JM, Hayes DJ, Chaliss RAJ, Morgan-hughes JA, Clarck JB. Treatment of experimental NADH ubiquinone reductase deficiency with menadione. Brain 1992,115:991-1000.

[5] 5. Di Mauro S. Bonilla F, Zeviani M, Nakagawa M, De Vivo D. Mitochondrial myopathies. Ann Neurol. 1985:17:521-538.

[6] 6. Hall RE, Henrikson KG, Lewis SF, Haller RG, Kennaway NG. Mitochondrial myopathy with succinate dehydrogenase and aconilase deficiency: abnormalities of several iron-sulfur proteins. J Clin Invest, 1993;92:2660-2666.

[7] 7. Ichiki T. Tanaka M, Nishirimi M, Suziki H, Ozawa T, Kobayashi M, Wada Y. Deficiency of subunils of complex I and mitochondrial cncephalomyopathy. Ann Neurol 1988;23:287-294.

[8] 8. Jung K, Reinholdt C, Scholz D. Inhibited efficiency of kidney mitochondria isolated from rats treated with ciclosporin A. Nephron 1987:45:43-45.

[9] 9. Kim SJ, Lee KO, Takamiya S, Capaldi RA. Mitochondrial myopathy involving ubiquiiiol-cytochrome e oxidoreductase (complex III) identified by immunoelectron microscopy. Biochim Biophys Acta 1987;92:270-276.

[10] 10. Lee CP. Biochemical studies of isolated mitochondria from normal and diseased tissues. Biochim Biophys Acta 1995:1271:21-28.

[11] 11. Lowry OH, Rosebrough NJ, Rut AC, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-275.

[12] 12. Luft R, Ikkos D, Palmieri G, Ernester L, Afzelius B. A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondria! respiratory control: a correlated clinical, biochemical, and morphological study. J Clin Invest 1962:41:1776-1804.

[13] 13. Mason TL, Poyton RO, Wharton DC, Shatz G. Cytochrome c oxidase from bakers yeast. J Biol Chem 1973;248:1346-1354.

[14] 14. Riggs JE, Schochet SS Jr., Fakadej AV, Papadimitriou A, Di Mauro S, Crosby TW, Gutmann L, Moxley III RT. Mitochondrial encephalomyopathy with decreased succinate-cytochrome c reductase activity. Neurology 1984;34:48-53.

[15] 15. Rivner MH, Shamsnia M, Hommes FA. Kearns-Sayre syndrome and complex II deficiency. Neurology 1989;39:693-696.

[16] 16. Schapira AHV, Cooper JM, Morgan-Hughes JA, Landon DN, Clark JB. Mitochondrial myopathy with a defect of mitochondrial-protein transport. N Engl J Med 1990;323:37-42.

[17] 17. Scholte HR, Agstcribbe E, Busch HFM, Hoogenraad TU, Jennekens FGI, Van Linge B, Luyt-Houwen IEM, Ross JD, Riters MHJ, Vcrduin MHM. Oxidative phosphorylation in human muscle in patients with ocular myopathy and after general anaesthesia. Biochim Biophys Acta 1990; 1018:211-216.

[18] 18. Singer TP, Ramsay RR, Ackrell BAC. Deficiencies of NADH and succinate dehydrogenases in degenerative diseases and myopathies. Biochim Biophys Acta 1995; 1271:211 -219.

[19] 19. Singer TP. Determination of the activity of succinate, NADH, choline and a-glycerophosphate dehydrogenase. Methods Biochem Anal 1974;22:125-175.

[20] 20. Somlo M. Induction des lactico-citochrome e redutases (D- et L-) de la levure aerobie par des lactates (D- et L-). Biochim Biophys Acta 1965;97:183-201.

[21] 21. Srere PA. Citrate synthase. Methods Enzymol 1969; 13:3-11.

[22] 22. Yonetani T, Ray GS. Kinetics of the aerobic oxidation of ferrocytochrome e by cytochrome e oxidase. J Biol Chem 1965;240:3392-3398.

[23] 23. Yorifuji S, Ogasahara S, Takahashi M, Tarui S. Decreased activities in mitochondrial inner membrane electron transport system in muscle from patients with Kearns-Sayre syndrome. J Neurol 1985;71:65-75.

[24] 24. Zheng X, Shoffner JM, Voljavec AS, Wallace DC. Evaluation of procedures for assaying oxidative phosphorylation enzyme activities in mitochondrial myopathy muscle biopsies. Biochim Biophys Acta, 1990.1019:1-10.