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Cytochrome c oxidase deficiency in menkes kinky hair disease
Cytochrome c Oxidase Deficiency in Menkes Kinky Hair Disease Mitsuo Maehara, MD, Nobuaki Ogasawara, MD, Naoki Mizutani, MD, Kazuyoshi Watanabe, MD and Sakae Suzuki, MD In a 4-year-old male with Menkes kinky hair disease (MKHD) treated with copper supplement therapy, reduced cytochrome a + a3 contents in liver was demonstrated to be 0.029 against 0.128 nmol/mg protein in the control. Cytochrome c oxidase activities in brain, liver, skeletal muscle, and heart were 47, 22, 54 and 59% of the control, respectively. The copper contents in brain and liver were decreased. In spite of increased serum levels of copper and ceruloplasmin, the decreased cytochrome c oxidase activities in v.arious organs were not corrected by copper supplement therapy. A search for a therapeutic method which can normalize copper enzymes in brain and liver, would seem to be a prerequisite for the treatment of MKHD. Maehara M, Ogasawara N, Mizutani N, Watanabe K, Suzuki S. Cytochrome c oxidase deficiency in Menkes kinky hair disease. Brain Dev 1983;5:533-40
Menkes kinky hair disease (MKHD) is an X- per in cells and withhold it from incorporation linked recessive disorder of the copper metabo- into copper metalloenzymes, and that unavaillism, characterized by failure to thrive, mental ability of copper in cells, or in the body, may retardation, peculiar hair, seizures, hypother- produce clinical manifestations of copper mia, defective vascular structure and low serum deficiency. The clinical symptoms of MKHD levels of copper and ceruloplasmin [1-3]. The may be mostly attributed to reduced activities primary biochemical lesion is unknown, but the of copper metalloenzymes, e.g., cytochrome c sequestration of copper within cells due to oxidase, dopamine /3-hydroxylase, tyrosinase, abnormal binding to the large amount of lysyl oxidase and ascorbate oxidase [2]. The metallothionein may be the underlying cause . pathogenesis of the central nervous system of the disease [4, 5]. In MKHD, it can be dysfunction in MKHD has been related to postulated that metallothionein may trap cop- deficient energy production due to cytochrome c oxidase deficiency, and also to an altered catecholamine metabolism due to dopamine From the Department of Pediatrics, School of Medi- /3-hydroxylase deficiency [6-9] . cine, Nagoya University, Nagoya (MM, NM, KW, SS); On the other hand, an organ-specific acDepartment of Biochemistry, Institute for Developcumulation and deficiency of copper were mental Research, Aichi Prefectural Colony (NO). found in a patient with MKHD [10-12]. This Received for pUblication: August 15, 1983. abnormal copper storage pattern apparently Accepted for publication: November 8, 1983. gives rise to a copper deficiency elsewhere in Key words: Menkes kinky hair disease, cytochrome c the body, particularly in brain, where it may oxidase, copper metabolism. cause irreversible damage through deficient Correspondence address: Dr. Mitsuo Maehara, Deof copper-dependent enzymes. Alfunction partment of Pediatrics, School of Medicine, Nagoya though cytochrome c oxidase activities in brain, University, 65 Tsuruma-cho, Showa-ku, Nagoya 466, liver, muscle and leukocytes have been deJapan.
scribed [7, 11, 13], relatively little is known about the relationship between cytochrome c oxidase and tissue copper content in MKHD. In this communication, a deficiency of cytochrome c oxidase and maldistribution of tissue copper are reported in various organs in a patient with MKHD, who had been treated with cupric sulfate for 4 years. The effects of copper supplement therapy on the clinical course of the disease and on the activity of cytochrome c oxidase are also discussed. Case Report The boy (MO) under study was the 2,300 g product of 40 weeks gestation and uneventful delivery. Throughout pregnancy, his mother had no complications. No consanguinity was found in the family, but his older brother died at the age of 11 months of a progressive neurodegenerative disease simulating MKHD. At the age of 3 months, the infant boy developed frequent generalized seizures. Convulsive attacks were not controlled completely with anticonvulsants. Marked hypotonia and hypothermia also persisted. His scalp hair was fair and sparse. Serum levels of copper and ceruloplasmin were markedly decreased (12 ,ug/ dl and 4 mg/ dl, respectively). The EEG revealed multifocal seizure discharges with poorly organized background activities. The cerebral angiography showed marked tortuosity
of vessels without any vascular occlusions and anomalies. MKHD was suspected from these clinical observations and laboratory findings at this time. Thereafter, he was treated with intravenous cupric sulphate (20-100,ug/kg/day) once a week for 6 months. Although his hair gradually darkened, no other clinical improvements occurred during copper supplement therapy. At the age of 1 year, his development quotient was 30, and severe growth failure was noted. After age 2 years, cupric sUlphate (0.8-3.0 mg/kg/ day) was administered orally with an intermittent intravenous injection of cupric sulphate (100 ,ug/kg) at least. once a month. Serum levels of copper and ceruloplasmin were increased and could be maintained at 40-70 ,ug/dl and 10-20 mg/dl, respectively, but epileptic seizures, severe mental and physical retardation and hypothermia remained during copper therapy. On computed cranial tomography, marked dilatation of subarachnoidal spaces and ventricles were noted. He was susceptible to upper respiratory infections. At the age of 4 years, he developed pneumonia and died from respiratory insufficiency. Autopsy disclosed severe atrophy of brain, marked muscle wasting and scorbutic skeletal changes. No cardiomegaly and hepatosplenomegaly were found. Serum levels of copper and ceruloplasmin during copper supplement therapy were shown in Fig 1.
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Fig 1 T~e effec~s of c~pper administration on serum levels of copper ( ___ ) and ceruloplasmin (O---D ~ In a patient WIth MKHD. Serum copper and ceruloplasmin were determined by atomic absorptIOn spectrophotometry and single radial immunodiffusion, respectively. Arrows indicated intravanous injections of cupric sulphate, and shaded area denoted oral doses of cupric sulphate.
