Biomaterial Database

Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples. 2002

Rolf Wibom, and Lars Hagenfeldt, and Ulrika von Döbeln

Centre for Inherited Metabolic Diseases, Department of Medical Laboratory Sciences and Technology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, Sweden. rolf.wibom@cmms.hs.sll.se

A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children.

UI	MeSH Term	Description	Entries
D008163	Luminescent Measurements	Techniques used for determining the values of photometric parameters of light resulting from LUMINESCENCE.	Bioluminescence Measurements,Bioluminescent Assays,Bioluminescent Measurements,Chemiluminescence Measurements,Chemiluminescent Assays,Chemiluminescent Measurements,Chemoluminescence Measurements,Luminescence Measurements,Luminescent Assays,Luminescent Techniques,Phosphorescence Measurements,Phosphorescent Assays,Phosphorescent Measurements,Assay, Bioluminescent,Assay, Chemiluminescent,Assay, Luminescent,Assay, Phosphorescent,Assays, Bioluminescent,Assays, Chemiluminescent,Assays, Luminescent,Assays, Phosphorescent,Bioluminescence Measurement,Bioluminescent Assay,Bioluminescent Measurement,Chemiluminescence Measurement,Chemiluminescent Assay,Chemiluminescent Measurement,Chemoluminescence Measurement,Luminescence Measurement,Luminescent Assay,Luminescent Measurement,Luminescent Technique,Measurement, Bioluminescence,Measurement, Bioluminescent,Measurement, Chemiluminescence,Measurement, Chemiluminescent,Measurement, Chemoluminescence,Measurement, Luminescence,Measurement, Luminescent,Measurement, Phosphorescence,Measurement, Phosphorescent,Measurements, Bioluminescence,Measurements, Bioluminescent,Measurements, Chemiluminescence,Measurements, Chemiluminescent,Measurements, Chemoluminescence,Measurements, Luminescence,Measurements, Luminescent,Measurements, Phosphorescence,Measurements, Phosphorescent,Phosphorescence Measurement,Phosphorescent Assay,Phosphorescent Measurement,Technique, Luminescent,Techniques, Luminescent
D008297	Male		Males
D008931	Mitochondria, Muscle	Mitochondria of skeletal and smooth muscle. It does not include myocardial mitochondria for which MITOCHONDRIA, HEART is available.	Sarcosomes,Mitochondrion, Muscle,Muscle Mitochondria,Muscle Mitochondrion,Sarcosome
D009247	NADH, NADPH Oxidoreductases	A group of oxidoreductases that act on NADH or NADPH. In general, enzymes using NADH or NADPH to reduce a substrate are classified according to the reverse reaction, in which NAD+ or NADP+ is formally regarded as an acceptor. This subclass includes only those enzymes in which some other redox carrier is the acceptor. (Enzyme Nomenclature, 1992, p100) EC 1.6.	Oxidoreductases, NADH, NADPH,NADPH Oxidoreductases NADH,Oxidoreductases NADH, NADPH
D002458	Cell Fractionation	Techniques to partition various components of the cell into SUBCELLULAR FRACTIONS.	Cell Fractionations,Fractionation, Cell,Fractionations, Cell
D002950	Citrate (si)-Synthase	Enzyme that catalyzes the first step of the tricarboxylic acid cycle (CITRIC ACID CYCLE). It catalyzes the reaction of oxaloacetate and acetyl CoA to form citrate and coenzyme A. This enzyme was formerly listed as EC 4.1.3.7.	Citrate Synthase,Synthase, Citrate
D004579	Electron Transport	The process by which ELECTRONS are transported from a reduced substrate to molecular OXYGEN. (From Bennington, Saunders Dictionary and Encyclopedia of Laboratory Medicine and Technology, 1984, p270)	Respiratory Chain,Chain, Respiratory,Chains, Respiratory,Respiratory Chains,Transport, Electron
D004796	Clinical Enzyme Tests	Analyses for a specific enzyme activity, or of the level of a specific enzyme that is used to assess health and disease risk, for early detection of disease or disease prediction, diagnosis, and change in disease status.	Enzyme Tests, Clinical,Clinical Enzyme Test,Enzyme Test, Clinical,Test, Clinical Enzyme,Tests, Clinical Enzyme
D005260	Female		Females
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man