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Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.

ABSTRACT: Extracellular flux (XF) analysis has become a mainstream method for measuring mitochondrial function in cells and tissues. Although this technique is commonly used to measure bioenergetics in intact cells, we outline here a detailed XF protocol for measuring respiration in permeabilized cells. Cells are permeabilized using saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), and they are provided with specific substrates to measure complex I- or complex II-mediated respiratory activity, complex III+IV respiratory activity or complex IV activity. Medium- and long-chain acylcarnitines or glutamine may also be provided for measuring fatty acid (FA) oxidation or glutamine oxidation, respectively. This protocol uses a minimal number of cells compared with other protocols and does not require isolation of mitochondria. The results are highly reproducible, and mitochondria remain well coupled. Collectively, this protocol provides comprehensive and detailed information regarding mitochondrial activity and efficiency, and, after preparative steps, it takes 6-8 h to complete.

SUBMITTER: Salabei JK

PROVIDER: S-EPMC4063296 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.

Salabei Joshua K JK Gibb Andrew A AA Hill Bradford G BG

Nature protocols 20140123 2

Extracellular flux (XF) analysis has become a mainstream method for measuring mitochondrial function in cells and tissues. Although this technique is commonly used to measure bioenergetics in intact cells, we outline here a detailed XF protocol for measuring respiration in permeabilized cells. Cells are permeabilized using saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), and they are provided with specific substrates to measu ...[more]

PMID: 24457333

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Project description:Amino acid (AA) metabolism is severely disturbed in critically ill ICU patients. To be able to make a more scientifically based decision on the type of protein or AA nutrition to deliver in ICU patients, comprehensive AA phenotyping with measurements of plasma concentrations and whole body production (WBP) is needed. Therefore, we studied ICU patients and matched control subjects using a novel pulse isotope method to obtain in-depth metabolic analysis. In 51 critically ill ICU patients (SOFA~6.6) and 49 healthy controls, we measured REE and body composition/phase-angle using BIA. In the postabsorptive state, we collected arterial (ized) blood for CRP and AA. Then, we administered an 8 mL solution containing 18 stable AA tracers as a pulse and calculated WBP. Enrichments: LC-MS/MS and statistics: t-test, ANCOVA. Compared to healthy, critically ill ICU patients had lower phase-angle (p < 0.00001), and higher CRP (p < 0.0001). Most AA concentrations were lower in ICU patients (p < 0.0001), except tau-methylhistidine and phenylalanine. WBP of most AA were significantly (p < 0.0001) higher with increases in glutamate (160%), glutamine (46%), and essential AA. Remarkably, net protein breakdown was lower. There were only weak relationships between AA concentrations and WBP. Critically ill ICU patients (SOFA 8-16) had lower values for phase angle (p = 0.0005) and small reductions of most plasma AA concentrations, but higher tau-methylhistidine (p = 0.0223) and hydroxyproline (p = 0.0028). Remarkably, the WBP of glutamate and glutamine were lower (p < 0.05), as was their clearance, but WBP of tau-methylhistidine (p = 0.0215) and hydroxyproline (p = 0.0028) were higher. Our study in critically ill ICU patients shows that comprehensive metabolic phenotyping was able to reveal severe disturbances in specific AA pathways, in a disease severity dependent way. This information may guide improving nutritional compositions to improve the health of the critically ill patient. CLINICAL TRIAL REGISTRY: Data are from the baseline measurements of study NCT02770092 (URL: https://clinicaltrials.gov/ct2/show/NCT02770092) and NCT03628365 (URL: https://clinicaltrials.gov/ct2/show/NCT03628365).

Dataset Information

Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.

Publications

Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.

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