534 Brain & Development, Vol 5, No 6, 1983
Materials and Methods Autopsy was performed within 4 hours after the patient's death. Brain, liver, kidney, heart and skeletal muscle were removed from the patient and washed with 0.154 M NaCl twice and stocked at _80°C until analysis. The tissues removed from a 3-month-old male who died of pneumonia were prepared in the same way and used as the control, because control materials in good analytical condition were not available from children of the same age. Mitochondria were isolated from brain and liver within 12 hours after resection according to the method of Hogeboom [14], with some modifications. Small portions of the tissues were minced finely and homogenized in 10 volumes ofa solution of 0.01 M Tris-HCI buffer, pH 7.4, containing 0.21 M mannitol, 0.07 M sucrose and 0.1 mM ethylenediamine tetraacetic acid in a Potter-Elvehjem homogenizer fitted with a Teflon pestle. The homogenate was then centrifuged at 800 g for 10 minutes. The supernatant was collected and centrifuged at 8,000 g for 10 minutes. The fluffy layer was discarded and the remaining pellet was resuspended in buffer and centrifuged again at 8,000 g for 10 minutes. This pellet was used as mitochondrial fraction. The entire procedures were carried out at 4°C. Estimation of the contents of cytochrome a + a3, b, c and c1 of mitochondria was made following the calculation of Williams [15] . Isolated mitochondria were treated with deoxycholic acid and reduced by a few milligrams of sodium dithionite. Difference absorption spectrum of reduced form of cytochromes against cytochromes oxidized by potassium ferricyanide were taken with Shimadzu MPS 50 L spectrophotometer. Determination of cytochrome c oxidase activities was carried out by the method of Wharton and Tzagoloff [16]. Tissue was homogenized in 10 volumes of a solution of 0.01 M potassium phosphate buffer, pH 7.0, containing 0.25 M sucrose in a Potter-Elvehjem homogenizer, and the homogenate was centrifuged at 1,000 g for 10 minutes. The resultant supernatant was sonicated for 10 seconds three times with a Branson sonifier B-12, and was used for assay of enzymatic activities. The reaction mixture was composed of 50 uM ferrocytochrome c in 0.01 M potassium phosphate buffer, pH 7.0. Ferrocytochrome c was pre-
pared by adding a few grains of ascorbic acid to a 1% solution of cytochrome c followed by dialysis to remove excess ascorbate. One ml of the reaction mixture was preincubated in a cuvette at 37°C for 5 minutes, and the reaction was started by adding 10 to 40 ul of sonicated supernatant. The change of absorbance at 550 nm was recorded with Hitachi 124 spectrophotometer continuously for I minute. Activity of cytochrome c oxidase was expressed as the first order rate constant. An extinction coefficient, Esso run , of 18.5 mM-1.cm- 1 was assumed [17]. Inhibition by sodium azide was performed as blank following every sample. Determination of succinate dehydrogenase (SDH) activities was performed by the method of Pennington [18]. One hundred JLl of the supernatant of the homogenate used in determination of cytochrome c oxidase activities was incubated with 1 ml of the reaction mixture containing 0.05 M phosphate buffer, pH 7.4, 0.1 % 2-(p-indophenyl)-3-(p-nitrophenyl)-5phenyltetrazolium (INT) , 25 mM sucrose, and 50 mM sodium succinate at 37°C for 30 minutes. The' reaction was terminated by adding I ml of 10% trichloroacetic acid. The formed formazan was extracted by 4 ml of ethyl acetate and quantified spectrophotometrically at 540 nm. Zero time incubation was used as blank. Copper content of tissue was determined by flame atomic absorption spectrophotometry with wet ashing method [19]. Some 300-500 mg of tissue was weighed precisely,. and ashing of tissue was carried out on a hot plate at 150°C by adding 1 ml of concentrated nitric acid followed by 2 ml of perchloric acid until clear solution was obtained. The resultant solution was transfered to volumetric flask and adequately diluted with distilled water for determination of copper content. Copper determination was performed using a Hitachi 170-10 atomic absorption spectrophotometer. Fibroblast derived from skin biopsied from the patient with MKHD and patients with other neurological diseases was grown in Eagle's essential medium (MEM) containing 10% fetal calf serum .. The cells were harvested at confluent stage by treatment with trypsin. The cells were suspended in 0.01 M phosphate buffer, pH 7.0 and sonicated for 10 seconds three times, and used for analysis of enzymatic activities as described above. For determination Maehara et al: Menkes kinky hair disease 535
of copper content of fibroblasts, harvested cells were suspended in deionized water and sonically disrupted. The lysate was centrifuged at 1,500 g for 20 minutes. The supernatant was assayed for copper and protein content. Protein was determined by the method of Lowry et al [20] , with bovine serum albumin as standard.
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Results The difference absorption spectra of liver mitochondria from the patient with MKHD and the control were shown in Fig 2. In MKHD, the absorbance at 605 nm, the alpha region of spectrum of cytochrome a + a3 (Le., cytochrome c oxidase), was markedly diminished compared to the control. Also the peak of the gamma region of spectrum was shifted left, which also indicated diminished content of cytochrome a + a3 in mitochondria. The decreased absorbance of cytochrome a + a3 was also noted in brain mitochondria from the patient (data not shown). Cytochrome contents of liver mitochondria calculated spectrophotometrically were shown in Table 1. Cytochrome a + a3 was decreased to 22% of that found in the control. Although cytochrome c was also decreased in our patient with MKHD, it was not certain that the reduction of cytochrome c was a constant finding for MKHD. The activities of cytochrome c oxidase, SDH, and the tissue copper contents in various organs from the patient and control were summarized in Table 2. Statistical analysis could not be performed because of unavailability of more control materials from agematched subjects. In brain, liver, skeletal muscle and heart of the patient, the activities of cytochrome c oxidase were reduced to about 47%, 22%, 54% and 59% of that of the control, respectively. On the other hand, its activity in kidney was increased to 168% of that of the control. Km value of cytochrome c oxidase for ferrocytochrome c in kidney from the patient was 6.5 x 10-6 M against 8.0 x 10-6 M in the control. It seemed that the property of the enzyme was not changed significantly in MKHD. The activities of SDH, an inner mitochondrial marker, in various organs from the patient were slightly decreased compared to that of the control except for kidney. In connection with 536 Brain & Development, Vol 5, No 6,1983
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600
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450
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500
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WAVELENGTH(nm) Fig 2 Difference absorption spectra of liver mitochondria from a patient with MKHD (upper) and the control (bottom). The peak of cytochrome a + a3 at 605 nm was diminished. OI.(a + a3) and 'Y (a + a3) indicate the alpha and gamma regions of spectrum of cytochrome a + a3, respectively.
