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Respiratory Chain Defectsin the Mitochondria of Cultured Skin

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Respiratory Chain Defectsin the Mitochondria of Cultured Skin Respiratory Chain Defects in the Mitochondria of Cultured Skin Fibroblasts from Three Patients with Lacticacidemia Brian H. Robinson,* Jewel Ward,4 Paul Goodyer,§ and A. Baudetil *Department ofBiochemistry and Pediatrics, University of Toronto, and The Research Institute, The Hospitalfor Sick Children, Toronto, Ontario; tChild Development Center, Memphis, Tennessee; §Montreal Children's Hospital, Montreal, Quebec, Canada; 1Department ofPediatrics, Baylor College ofMedicine; and Texas Children's Hospital, Houston, Texas Abstract have defects in the mitochondrial respiratory chain. In one of these patients we have published a preliminary investigation The cultured skin fibroblasts from three patients with lacticac- suggesting the respiratory chain as the site of the enzyme de- idemia were found to have low rates of 1-1[4Cjpyruvate oxidation fect (7). in the face of normal pyruvate-dehydrogenase activity. After in- cubation with 1 mM glucose, these three cell strains also exhib- Case reports ited lactate/pyruvate ratios which were three times greater than Patient 1. K.V. was a small-for-gestational-age infant who, after those of controls. In two of the patients, both ATP and oxygen a term delivery, fed poorly for the first week of life. At 10 d of consumption in fibroblast mitochondrial preparations was defi- age the infant developed an acute acidotic episode with tachy- cient with NAD-linked substrates but normal with succinate and pnea. By the 12th d the serum lactate was 30 mM with a serum ascorbate/N'N'N'N' tetramethyl phenylene diamine. In the third pyruvate of 0.22 mM. Serum 3-hydroxybutyrate was 1.5 mM patient, ATP synthesis in mitochondrial preparations was defi- and acetoacetate 0.04 mM. On day 13 the child expired due to cient with all substrates tested. Measurement of Rotenone-sen- overwhelming acidosis. Two previous sibs had died in the neo- sitive NADH-cytochrome c reductase in mitochondrial prepa- natal period after a similar course. rations from skin fibroblasts showed that two of the patients Patient 2. I.W. presented at 15 d with anorexia, weakness, had 14 and 18%, respectively, of control activity. In the third irritability and hypertonic posturing. An electroencephalogram patient, cytochrome oxidase activity was 15% of that in controls. showed focal seizure discharge indicative of diffuse encephalop- We conclude that respiratory chain defects can be demonstrated athy. In the next two days he deteriorated with periods of apnea in cultured skin fibroblasts with consistency using a number of and then total respiratory failure for which he was intubated. different techniques. He was found to have lacticacidemia with a blood lactate of 7.3 mM and a blood pyruvate of 0.12 mM. The patient expired Introduction at I mo of age, and at post-mortem the central nervous system Reported defects in the mitochondrial respiratory chain both in was seen to be rarefied with diffuse necrosis of the subcortical children and in adults have often been associated with chronic white matter and dysmyelination. The parents were first cousins. lacticacidemia. These defects include deficiencies of NADH- Patient 3. W.F. was healthy at birth except for the presence Coenzyme Q reductase (1-7), cytochrome b (8-10), succinate of hypospadias and a micropenis. He had only one testicle, which cytochrome c reductase (1 1, 12) and cytochrome oxidase (13- was undescended. The frontal bones of the forehead were un- 25). Many of these defects are reported as being tissue-specific derdeveloped, giving a narrow forehead. At 8 mo the patient in that only certain tissues, usually muscle, exhibit the enzyme was readmitted to hospital with hypoglycemia and acidosis after deficiencies; but except for two sibs with cytochrome oxidase a urinary tract infection. He was also not thriving and had in- deficiency (24), the deficit in enzyme activity is not demonstrable creased blood lactic acid. Blood lactic acid continued to be el- in fibroblasts. Clinical phenotypes vary from muscular weakness evated at levels between 3.0 and 6.9 mM. By 18 mo he could in adults to overwhelming lactic acidosis in infants, with a spec- sit up supported, but his development, both physical and mental, trum of neurological and neuromuscular presenting features in was delayed. between, and it is not clear why such basic defects should exhibit such heterogeneity. Methods In the course of screening a population of pediatric patients Chemicals. All chemicals used were analytical grade. Cytochrome e and with chronic lacticacidemia for observable defects in cultured TMPD were obtained from the Sigma Chemical Co. (St. Louis, Missouri). skin fibroblasts, we found a number of cell strains that exhibited Skin fibroblast cultures. Human skin fibroblasts were grown from low rates of l-_'4C]pyruvate oxidation to '4CO2 in the face of explants of forearm skin biopsies taken with informed