Oxygen consumption was measured polarographically in water-jacketed respiration chambers maintained at 37 °C (Oxygraph, Hanstech Instruments, King’s Lynn, UK) as previously detailed [56 (link)]. Following daily calibration with Buffer Z [50 mM K-MES (Sigma M0895), 30 mM KCl (Sigma P4504), 10 mM K2HPO4 (Fisher, Hampton, NH, USA, P290), 1 mM EGTA (Sigma E4378), 5 mM MgCl2-6H2O (Sigma M2670), 0.005 mM Glutamate (Sigma G8415), 0.002 mM Malate (Sigma M6773), and 0.05% BSA (Sigma A6003), pH to 7.1 at 4 °C] and Na2SO4, 10 µL of isolated SS or IMF mitochondria was independently resuspended in 965 µL of buffer Z, containing 20 mM creatine (Sigma C0708) warmed to 37 °C within the oxygraph chamber. Mitochondria were allowed to equilibrate before the addition of 10 µL Malate (272.21 mM; Sigma M7397) and 10 µL pyruvate (500 mM; Sigma P5280) followed by the addition of 5 µL of ADP (48.09 mM; MP Biomedicals, Irvine, CA, USA, 150259) to determine state 3 and state 4 respiration. Respiratory control ratio (RCR) was designated as state 3 respiration divided by state 4 respiration. Values were normalized post hoc to protein content by the Bradford method.
Malate
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Macao
Malate is a laboratory instrument used to measure the concentration of malate, a dicarboxylic acid, in various samples. It provides quantitative analysis of malate levels in biological, food, and environmental samples.
Automatically generated - may contain errors
Lab products found in correlation
Succinate, by Merck Group (52 mentions)
Pyruvate, by Merck Group (43 mentions)
Glutamate, by Merck Group (38 mentions)
Rotenone, by Merck Group (36 mentions)
Adenosine diphosphate (adp), by Merck Group (32 mentions)
Antimycin a, by Merck Group (26 mentions)
Oligomycin, by Merck Group (22 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (14 mentions)
Ascorbate, by Merck Group (12 mentions)
Sucrose, by Merck Group (12 mentions)
Mannitol, by Merck Group (12 mentions)
Bovine serum albumin (bsa), by Merck Group (10 mentions)
Digitonin, by Merck Group (9 mentions)
Potassium chloride (kcl), by Merck Group (9 mentions)
Adenosine triphosphate (atp), by Merck Group (8 mentions)
Taurine, by Merck Group (7 mentions)
Kh2po4, by Merck Group (7 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (7 mentions)
Sodium pyruvate, by Merck Group (7 mentions)
Cytochrome c, by Merck Group (6 mentions)
93 protocols using malate
1
Mitochondrial Respiration Measurement
2
Mitochondrial Respiration and Fatty Acid Oxidation
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Mitochondrial respiration was evaluated as O2 consumption in isolated liver as previously described (28 (link)). Mitochondria were supplemented with substrates 10 mM glutamate/2 mM malate (Sigma-Aldrich) and 10 mM succinate/0.5 mM rotenone (Sigma-Aldrich), to measure adenosine diphosphate (ADP)–independent respiration activity (state 4). After addition of 1 mM ADP (Sigma-Aldrich), state 3 respiration activity was measured. Respiration was uncoupled by successive addition of carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) up to 3 mM to reach maximal respiration.
Fatty acid oxidation was measured from isolated liver mitochondria from mice fasted for 5 hours before sacrifice using the Oxygraph 2K respirometer (Oroboros Oxygraph-2K, Oroboros Instruments) as described in (51 (link)) and reviewed in (52 ). State 4 respiration was measured using 5 mM malate (Sigma-Aldrich) and 1 mM palmitoyl carnitine (Sigma-Aldrich), and state 3 respiration was measured using 1 mM K-ADP (Sigma-Aldrich) and 10 mM K-succinate (Sigma-Aldrich). Inhibitors used included 0.5 mM oligomycin (Sigma-Aldrich) and 2.5 μM antimycin. FCCP was used as a measure of uncoupling and maximal respiration rate in titrations up to 3 μM.