Table 1 Contents of cytochromes in liver mitochondria
Cytochrome (nmol/mg protein) a + a3 b
MKHD
Control
0.029 0.283 0.055 0.014
0.128 0.285 0.027 0.062
cytochrome c oxidase deficiency, the reduced activity of SDH might suggest that the mitochondrial abnormalities were present in MKHD. These results would support the study of Yoshimura et al [20], which showed the morphological abnormalities of the mitochondria in MKHD. In kidney, the activities of cytochrome c oxidase and SDH were increased in MKHD compared to the control. As the large amount of copper was accumulated after copper supplement therapy, especially in kidney, we assumed that the increase of the enzymes was one of
Table 2 Activities of cytochrome c oxidase and SDH, and tissue copper contents in various organs from a patient with MKHD
Copper (pg/g wet tissue) Brain Liver Heart Kidney Skeletal muscle Cultured fibroblast
MKHD
Control
0.59 6.06 7.60 89.20 2.84 415*
0.80 66.70 1.90 3.20 1.20 123* 117*
Cytochrome c oxidase (nmol/min/mg protein)
SDH (nmol/min/mg protein)
MKHD
Control
MKHD
Control
13.2 24.2 70.9 66.7 21.1 29.1
28.0 112.0 120.0 39.6 39.1 20.5 27.5
2.0 22.4 19.0 20.7 2.1 4.3
2.6 24.8 25.8 6.0 18.2 5.8
*: (ng/mg protein)
the effects of the therapy. However, more work was needed to elucidate the effects of copper supplement therapy on the copperdependent enzymes in various organs.in MKHD. Tissue copper contents of brain and liver were decreased, but increased in heart, skeletal muscle, and kidney. The large deposition of copper in kidney was probably accentuated by the long-term copper supplement therapy. The uneven distribution of copper in the' body, reported recently [10-12] as characteristic of MKHD, was also observed in the present patient. In fibroblasts from MKHD, copper content was increased to three times the control level. Activities of cytochrome c oxidase and SDH in fibroblasts were not significantly different from those of the controls (Table 2). The relationship between tissue copper content and cytochrome c oxidase activity was shown in Fig 3. It was suggested that the severity of cytochrome c oxidase deficiency was not correlated with tissue copper contents in MKHD. Fibroblasts that had increased copper content showed normal activity of cytochrome c oxidase in MKHD. In conclusion, cytochrome c oxidase deficiency found in the several organs except for kidney from the patient with MKHD was considered to be a secondary phenomenon due to abnormal copper metabolism. However, its deficiency could not be normalized by longterm copper supplement therapy, presumably resulting from the inability to incorporate serum copper into the cells of brain and liver, and from the defects of intracellular transport
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Cytochrome c oxidase activity (nmol/min/mg protein)
Fig 3 The relationship between cytochrome c oxidase activity and tissue copper content in MKHD.
of copper to t.he copper-enzymes in skeletal muscle and heart. Cytochrome c oxidase deficiency might result in abnormal development of the central nervous system in MKHD through failure of tissue energetics. Discussion Menkes kinky hair disease (MKHD) was formerly considered to be due to copper deficiency because of depressed levels of the metal in serum and liver, and many attempts have been made to influence the course of the disease by parenteral administration of copper derivatives [1-3]. Recently, however, the elevated tissue copper contents in most extrahepatic tissues, except for brain, have been reported in MKHD [10-12]. The retention of excess copper in Maehara et al: Menkes kinky hair disease 537
tissue, which is also reflected in cultured skin growth failure and neurodegenerative changes fibroblasts, seems to be a basic biochemical in our patient may chiefly be ascribed to the effect determined genetically [22]. On the deficiency of this vital enzyme [3, 7]. other hand, all the clinical features of MKHD The present results showed that the reduchave been explained by the reduced activities tion of cytochrome c oxidase activity in MKHD of copper-dependent enzymes resulting from was not correlated with tissue copper contents copper-deficiency [3, 6,9] . According to these in various organs. The activity of the enzyme recent studies, it seems that organ-specific was decreased in brain and liver with the low maldistribution of copper may have influence copper contents. It was decreased in muscle on the activities of copper-dependent enzymes despite the increased copper content. In in various organs in MKHD. So, we have at- kidney, both the enzyme activity and the tempted to elucidate the relationship between copper content were increased. Also, cytothe activities of copper dependent-enzymes chrome c oxidase deficiency was not expressed and the tissue copper contents in several organs in cultured skin fibroblasts from our patient. in MKHD. To our knowledge, there are only These observations confirmed that cytochrome a few studies dealing with the activity of c oxidase deficiency in brain, liver and muscle copper-dependent enzyme and the tissue in MKHD occurred secondarily due to the copper contents in patients with MKHD. uneven distribution of copper in the body and Cytochrome c oxidase (ferrocytochrome the defect in copper utilization within cells. c: oxygen oxidoreductase, EC.1.9.3.l) is the We assumed that not only the defect of interterminal element of the electron transport cellular transport of copper but also that of system located in the inner membrane of intracellular transport of copper played the mitochondria, and can readily reduce molecular important roles in the