High-performance liquid chromatography-based methods of enzymatic analysis: electron transport chain activity in mitochondria from human skeletal muscle
Abstract
This study addresses an application of pyridine nucleotide enzymatic analyses to evaluate the activity of the mitochondrial electron transport chain (reduced nicotinamide adenine dinucleotide (NADH) oxidase) and Complexes I and II in samples of human muscle as small as approximately 10 mg wet weight. Key aspects in this adaptation are the use of high-performance liquid chromatography with fluorescence detection of NADH and use of alamethicin, a channel-forming antibiotic that enables an unrestricted access of substrates into the mitochondrial matrix. The procedure includes disintegration of tissue by Polytron homogenizer, extraction of myosin from myofibrillar fragments by KCl/pyrophosphate to facilitate release of mitochondria, and preparation of fractions of subsarcolemmal and intermyofibrillar mitochondria. Oxidation of NADH or succinate is assayed in the presence of 40 microg/ml alamethicin and the reaction is terminated by H(2)SO(4), which also destroys the remaining NADH. Nicotinamide adenine dinucleotide (NAD) or fumarate concentrations are measured using alcohol dehydrogenase or fumarase plus malic dehydrogenase reactions, respectively. Generation of NADH, assessed in auxiliary reactions in the presence of hydrazine, is strictly proportional to NAD or fumarate content across a concentration range of 1-20 microM. NADH is quantitatively analyzed with a detection limit of 3-5 pmol by HPLC using a reverse-phase Hypersil ODS column connected to a fluorescence detector.
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DNA was extracted from the same homogenate, and mtDNA was measured to adjust for differences in the content of mitochondria.
“DNA was extracted from the same homogenate (total nucleic acid extraction; Epicenter, Madison, WI), and mtDNA was measured to adjust for differences in the content of mitochondria (29).”
From A High-Fat Diet Coordinately Downregulates Genes Required for Mitochondrial Oxidative Phosphorylation in Skeletal Muscle (2005) · cites this paper as [29]
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- Authors
- Ritov, V. B., Menshikova, E. V., & Kelley, D. E.
- Affiliations
- University of Pittsburgh (US)
- Corresponding author
- Ritov, Vladimir B.
- ORCID
- Kelley, David E.
- Venue
- Analytical Biochemistry
- Publisher
- Elsevier BV
- Published
- 2004-10
- Volume
- 333
- Issue
- 1
- Pages
- 27-38
- ISSN
- 0003-2697 (ISSN-L)
- OpenAlex
- W2062922016
- Cited by
- 52
- References
- 54
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 4 · 2022: 3 · 2021: 3 · 2020: 4 · 2019: 4 · 2018: 2 · 2017: 2 · 2016: 5 · 2015: 3 · 2014: 1
- FWCI
- 1.54
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Analytical Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- University of Pittsburgh
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK049200, R01 DK49200-06)
- NIH Clinical Center
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
- Physical Sciences › Chemistry › Spectroscopy › Analytical Chemistry and Chromatography
- Keywords
- mitochondrial electron transport chain, NADH oxidase, complex I, complex II, human skeletal muscle, alamethicin, subsarcolemmal mitochondria, interfibrillar mitochondria, succinate oxidation
- MeSH terms
- Alamethicin, Chromatography, High Pressure Liquid, Electron Transport, Humans, Mitochondria, Muscle, NAD, Oxidation-Reduction, Sensitivity and Specificity, Sulfuric Acids, Muscle, Skeletal, Succinic Acid, Electron Transport Complex II, Electron Transport Complex I
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