Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314980 | PMC |
| http://dx.doi.org/10.1016/j.mito.2012.01.001 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Expanding the molecular and phenotypic spectrum of truncating mutations.
Neurol Genet
February 2020
Department of Neuromuscular Diseases (E. Bugiardini, O.V.P, A.H., H.H., R.Q., M.G.H., R.D.S.P.), UCL Queen Square Institute of Neurology and The National Hospital for Neurology and Neurosurgery, London, United Kingdom; Mitochondrial Medicine Group (E. Bottani, C.B., M.Z.), Medical Research Council Mitochondrial Biology Unit, Cambridge, United Kingdom; Department of Molecular and Translational Medicine (E. Bottani, A.V.), University of Brescia; Medical Genetics and Neurogenetics Unit (S.M., E.L., C.L.), Fondazione IRCCS Istituto Neurologico, "C. Besta," Milan, Italy; Neurogenetics Unit (C.W.), and Neurometabolic Unit (A.L., I.H., A.C.), The National Hospital for Neurology and Neurosurgery; Division of Neuropathology (K.V., J.L.H.), UCL Queen Square Institute of Neurology; and Dubowitz Neuromuscular Centre (R.Q.), Great Ormond Street Hospital, London, United Kingdom.
Article Synopsis
- This study investigates the clinical effects of two truncating mutations related to mitochondrial encephalomyopathy in three patients, revealing a variety of symptoms including cerebellar ataxia, kidney issues, and cognitive decline.
- The researchers used cybrid cell studies to confirm the pathogenic nature of a novel mutation and examined how these mutations impact mitochondrial complex V structure and function.
- Findings indicate that these mutations can cause a diverse range of symptoms and display varying degrees of heteroplasmy in different tissues, emphasizing the importance of genetic counseling for affected individuals.
First-line Screening of OXPHOS Deficiencies Using Microscale Oxygraphy in Human Skin Fibroblasts: A Preliminary Study.
Int J Med Sci
February 2020
Univ. Lille, Inserm, UMR-S 1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, F-59000 Lille, France.
Article Synopsis
- The diagnosis of mitochondrial diseases is difficult due to their diverse symptoms and genetic variations, requiring clinical and genetic assessments alongside biochemical tests.
- Researchers tested microscale XF technology to measure oxygen consumption in skin fibroblasts from pediatric patients with mitochondrial disorders, establishing a reliable protocol for key respiratory parameters.
- They found a decrease in maximum respiration and spare respiratory capacity across all patients, suggesting microscale oxygraphy could be an effective initial screening method for OXPHOS deficiencies.
Clinical validity of biochemical and molecular analysis in diagnosing Leigh syndrome: a study of 106 Japanese patients.
J Inherit Metab Dis
September 2017
Department of Metabolism, Chiba Children's Hospital, 579-1 Heta-cho, Midori-ku, Chiba, 266-0007, Japan.
Leigh syndrome (LS) is a progressive neurodegenerative disorder of infancy and early childhood. It is clinically diagnosed by typical manifestations and characteristic computed tomography (CT) or magnetic resonance imaging (MRI) studies. Unravelling mitochondrial respiratory chain (MRC) dysfunction behind LS is essential for deeper understanding of the disease, which may lead to the development of new therapies and cure.
View Article and Find Full Text PDFMitochondrial dysfunction in osteoarthritis is associated with down-regulation of superoxide dismutase 2.
Arthritis Rheum
February 2013
Newcastle University, Newcastle upon Tyne, UK.
Objective: Superoxide dismutase 2 (SOD2) is down- regulated in osteoarthritis (OA). This study was undertaken to investigate the functional effects of this down-regulation in the context of oxidative damage and mitochondrial dysfunction.
Methods: Lipid peroxidation in articular cartilage from OA patients and from lesion-free control subjects with femoral neck fracture was assessed by measuring malondialdehyde levels using the thiobarbituric acid reactive substances assay.
Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells.
Mitochondrion
March 2012
Unit of Molecular Neurogenetics, Pierfranco and Luisa Mariani Center for the Study of Mitochondrial Disorders in Children, IRCCS Foundation Neurological Institute "C. Besta", Milan, Italy.
Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!