diminished cytochrome oxygen. Cytochrome c oxidase is generally c oxidase activity in the several organs of . considered to contain 2 heme A components MKHD. and 2 g atom copper/functional unit of the According to the recent studies [4, 5, 27] , enzyme. It is evident that copper is an integral it seems likely that the increased metallopart of the cytochrome c oxidase molecule thionein synthesis in MKHD may limit both [23]. In copper-deficient lambs or rats, the the intestinal transport and renal metabolism association of low tissue copper contents with of copper. Riordan et al suggested that the rereduced cytochrome c oxidase activities has tention of copper in Menkes' cell by such a high been reported [23-25]. The neurodegenerative affinity ligand as metallothionein would explain changes seen in these animals are related to the the depressed activity of copper-dependent enzymes in the presence of excess copper in deficient activity of the enzyme. French et al first described diminished cyto- Menkes' cells. On the other hand, our result chrome a + a3 contents of brain, liver, and that the activity of cytochrome c oxidase was muscle in a patient with MKHD [7]. They increased in kidney of MKHD might suggest considered that clinical, morphological and that the function of metallothionein to transfer neurochemical findings consistent with growth the metal to copper-dependent enzymes [28] failure in MKHD could result from failure of was not dearranged. There seemed to be a the energy metabolism. The present investi- unidentified metabolic alteration other than the gators also attempted to evaluate the effect increased synthesis of metallothionein which of copper supplement therapy on tissue copper might be responsible for the decreased activity of copper-dependent enzymes in various organs contents and on cytochrome c oxidase activity. The present study demonstrated the dimin- inMKHD. Copper supplement therapy in the patient ished contents of cytochrome a + a3 in liver and the reduction of cytochrome c oxidase under study failed to alter the progressive activity in brain, liver, skeletal muscle and neurodegenerative course of the disease, alheart in MKHD. Although the exact contribu- though serum levels of copper and ceruloplastion of depressed cytochrome c oxidase activity min were increased as described in previous to the development of the clinical manifesta- reports [12, 26]. The determination of tissue tion and pathological features of MKHD is copper contents in various organs also connot fully understood at this time, hypothermia, firmed the observation of William et al that the 538 Brain & Development, Vol 5, No 6, 1983
uneven distribution of copper in the body was not reversed by copper infusion [12] . The improvement of the oxidase activity of ceruloplasmin by copper infusion therapy has been reported, only a few data are available regarding the effect of copper therapy on the activities of the other copper-dependent enzymes in different organs in MKHD. Garnica et al noted a progressive deficit in leukocyte cytochrome c oxidase activity after cessation of parenteral copper administration [29]. Recently, Nooijen et al reported a 1.S-fold increase of cytochrome c oxidase activity in leukocytes after copper therapy in one of patients with MKHD [11]. Also, Loyola et al suggested some improvement in cytochrome c oxidase activity after parenteral treatment because the elevated lactic acid concentration in cerebrospinal fluid was reduced as the copper concentration increased in serum [30]. These studies suggested that the infused copper may enter cytoplasm and be utilized as the constituent of the enzyme. The effect of copper therapy on cytochrome c oxidase activity in brain, liver and muscle was unfavorable in our patient, although the enzyme activity seemed to be increased in kidney after the copper therapy. The copper concentration in brain and liver remained low in spite of long-term copper administration, whereas it was increased markedly in kidney. The availability of parenterally administered copper was presumably limited by organspecific mal distribution of copper in MKHD. Also, according to our result obtained in muscle, the intracellular transport mechanism of copper from the copper-carrier protein to the copper-dependent enzymes seemed to be disturbed in MKHD. A search for methods not only to transfer copper into the organs such as brain and liver but also to enhance the activities of copper-dependent enzymes within the cells is a prerequisite for the treatment of this devastating disorder.
Acknowledgments We wish to thank Prof. Takayuki Ozawa and Dr. Masashi Tanaka for their valuable instructions. This study was partly supported by Grant No 8111 from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan.
References 1. Menkes JH, Alter M, Steigleder GK, Weakley DR, Sung JH. A sex-linked recessive disorder with retardation of growth, peculiar hair, and focal cerebral and cerebellar degeneration. Pediatrics 1962;29:764-79. 2. French JH. X-chromo some-linked copper malabsorption (X-CLCM). In: Vinken PG, Bruyn GW, eds. Handbook of clinical neurology Vol 29. Amsterdam· New York· Oxford: North-Holland Publ Co, 1977:279-304. 3. Dankes DM, Campbell PE, Stevens BJ, Mayne V, Cartwright E. Menkes kinky hair syndrome. An inherited defect in copper absorption with wide spread effects. Pediatrics 1972;50: 188-201. 4. Labadie GU, Hirschhorn K, Katz S, Beratis NG. Increased copper metallothionein in Menkes' cultured skin fibroblasts. Pediatr Res 1981;15: 257-61. 5. Riordan JR, Jolicoeur-Paquet L. Metallothionein accumulation may account for intracellular copper retention in Menkes' disease. J Bioi Chem 1982;257:4639-45. 