Fatty acid oxidation was measured from isolated liver mitochondria from mice fasted for 5 hours before sacrifice using the Oxygraph 2K respirometer (Oroboros Oxygraph-2K, Oroboros Instruments) as described in (51 (link)) and reviewed in (52 ). State 4 respiration was measured using 5 mM malate (Sigma-Aldrich) and 1 mM palmitoyl carnitine (Sigma-Aldrich), and state 3 respiration was measured using 1 mM K-ADP (Sigma-Aldrich) and 10 mM K-succinate (Sigma-Aldrich). Inhibitors used included 0.5 mM oligomycin (Sigma-Aldrich) and 2.5 μM antimycin. FCCP was used as a measure of uncoupling and maximal respiration rate in titrations up to 3 μM.
3
Mitochondrial Dysfunction Evaluation Protocol
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Apocynin, apyrase, bovin serum albumin, butylated hydroxytoluene, cytochrome C, dichlorofluorescin diacetate (DCFH-DA), diphenyleneiodonium (DPI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), dithiotreitol (DTT), L-lactic dehydrogenase (EC 1.1.1.27), leupeptin, NAD+, NADH, PGE1, piceatannol, phenylmethylsulfonyl fluoride (PMSF), PP2 analogue (PP2), protease inhibitor cocktail (Cat. N° P8340), superoxide dismutase (SOD), thiobarbituric acid (TBA), digitonin, pyruvate, malate, succinate, ouabain, ampicillin, di-adenosine-5'penta-phosPHAte, rotenone, antimycin A, ADP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and all chemicals were from Sigma-Aldrich, USA. Lectins (WGA, PHA, LCA) and LY294002 were purchased from Merck Millipore, Germany. 2(trifluoromethyl)phenothiazine (2TFP), specific inhibitor of Nox1 [30 (link)], was a gift of Prof. Bruno Tasso Dept. PHArmacy, Genoa University, Genoa. Apocynin, DPI, DTT, LY294002, piceatannol, PP2, 2TFP and FCCP, were diluted in saline from a stock DMSO solution immediately before each experiment. MitoProbe™ Tetramethylrhodamine Methyl Ester (TMRM) from ThermoFisher Scientific was a gift from Dr. Paolo Degan, UO Mutagenesi e Prevenzione Oncologica, IRCCS Ospedale Policlinico San Martino, Genoa. ATP bioluminescence assay kit CLSII and ATP standard solution were from Roche, Switzerland.
4
Comprehensive Chemical Reagent Procurement
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Chemicals were mostly obtained from Sigma–Aldrich (Taufkirchen, Germany). Nitric acid, K2HPO4, KCl, malate, iodacetamide, multi-element standard IV, copper (II) sulfate pentahydrate, ethanol, and xylene were purchased from Merck (Darmstadt, Germany). Acetyl-CoA, reduced nicotinamide adenine dinucleotide (NADH), phosphoenolpyruvate, pyruvate kinase and lactate dehydrogenase were obtained from Roche Diagnostics (Mannheim, Germany). Bovine serum albumin (BSA) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Carl-Roth (Karlsruhe, Germany). Tris-(hydroxymethyl)aminomethane (TRIS) was obtained from VWR International GmbH (Ismaning, Germany). Gelatin was purchased from Grüssing (Filsum, Germany). Rhodium Inductively Coupled Plasma (ICP) standard solution was purchased from SCP Science (Baie D’Urfé, Canada). Osmium tetroxide and uranyl-less contrasting agent were obtained from Science Services GmbH (Munich, Germany). Propylene oxide and epoxy resin were purchased from SERVA Electrophoresis GmbH (Heidelberg, Germany). Lead citrate was purchased from Leica Biosystems (Wetzlar, Germany).
5
Mitochondrial Respiration Assay in Cells
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A fixed number of cells were resuspended in MiR05 respiratory medium [20 mM HEPES (Thermo Fisher Scientific), 0.5 mM EGTA (Merck), 3 mM MgCl2⋅6H2O (Merck), 10 mM KH2PO4 (Merck), 20 mM taurine (Merck), 1 mg/ml bovine serum albumin (Merck), 60 mM potassium lactobionate (Merck), and 110 mM sucrose (Thermo Fisher Scientific), pH7.1] at a density of 1.5 x 105 cells⋅mL-1, and the suspension transferred to Oxygraph-2K (Oroboros Instruments, Innsbruck, Austria) chambers. Cells were first permeabilized by injection of 1 μL of 10 mg⋅mL-1 digitonin (Sigma) in DMSO (final concentration of 5 μg⋅mL-1). A substrate-inhibitor titration was then performed, comprising sequential injection of 2 mM malate (Merck), 0.2 mM octanoyl l -carnitine (Tocris Bioscience, Bristol, UK), 10 mM ADP (Merck), 10 μM cytochrome c (Merck), 25 mM pyruvate (Merck), 10 mM glutamate (Merck), 10 mM succinate (Merck), 0.5 μM rotenone (Merck), and 2.5 μM antimycin A (Merck). Datlab v. 6.0 (Oroboros Instruments) was used for data acquisition and analysis.