parental consent normal activity of the pyruvate dehydrogenase complex. In fol- and cultured in a-minimal essential medium supplemented with 10% fetal calf serum. It is a characteristic of the respiratory chain-deficient lowing up the etiology of this malfunction we have shown in a cell strains that cell death occurs very rapidly after the glucose in the series of well-documented steps that three of these cell strains culture medium has been used up. We therefore had to refeed the cultures at 5-d intervals to keep them growing. In the later investigations, when Address reprint requests to Dr. Robinson, Research Institute, The Hos- large numbers of cells had to be grown for isolation of mitochondria, pital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada the a-medium was supplemented with an additional 5 mM glucose to M5G 1X8. give a final concentration of 10.5 mM glucose in the culture medium. Receivedfor publication 4 November 1985. Determination of lactate/pyruvate ratios. Confluent skin fibroblast cultures (I ing cell protein) were drained ofculture fluid and the medium J. Clin. Invest. replaced with a Krebs phosphate buffer (2 ml). The fibroblast cultures © The American Society for Clinical Investigation, Inc. were then incubated for 1 h at 370C to deplete substrate and glycogen 0021-9738/86/05/1422/06 $ 1.00 reserves. At this point the buffer was replaced by 2 ml of Krebs phosphate Volume 77, May 1986, 1422-1427 buffer containing I mM glucose. After I h, 0.1 ml 1.6 M perchloric acid 1422 B. H. Robinson, J. Ward, P. Goodyer, and A. Baudet was added. The cells were scraped from the dish with the acidified buffer, activity of the pyruvate dehydrogenase complex have low rates centrifuged, and separated into supernatant and pellet. The supernatant of 1-['4Clpyruvate oxidation to '4CO2 and an abnormal was assayed for lactate and pyruvate (26, 27) and the pellet for protein NAD/NADH ratio, determined either by direct measurement content. (33) or from the lactate/pyruvate ratio in cell cultures (7). Three Determination of enzyme activity. Pyruvate dehydrogenase was cell strains were selected for this study which had rates of measured by the method of Hyland and Leonard (28) and whole cell which were 38, 16, and 27% of control I-["C]pyruvate oxidation by the method of Robinson, Taylor, and Sher- 1-["4C]pyruvate rates, wood (29). Rotenone-sensitive NADH-cytochrome c reductase was respectively (Table I). The activities of the pyruvate dehydro- measured in sonicated mitochondrial preparations as described by Mo- genase complex were 88, 93, and 57%, respectively, of that in readith et al. (6), except that sodium azide (5 mM) rather than cyanide controls. For purposes of comparison, a pyruvate dehydroge- was used to inhibit reoxidation of cytochrome c by cytochrome oxidase. nase-deficient cell strain is included that has 2% of the normal Cytochrome oxidase was determined in mitochondria by the methods activity of the pyruvate dehydrogenase complex and 20% of the described by DiMauro et al. (19). Succinate cytochrome c reductase was normal rate of 1-['4Clpyruvate oxidation. measured by following the reduction of cytochrome c at 550 nm (30). Confluent cultures of skin fibroblasts of the patients being Measurement ofATP synthesis infibroblast mitochondria and dig- studied were incubated for 1 h in glucose-free phosphate-buffered itonin-treatedfibroblasts. The ability of fibroblast mitochondria to syn- saline (PBS). Following a further 1-h incubation in PBS with 1 thesize ATP was tested in two systems, one using isolated mitochondria prepared by the method of Millis and Pious (31) and the other using mM glucose, the accumulation oflactate and pyruvate was mea- digitonin-treated fibroblasts (7). In the latter procedure, confluent skin sured in the incubation medium. Control cells strains produced fibroblast cultures in 9-cm Petri dishes were drained of culture medium lactate at a rate of about 600 nmol/h per mg cell protein, and and the medium replaced by 1 ml ofa solution containing 0.25 M sucrose, this increases somewhat in the presence of the respiratory in- 20 mM morpholinopropane sulphonate (MOPS), pH 7.4, and 0.8 hibitor rotenone. Cell strains from the patients, while giving mg/ml digitonin. 3 min later this was removed and replaced with I ml slightly but not significantly increased rates of lactate production, of a buffer containing 0.25 M sucrose, 20 mM EDTA, and 20 mM gave much lower rates of pyruvate production. The resulting MOPS, pH 7.4. After 5 min this medium was replaced with I ml of a lactate/pyruvate ratio in the medium for these patients were medium containing 0.25 M sucrose, 25 mM MOPS, pH 7.4, 1 mM some three times greater than in control cell strains. Both the EDTA, 5 mM potassium phosphate, I mM ADP (no-substrate medium). amounts of lactate and pyruvate produced and the ratio of lactate To assess the potency of various substrates with respect to ATP produc- to pyruvate was normal in the pyruvate dehydrogenase deficient tion, various substrates were added to this basic medium.
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