6. Holtzman NA. Menkes' kinky hair syndrome; a genetic disease involving copper. Fed Proc 1976;35:2276-80. 7. French JH, Sherard ES, Lubell H, Brotz M, Moor CL. Trichopoliodystrophy. I. Report of a case and biochemical studies. Arch Neurol 1972;26:22944. 8. Hunt DM. Primary defect in copper transport underlies mottled mutants in the mouse. Nature 1974;249:852-4. 9. Grover 'WD, Henkin RI, Schwartz M, Brodsky N, Hobdell E, Stolk JM. A defect in catecholamine metabolism in kinky hair disease. Ann NeuroI1982;12:263-6. 10. Horn N, Keydorn K, Domsgaard E, Tygstrup I, Vestermark S. Is Menkes syndrome a copper storage disorder? Clin Genet 1978;14:186-7. 11. Nooijen JL, De Groot CJ, Van dem Hamer CJA, Monnens LAH, Willemse J, Niermeijer MF. Trace element studies in three patients and a fetus with Menkes' disease. Effect of copper therapy. Pedi4tr Res 1981;15:284-9. 12. William DM, Atkin CL. Tissue copper concentrations of patients with Menkes' kinky hair disease. Am J Dis Child 1981; 135:375-6. 13. Grover WD, Scrutton Me. Copper infusion therapy in trichopoliodystrophy. J Pediatr 1975;86:216-20. 14. Hogeboom GH. Fractionation of cell components of animal tissues. In: Colowick SP and Kaplan NO, eds. Methods in enzymology Vol 1. New York: Academic Press, 1955:16-19. 15. Williams IN. A method for the simultaneous quantitative estimation of cytochrome a, b, c, and cl in mitochondria. Arch Biochem Biophys 1964;107:537-43. 16. Wharton DC, Tzagoloff A. Cytochrome oxidase from beef heart mitochondria. In: Estabrook RW, ed. Methods in enzymology Vol 10. New York: Academic Press, 1962:245-50. 17. Rascati RJ, Persons D. Purification and characterization of cytochrome c oxidase from rat liver
Maehara et al: Menkes kinky hair disease 539
mitochondria. J BioI Chern 1979;254:1586-93. 18. Pennington RJ. Biochemistry of dystrophic muscle. Biochern J 1961;80:649-54. 19. Harrison WW, Netsky MG, Brown MD. Trace elements in human brain: copper, zinc, iron and magnesium. Clin Chern Acta 1968;21:55-60. 20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J BioI Chern 1951;193:265-75. 21. Yoshimura N, Kudo H. Mitochondrial abnormalities in Menkes' kinky hair disease (MKHD). Acta NeuropathoI1983;59:295-303. 22. Goka JT, Stevenson RE, Hefferan PM, Howell RR. Menkes disease: A biochemical abnormality in cultured human fibroblasts. Proc Nat Acad Sci1976;73:604-6. 23. Howell JM, Davidson AN. The copper content and cytochrome oxidase activity of tissue from normal and swayback lambs. Biochern J 1959; 72:365-8. 24. Prohaska J, Wells WW. Copper deficiency in the
540 Brain & Development, Vol 5, No 6, 1983
25.
26.
27. 28. 29.
30.
developing rat brain: Evidence for abnormal mitochondria. J Neurochern 1975;25:221-8. Mills CF, Williams RB. Copper concentration and cytochrome oxidase and ribonuclease activities in the brain of copper-deficient lambs. Biochern J 1962;85:629-32. Deish P, Wheeler EM, Roberts PF, Jones RD. Menkes' syndrome: report of a patient treated from 21 days of age with parenteral copper. Arch Dis Child 1978;53:956-61. Webb M, Cain K. Functions of metallothionein. Biochern Pharrnac 1982;137-42. Brady OF. The physiological function of metallothionein. Trends Biochern Sci 1982;7:143-5. Garnica AD, Frias JL, Easley JF, Rennert DM. Menkes' kinky hair disease. A defect in metallothionein metabolism? Birth Defect 1974;10: 149-55. Loyola MA, Dodson WE. Metabolic consequence of trichopoliodystrophy. J Pediatr 1981 ;98: 588-91.
Menkes kinky hair disease (MKHD) is an X- per in cells and withhold it from incorporation linked recessive disorder of the copper metabo- into copper metalloenzymes, and that unavaillism, characterized by failure to thrive, mental ability of copper in cells, or in the body, may retardation, peculiar hair, seizures, hypother- produce clinical manifestations of copper mia, defective vascular structure and low serum deficiency. The clinical symptoms of MKHD levels of copper and ceruloplasmin [1-3]. The may be mostly attributed to reduced activities primary biochemical lesion is unknown, but the of copper metalloenzymes, e.g., cytochrome c sequestration of copper within cells due to oxidase, dopamine /3-hydroxylase, tyrosinase, abnormal binding to the large amount of lysyl oxidase and ascorbate oxidase [2]. The metallothionein may be the underlying cause . pathogenesis of the central nervous system of the disease [4, 5]. In MKHD, it can be dysfunction in MKHD has been related to postulated that metallothionein may trap cop- deficient energy production due to cytochrome c oxidase deficiency, and also to an altered catecholamine metabolism due to dopamine From the Department of Pediatrics, School of Medi- /3-hydroxylase deficiency [6-9] . cine, Nagoya University, Nagoya (MM, NM, KW, SS); On the other hand, an organ-specific acDepartment of Biochemistry, Institute for Developcumulation and deficiency of copper were mental Research, Aichi Prefectural Colony (NO). found in a patient with MKHD [10-12]. This Received for pUblication: August 15, 1983. abnormal copper storage pattern apparently Accepted for publication: November 8, 1983. gives rise to a copper deficiency elsewhere in Key words: Menkes kinky hair disease, cytochrome c the body, particularly in brain, where it may oxidase, copper metabolism. cause irreversible damage through deficient Correspondence address: Dr. Mitsuo Maehara, Deof copper-dependent enzymes. Alfunction partment of Pediatrics, School of Medicine, Nagoya though cytochrome c oxidase activities in brain, University, 65 Tsuruma-cho, Showa-ku, Nagoya 466, liver, muscle and leukocytes have been deJapan.
