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Show Journal of Neuro- Ophlhalmology 18( 2): 84- 85, 1998. © 1998 Uppincott- Ravcn Publishers, Philadelphia Energy Charge Is Not Decreased in Lymphocytes of Patients With Leber's Hereditary Optic Neuropathy With the 11778 Mutation May- Yung Yen, M. D., Jian- Fu Lee, B. s., Jorn- Hon Liu, M. D., and Yau- Huei Wei, Ph. D. Objectives: A defect in mitochondrial energy conservation is strongly suggested to be involved in the pathogenesis of Leber's hereditary optic neuropathy ( LHON). The authors therefore compared the energy charge in lymphocytes among patients with LHON, their asymptomatic maternal lineages, and normal control subjects. Materials and Methods: Blood samples were obtained from 7 patients, 10 asymptomatic maternal relatives, and 16 normal subjects. Molecular analysis confirmed that all had the ho-moplasmic 11778 point mutation in the mtDNA of their blood cells. The concentrations of adenosine triphosphate ( ATP), diphosphate ( ADP), and monophosphate ( AMP) were determined by high- performance liquid chromatography. The energy charge was calculated as ( ATP + Vi ADP)/( ATP + ADP + AMP). Results: The mean energy charges of lymphocytes were 0.871 ± 0.049 in patients with LHON, 0.884 ± 0.061 in their asymptomatic maternal relatives, and 0.885 ± 0.061 in normal controls, respectively. No statistically significant difference was found among the three groups. Conclusions: Although the study did not find the anticipated change in energy charge in peripheral blood cells, this neither confirms nor rejects the notion that a defect in the mitochondrial oxidative phosphorylation system is involved in the pathogenesis of LHON. Key Words: Leber's hereditary optic neuropathy- Mitochondrial DNA mutation- Oxidative phosphorylation- Energy charge. Leber's hereditary optic neuropathy ( LHON) is a maternally inherited disease that results in acute bilateral visual loss predominantly in healthy young men. In 1988, Wallace et al. ( 1) first reported a point mutation at nucleotide position 11778 of the mitochondrial DNA ( mtDNA) in patients with LHON. Manuscript received March 25, 1997; accepted February 20, 1998. From the Department of Ophthalmology, Taipei Veterans General Hospital ( M. Y. Y., J. H. L.), and the Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang- Ming University ( J. F. L., Y. H. W.), Taipei, Taiwan, Republic of China. Address correspondence and reprint requests to Dr. May- Yung Yen, Department of Ophthalmology, Taipei Veterans General Hospital, National Yang- Ming University, Taipei, 11217, Taiwan, ROC. The pathogenetic mechanism for this disease remains unknown, but a defect in mitochondrial energy conservation is suggested to be involved ( 2,3). Thus, we compared the energy charge in lymphocytes among patients with LHON, their asymptomatic maternal lineages, and normal control subjects. METHODS Blood samples were obtained from 7 patients with LHON and 10 of their asymptomatic maternal relatives. Molecular analysis confirmed that all had the homoplas-mic 11778 point mutation in the mtDNA of their blood cells. Sixteen normal control subjects were also studied. The lymphocytes were separated using 5 ml of Poly-morphprep ( Nycomed Pharma AS, Oslo, Norway) and were diluted to a cell density of 106- 107/ ml. The adenine nucleotides in the freshly prepared lymphocytes were extracted with trichloroacetic acid and analyzed with a high- performance liquid chromatography system. The concentrations of adenosine triphosphate ( ATP), diphosphate ( ADP), and monophosphate ( AMP) were determined from the area of their respective peaks in the chromatogram. The energy charge of the lymphocytes was calculated as ( ATP + Vi ADP)/( ATP + ADP + AMP) ( 4). RESULTS The mean energy charges of lymphocytes were 0.871 ± 0.049 in patients with LHON, 0.884 ± 0.061 in asymptomatic maternal relatives, and 0.885 ± 0.061 in normal control subjects ( Table 1). No statistically significant difference in the energy charge was found among the three groups ( Kruskal- Wallis test, P > 0.05). DISCUSSION In the past few years, more than 15 mtDNA point mutations involving different subunits of complexes I, III, and IV of the respiratory chain have been reported to be associated with LHON. It is still unknown why different mtDNA mutations 84 ENERGY CHARGE IN LHON 85 TABLE 1. Mean energy charge of lymphocytes from Leber's hereditary optic neuropathy patients with 11778 mtDNA imitation, asymptomatic maternal relatives and normal controls Standard Study subjects Mean deviation Patients ( n = 7) 0.871 0.049 Relatives ( n = 10) 0.884 0.061 Controls ( n = 16) 0.885 0.061 Kruskal- Wallis test, /; > 0.05. cause the same clinical manifestation- acute visual loss. One of the possible explanations is that the phenotype results from a general reduction in mitochondrial energy production. The energy production may be controlled by the relative amounts of ATP, ADP, and AMP in the cell, which are frequently expressed as the energy charge of the cell. Energy charge is an index of the energy status of the cell ( 4). When the energy charge is high, ATP-generating ( catabolic) reactions are inhibited, whereas ATP- utilizing ( anabolic) pathways are favored. The normal energy charge of most cells lies between 0.80 and 0.95 ( 4). Although a defect in energy conservation is suggested as a pathogenesis of LHON, our results do not support the notion that patients with the 11778 mtDNA mutation have lower energy production in their blood cells. However, it is important to point out that lymphocytes do not represent optic nerve tissues, which are the target of LHON. Fibers in the prelaminar and laminar optic nerves are particularly sensitive to insults to cytochrome oxidase of the electron transport chain ( 5). Axons in these regions have a higher energy demand because the unmyelinated segments have the increased energy needs to generate nonsaltatory action potentials ( 6). Slow as well as fast axonal transport have been described as being extremely ATP dependent ( 7). Interference with the production of ATP might lead to reduced axon transport in the smallest fibers undergoing the sharpest turns, as found in the pap-illomacular bundle ( 8). The outer segments of the retinal receptors in a patient with LHON were found to contain a relatively low density of mitochondria ( 9). It was demonstrated that in patients with LHON, mitochondrial functional insufficiency leads to blocked axonal transport in the smallest, longest, and most active fibers, and that this injury in the optic nerve begins at the prelaminar optic disc ( 10). These findings indicate that a deficiency of energy production in the optic nerve tissue is still the most plausible explanation of the onset of visual loss in patients with LHON. Although our results suggest that the molecular basis of LHON caused by the 11778 mtDNA mutation is not related to the energy metabolism of peripheral blood cells, this neither confirms nor rejects the notion that a defect in the mitochondrial oxidative phosphorylation system is involved in the pathogenesis of LHON. Acknowledgment: This study was supported by grant no. 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Local supply of energy required for fast axoplasmic transport. Proc Natl Acad Sci USA 1971; 68: 1279- 82. 8. Sadun AA, Martone JF, Muci- Mendoza R, et al. Epidemic optic neuropathy in Cuba. Arch Ophthalmol 1994; 1 12: 691- 9. 9. Joshi RC, Hsu HY, Kashima Y, Heller KB, Sherman .1, Sadun AA. Mitochondrial changes in retinal receptor outer segments in a case of Leber's hereditary optic neuropathy. Invest Ophthalmol Vis Sci 1996; 37( Suppl): 2271. 10. Sadun AA, Joshi RC, Sherman J. Comparing mitochondrial optic neuropathies: Leber's hereditary and acquired optic neuropathies. Invest Ophthalmol Vis Sci 1996; 37( Suppl): 4239. J Neuro- Ophlluilmol, Vol. 18, No. 2, 1998 |