6
Mitochondrial Respiration Analysis
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Respirometric analysis on mitochondria and differentiated cultured primary myotubes was performed by high-resolution respirometry (O2-K; Oroboros). Basal cell respiration was determined in respiration medium (contents as described earlier). After basal cell respiration, the cells were permeabilized with digitonin. Saturating levels of pyruvate (5 mM), malate (1 mM), and succinate (10 mM) were used as substrates (Sigma-Aldrich), together with ADP (2.5 mM). The cells were exposed to H 2 O 2 at various concentrations (20-200 mM) while oxygen consumption was measured.
7
Earthworm Exposure to Pharmaceuticals
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Fluoxetine (≥98%), pepsin, pancreatin, malate, bile extract and sodium bicarbonate extract were obtained from Sigma Aldrich (Dorset, UK). Lactic acid, citric acid, acetic acid and methanol (High Performance Liquid Chromatography [HPLC] grade 99.9%) were obtained from Fisher Scientific (Loughborough, UK).
A sandy loam (pH 6.47) soil was collected from an unpolluted site (N 53.957045, W -1.137880) for use in the earthworm exposures. Roots and stones were first removed by hand. The soil was air dried for 24 h before passing through a 2 mm sieve. Details of how moisture content and maximum water holding capacity were determined are given in Supplemental Data.
A sandy loam (pH 6.47) soil was collected from an unpolluted site (N 53.957045, W -1.137880) for use in the earthworm exposures. Roots and stones were first removed by hand. The soil was air dried for 24 h before passing through a 2 mm sieve. Details of how moisture content and maximum water holding capacity were determined are given in Supplemental Data.
8
Mitochondrial Respiration Analysis
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OCR was determined using an XF24 Extracellular Flux Analyzer (Agilent Technologies, Seahorse). Isolated quadricep mitochondria (100 μg) were loaded onto XF24 cell culture microplates in KCl buffer containing 7 mM pyruvate (Sigma-Aldrich) and 1 mM malate (Sigma-Aldrich) and subjected to the Mito Stress Test Kit using the standard protocol provided (Agilent Technologies; 103015-100). Briefly, after basal respiration was measured, the mitochondria were treated sequentially with 10 mM adenosine diphosphate (Sigma-Aldrich), 2 μM oligomycin (Agilent Technologies), 5 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (Agilent Technologies), and 0.5 μM of a mixture of rotenone and antimycin A (Agilent Technologies).
9
Mitochondrial Electron Flow Analysis
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Mitochondria were isolated from the soleus muscle and heart, and prepared based on the previously described procedure [36 (link),39 (link)]. Analysis was performed by Seahorse Metabolic Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA). For electron flow experiments, isolated mitochondria were diluted in cold MAS buffer (enriched with 10 mM pyruvate (Sigma-Aldrich, St. Louis, MO, USA)2 mM malate (Sigma-Aldrich, St. Louis, MO, USA), and 4 µM FCCP (Sigma-Aldrich, St. Louis, MO, USA). A mitochondrial suspension of 25 µL was placed into Seahorse plate wells and centrifuged at 2000× g for 15 min at 4 °C. The concentration of mitochondrial protein was 6 µg per well. After centrifugation, 180 µL of prewarmed MAS buffer supplemented with pyruvate, malate, and FCCP was added to each well, and the plate was then placed into a non-CO2 incubator for 8 min. The Seahorse cartridge was filled with the following reagents: 2 µM Rotenone (Sigma-Aldrich, St. Louis, MO, USA), 2 mM succinate (Sigma-Aldrich, St. Louis, MO, USA), 4 µM Antimycin (Sigma-Aldrich, St. Louis, MO, USA), and a mix of 10 mM ascorbate (Sigma-Aldrich, St. Louis, MO, USA) and 100 µM TMPD (Sigma-Aldrich, St. Louis, MO, USA).
10
Metabolite Profiling of Isotope-Labeled Compounds
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