scribed [7, 11, 13], relatively little is known about the relationship between cytochrome c oxidase and tissue copper content in MKHD. In this communication, a deficiency of cytochrome c oxidase and maldistribution of tissue copper are reported in various organs in a patient with MKHD, who had been treated with cupric sulfate for 4 years. The effects of copper supplement therapy on the clinical course of the disease and on the activity of cytochrome c oxidase are also discussed. Case Report The boy (MO) under study was the 2,300 g product of 40 weeks gestation and uneventful delivery. Throughout pregnancy, his mother had no complications. No consanguinity was found in the family, but his older brother died at the age of 11 months of a progressive neurodegenerative disease simulating MKHD. At the age of 3 months, the infant boy developed frequent generalized seizures. Convulsive attacks were not controlled completely with anticonvulsants. Marked hypotonia and hypothermia also persisted. His scalp hair was fair and sparse. Serum levels of copper and ceruloplasmin were markedly decreased (12 ,ug/ dl and 4 mg/ dl, respectively). The EEG revealed multifocal seizure discharges with poorly organized background activities. The cerebral angiography showed marked tortuosity
of vessels without any vascular occlusions and anomalies. MKHD was suspected from these clinical observations and laboratory findings at this time. Thereafter, he was treated with intravenous cupric sulphate (20-100,ug/kg/day) once a week for 6 months. Although his hair gradually darkened, no other clinical improvements occurred during copper supplement therapy. At the age of 1 year, his development quotient was 30, and severe growth failure was noted. After age 2 years, cupric sUlphate (0.8-3.0 mg/kg/ day) was administered orally with an intermittent intravenous injection of cupric sulphate (100 ,ug/kg) at least. once a month. Serum levels of copper and ceruloplasmin were increased and could be maintained at 40-70 ,ug/dl and 10-20 mg/dl, respectively, but epileptic seizures, severe mental and physical retardation and hypothermia remained during copper therapy. On computed cranial tomography, marked dilatation of subarachnoidal spaces and ventricles were noted. He was susceptible to upper respiratory infections. At the age of 4 years, he developed pneumonia and died from respiratory insufficiency. Autopsy disclosed severe atrophy of brain, marked muscle wasting and scorbutic skeletal changes. No cardiomegaly and hepatosplenomegaly were found. Serum levels of copper and ceruloplasmin during copper supplement therapy were shown in Fig 1.
CUS04, I.V. j.l
g/ dl mg/ dl 100 20
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C. 0 ::J
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3(months)
2 Age(years)
Fig 1 T~e effec~s of c~pper administration on serum levels of copper ( ___ ) and ceruloplasmin (O---D ~ In a patient WIth MKHD. Serum copper and ceruloplasmin were determined by atomic absorptIOn spectrophotometry and single radial immunodiffusion, respectively. Arrows indicated intravanous injections of cupric sulphate, and shaded area denoted oral doses of cupric sulphate.
534 Brain & Development, Vol 5, No 6, 1983
Materials and Methods Autopsy was performed within 4 hours after the patient's death. Brain, liver, kidney, heart and skeletal muscle were removed from the patient and washed with 0.154 M NaCl twice and stocked at _80°C until analysis. The tissues removed from a 3-month-old male who died of pneumonia were prepared in the same way and used as the control, because control materials in good analytical condition were not available from children of the same age. Mitochondria were isolated from brain and liver within 12 hours after resection according to the method of Hogeboom [14], with some modifications. Small portions of the tissues were minced finely and homogenized in 10 volumes ofa solution of 0.01 M Tris-HCI buffer, pH 7.4, containing 0.21 M mannitol, 0.07 M sucrose and 0.1 mM ethylenediamine tetraacetic acid in a Potter-Elvehjem homogenizer fitted with a Teflon pestle. The homogenate was then centrifuged at 800 g for 10 minutes. The supernatant was collected and centrifuged at 8,000 g for 10 minutes. The fluffy layer was discarded and the remaining pellet was resuspended in buffer and centrifuged again at 8,000 g for 10 minutes. This pellet was used as mitochondrial fraction. The entire procedures were carried out at 4°C. Estimation of the contents of cytochrome a + a3, b, c and c1 of mitochondria was made following the calculation of Williams [15] . Isolated mitochondria were treated with deoxycholic acid and reduced by a few milligrams of sodium dithionite. Difference absorption spectrum of reduced form of cytochromes against cytochromes oxidized by potassium ferricyanide were taken with Shimadzu MPS 50 L spectrophotometer. Determination of cytochrome c oxidase activities was carried out by the method of Wharton and Tzagoloff [16]. Tissue was homogenized in 10 volumes of a solution of 0.01 M potassium phosphate buffer, pH 7.0, containing 0.25 M sucrose in a Potter-Elvehjem homogenizer, and the homogenate was centrifuged at 1,000 g for 10 minutes. The resultant supernatant was sonicated for 10 seconds three times with a Branson sonifier B-12, and was used for assay of enzymatic activities. The reaction mixture was composed of 50 uM ferrocytochrome c in 0.01 M potassium phosphate buffer, pH 7.0. Ferrocytochrome c was pre-
pared by adding a few grains of ascorbic acid to a 1% solution of cytochrome c followed by dialysis to remove excess ascorbate. One ml of the reaction mixture was preincubated in a cuvette at 37°C for 5 minutes, and the reaction was started by adding 10 to 40 ul of sonicated supernatant. The change of absorbance at 550 nm was recorded with Hitachi 124 spectrophotometer continuously for I minute. Activity of cytochrome c oxidase was expressed as the first order rate constant. An extinction coefficient, Esso run , of 18.5 mM-1.cm- 1 was assumed [17]. Inhibition by sodium azide was performed as blank following every sample. Determination of succinate dehydrogenase (SDH) activities was performed by the method of Pennington [18]. One hundred JLl of the supernatant of the homogenate used in determination of cytochrome c oxidase activities was incubated with 1 ml of the reaction mixture containing 0.05 M phosphate buffer, pH 7.4, 0.1 % 2-(p-indophenyl)-3-(p-nitrophenyl)-5phenyltetrazolium (INT) , 25 mM sucrose, and 50 mM sodium succinate at 37°C for 30 minutes. The' reaction was terminated by adding I ml of 10% trichloroacetic acid. The formed formazan was extracted by 4 ml of ethyl acetate and quantified spectrophotometrically at 540 nm. Zero time incubation was used as blank. Copper content of tissue was determined by flame atomic absorption spectrophotometry with wet ashing method [19]. Some 300-500 mg of tissue was weighed precisely,. and ashing of tissue was carried out on a hot plate at 150°C by adding 1 ml of concentrated nitric acid followed by 2 ml of perchloric acid until clear solution was obtained. The resultant solution was transfered to volumetric flask and adequately diluted with distilled water for determination of copper content. Copper determination was performed using a Hitachi 170-10 atomic absorption spectrophotometer. Fibroblast derived from skin biopsied from the patient with MKHD and patients with other neurological diseases was grown in Eagle's essential medium (MEM) containing 10% fetal calf serum .. The cells were harvested at confluent stage by treatment with trypsin. The cells were suspended in 0.01 M phosphate buffer, pH 7.0 and sonicated for 10 seconds three times, and used for analysis of enzymatic activities as described above. For determination Maehara et al: Menkes kinky hair disease 535
of copper content of fibroblasts, harvested cells were suspended in deionized water and sonically disrupted. The lysate was centrifuged at 1,500 g for 20 minutes. The supernatant was assayed for copper and protein content. Protein was determined by the method of Lowry et al [20] , with bovine serum albumin as standard.
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0.02 OD unit
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Results The difference absorption spectra of liver mitochondria from the patient with MKHD and the control were shown in Fig 2. In MKHD, the absorbance at 605 nm, the alpha region of spectrum of cytochrome a + a3 (Le., cytochrome c oxidase), was markedly diminished compared to the control. Also the peak of the gamma region of spectrum was shifted left, which also indicated diminished content of cytochrome a + a3 in mitochondria. The decreased absorbance of cytochrome a + a3 was also noted in brain mitochondria from the patient (data not shown). Cytochrome contents of liver mitochondria calculated spectrophotometrically were shown in Table 1. Cytochrome a + a3 was decreased to 22% of that found in the control. Although cytochrome c was also decreased in our patient with MKHD, it was not certain that the reduction of cytochrome c was a constant finding for MKHD. The activities of cytochrome c oxidase, SDH, and the tissue copper contents in various organs from the patient and control were summarized in Table 2. Statistical analysis could not be performed because of unavailability of more control materials from agematched subjects. In brain, liver, skeletal muscle and heart of the patient, the activities of cytochrome c oxidase were reduced to about 47%, 22%, 54% and 59% of that of the control, respectively. On the other hand, its activity in kidney was increased to 168% of that of the control. Km value of cytochrome c oxidase for ferrocytochrome c in kidney from the patient was 6.5 x 10-6 M against 8.0 x 10-6 M in the control. It seemed that the property of the enzyme was not changed significantly in MKHD. The activities of SDH, an inner mitochondrial marker, in various organs from the patient were slightly decreased compared to that of the control except for kidney. In connection with 536 Brain & Development, Vol 5, No 6,1983
w
() Z
I
,
450
500
r(a+a3)
550
600
J,
1
NORMAL
0.02 OD unit
c:(
ID
c::
oU) ID
c:(
400
I
450
I
500
I
550
I
600
WAVELENGTH(nm) Fig 2 Difference absorption spectra of liver mitochondria from a patient with MKHD (upper) and the control (bottom). The peak of cytochrome a + a3 at 605 nm was diminished. OI.(a + a3) and 'Y (a + a3) indicate the alpha and gamma regions of spectrum of cytochrome a + a3, respectively.
Table 1 Contents of cytochromes in liver mitochondria
Cytochrome (nmol/mg protein) a + a3 b
MKHD
Control
0.029 0.283 0.055 0.014
0.128 0.285 0.027 0.062
cytochrome c oxidase deficiency, the reduced activity of SDH might suggest that the mitochondrial abnormalities were present in MKHD. These results would support the study of Yoshimura et al [20], which showed the morphological abnormalities of the mitochondria in MKHD. In kidney, the activities of cytochrome c oxidase and SDH were increased in MKHD compared to the control. As the large amount of copper was accumulated after copper supplement therapy, especially in kidney, we assumed that the increase of the enzymes was one of
Table 2 Activities of cytochrome c oxidase and SDH, and tissue copper contents in various organs from a patient with MKHD
Copper (pg/g wet tissue) Brain Liver Heart Kidney Skeletal muscle Cultured fibroblast
MKHD
Control
0.59 6.06 7.60 89.20 2.84 415*
0.80 66.70 1.90 3.20 1.20 123* 117*
Cytochrome c oxidase (nmol/min/mg protein)
SDH (nmol/min/mg protein)
MKHD
Control
MKHD
Control
13.2 24.2 70.9 66.7 21.1 29.1
28.0 112.0 120.0 39.6 39.1 20.5 27.5
2.0 22.4 19.0 20.7 2.1 4.3
2.6 24.8 25.8 6.0 18.2 5.8
*: (ng/mg protein)
the effects of the therapy. However, more work was needed to elucidate the effects of copper supplement therapy on the copperdependent enzymes in various organs.in MKHD. Tissue copper contents of brain and liver were decreased, but increased in heart, skeletal muscle, and kidney. The large deposition of copper in kidney was probably accentuated by the long-term copper supplement therapy. The uneven distribution of copper in the' body, reported recently [10-12] as characteristic of MKHD, was also observed in the present patient. In fibroblasts from MKHD, copper content was increased to three times the control level. Activities of cytochrome c oxidase and SDH in fibroblasts were not significantly different from those of the controls (Table 2). The relationship between tissue copper content and cytochrome c oxidase activity was shown in Fig 3. It was suggested that the severity of cytochrome c oxidase deficiency was not correlated with tissue copper contents in MKHD. Fibroblasts that had increased copper content showed normal activity of cytochrome c oxidase in MKHD. In conclusion, cytochrome c oxidase deficiency found in the several organs except for kidney from the patient with MKHD was considered to be a secondary phenomenon due to abnormal copper metabolism. However, its deficiency could not be normalized by longterm copper supplement therapy, presumably resulting from the inability to incorporate serum copper into the cells of brain and liver, and from the defects of intracellular transport
•
20).
o • MKHD
o Control
•
• o
•
0
20
•
o
o
o
40
60
80 100 120
Cytochrome c oxidase activity (nmol/min/mg protein)
Fig 3 The relationship between cytochrome c oxidase activity and tissue copper content in MKHD.
of copper to t.he copper-enzymes in skeletal muscle and heart. Cytochrome c oxidase deficiency might result in abnormal development of the central nervous system in MKHD through failure of tissue energetics. Discussion Menkes kinky hair disease (MKHD) was formerly considered to be due to copper deficiency because of depressed levels of the metal in serum and liver, and many attempts have been made to influence the course of the disease by parenteral administration of copper derivatives [1-3]. Recently, however, the elevated tissue copper contents in most extrahepatic tissues, except for brain, have been reported in MKHD [10-12]. The retention of excess copper in Maehara et al: Menkes kinky hair disease 537
tissue, which is also reflected in cultured skin growth failure and neurodegenerative changes fibroblasts, seems to be a basic biochemical in our patient may chiefly be ascribed to the effect determined genetically [22]. On the deficiency of this vital enzyme [3, 7]. other hand, all the clinical features of MKHD The present results showed that the reduchave been explained by the reduced activities tion of cytochrome c oxidase activity in MKHD of copper-dependent enzymes resulting from was not correlated with tissue copper contents copper-deficiency [3, 6,9] . According to these in various organs. The activity of the enzyme recent studies, it seems that organ-specific was decreased in brain and liver with the low maldistribution of copper may have influence copper contents. It was decreased in muscle on the activities of copper-dependent enzymes despite the increased copper content. In in various organs in MKHD. So, we have at- kidney, both the enzyme activity and the tempted to elucidate the relationship between copper content were increased. Also, cytothe activities of copper dependent-enzymes chrome c oxidase deficiency was not expressed and the tissue copper contents in several organs in cultured skin fibroblasts from our patient. in MKHD. To our knowledge, there are only These observations confirmed that cytochrome a few studies dealing with the activity of c oxidase deficiency in brain, liver and muscle copper-dependent enzyme and the tissue in MKHD occurred secondarily due to the copper contents in patients with MKHD. uneven distribution of copper in the body and Cytochrome c oxidase (ferrocytochrome the defect in copper utilization within cells. c: oxygen oxidoreductase, EC.1.9.3.l) is the We assumed that not only the defect of interterminal element of the electron transport cellular transport of copper but also that of system located in the inner membrane of intracellular transport of copper played the mitochondria, and can readily reduce molecular important roles in the diminished cytochrome oxygen. Cytochrome c oxidase is generally c oxidase activity in the several organs of . considered to contain 2 heme A components MKHD. and 2 g atom copper/functional unit of the According to the recent studies [4, 5, 27] , enzyme. It is evident that copper is an integral it seems likely that the increased metallopart of the cytochrome c oxidase molecule thionein synthesis in MKHD may limit both [23]. In copper-deficient lambs or rats, the the intestinal transport and renal metabolism association of low tissue copper contents with of copper. Riordan et al suggested that the rereduced cytochrome c oxidase activities has tention of copper in Menkes' cell by such a high been reported [23-25]. The neurodegenerative affinity ligand as metallothionein would explain changes seen in these animals are related to the the depressed activity of copper-dependent enzymes in the presence of excess copper in deficient activity of the enzyme. French et al first described diminished cyto- Menkes' cells. On the other hand, our result chrome a + a3 contents of brain, liver, and that the activity of cytochrome c oxidase was muscle in a patient with MKHD [7]. They increased in kidney of MKHD might suggest considered that clinical, morphological and that the function of metallothionein to transfer neurochemical findings consistent with growth the metal to copper-dependent enzymes [28] failure in MKHD could result from failure of was not dearranged. There seemed to be a the energy metabolism. The present investi- unidentified metabolic alteration other than the gators also attempted to evaluate the effect increased synthesis of metallothionein which of copper supplement therapy on tissue copper might be responsible for the decreased activity of copper-dependent enzymes in various organs contents and on cytochrome c oxidase activity. The present study demonstrated the dimin- inMKHD. Copper supplement therapy in the patient ished contents of cytochrome a + a3 in liver and the reduction of cytochrome c oxidase under study failed to alter the progressive activity in brain, liver, skeletal muscle and neurodegenerative course of the disease, alheart in MKHD. Although the exact contribu- though serum levels of copper and ceruloplastion of depressed cytochrome c oxidase activity min were increased as described in previous to the development of the clinical manifesta- reports [12, 26]. The determination of tissue tion and pathological features of MKHD is copper contents in various organs also connot fully understood at this time, hypothermia, firmed the observation of William et al that the 538 Brain & Development, Vol 5, No 6, 1983
uneven distribution of copper in the body was not reversed by copper infusion [12] . The improvement of the oxidase activity of ceruloplasmin by copper infusion therapy has been reported, only a few data are available regarding the effect of copper therapy on the activities of the other copper-dependent enzymes in different organs in MKHD. Garnica et al noted a progressive deficit in leukocyte cytochrome c oxidase activity after cessation of parenteral copper administration [29]. Recently, Nooijen et al reported a 1.S-fold increase of cytochrome c oxidase activity in leukocytes after copper therapy in one of patients with MKHD [11]. Also, Loyola et al suggested some improvement in cytochrome c oxidase activity after parenteral treatment because the elevated lactic acid concentration in cerebrospinal fluid was reduced as the copper concentration increased in serum [30]. These studies suggested that the infused copper may enter cytoplasm and be utilized as the constituent of the enzyme. The effect of copper therapy on cytochrome c oxidase activity in brain, liver and muscle was unfavorable in our patient, although the enzyme activity seemed to be increased in kidney after the copper therapy. The copper concentration in brain and liver remained low in spite of long-term copper administration, whereas it was increased markedly in kidney. The availability of parenterally administered copper was presumably limited by organspecific mal distribution of copper in MKHD. Also, according to our result obtained in muscle, the intracellular transport mechanism of copper from the copper-carrier protein to the copper-dependent enzymes seemed to be disturbed in MKHD. A search for methods not only to transfer copper into the organs such as brain and liver but also to enhance the activities of copper-dependent enzymes within the cells is a prerequisite for the treatment of this devastating disorder.
Acknowledgments We wish to thank Prof. Takayuki Ozawa and Dr. Masashi Tanaka for their valuable instructions. This study was partly supported by Grant No 8111 from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan.
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