How do you test mitochondrial function?

Methods to Test Mitochondrial Function

The gold standard for determining underlying mitochondrial dysfunction is the measurement of oxygen consumption rate (OCR), mitochondrial membrane potential assessment, and lactate/pyruvate ratio. 1

Primary Assessment Methods

Oxygen Consumption Rate (OCR)

• Seahorse XF Analyzer: Measures cellular respiration in real-time
• Oxygen polarography: Applied in isolated mitochondria, cultured cells, and permeabilized fibers from tissue biopsies 2
• Parameters measured:
  • Basal respiration
  • ATP turnover
  • Maximal respiration
  • Proton leak
  • Spare respiratory capacity

Mitochondrial Membrane Potential

• Fluorescent probes:
  • TMRM (Tetramethylrhodamine methyl ester)
  • MitoTrackers
  • Nonyl acridine orange 3
• Technical limitations: Non-specific binding, sensitivity to membrane potential changes, photo-toxicity 1

ATP Production Assessment

• Luminometric methods: Measure mitochondrial ATP production rate (MAPR) 4
• Phosphorus magnetic resonance spectroscopy (31PMRS): Quantifies ATP production in vivo 2
• FRET-based fluorescent reporters:
  • ATeam variants (mitoATeam) for mitochondrial ATP
  • Measure ATP/ADP ratio with PercevalHR 3

Secondary Assessment Methods

Respiratory Chain Complex Activity

• Spectrophotometric assays: Measure activity of complexes I-IV and ATP synthase
• Sample requirements: Tissues, cells, mitochondria-enriched fractions, or isolated mitochondria
• Calculation: Using Beer-Lambert law with appropriate extinction coefficients 3
• Normalization: Activities normalized to protein amount or citrate synthase activity

Reactive Oxygen Species (ROS) Detection

• Fluorescent probes:
  • MitoSOX Red: Detects mitochondrial superoxide
  • MitoPY1: Detects hydrogen peroxide
  • Mito-roGFP: Ratiometric reporter of redox state 3
• Enzymatic assays:
  • Glutathione concentration measurement
  • Aconitase activity (inhibited by ROS) 3

Mitochondrial Morphology Assessment

• Imaging techniques:
  • Fluorescent labeling with targeted proteins
  • Transmission electron microscopy
• Parameters to measure:
  • Size and number of mitochondria
  • Form factor and aspect ratio (branching and elongation) 3

Biomarkers for Clinical Assessment

• Coenzyme Q10: May be a better biomarker of mitochondrial respiratory chain enzyme activities than citrate synthase 5
• Lactate/pyruvate ratio: Elevated in mitochondrial dysfunction
• Citrate synthase activity: Traditional reference enzyme for mitochondrial content

Sample Collection Considerations

• Muscle biopsy: Provides the best opportunity to examine mitochondrial function (25mg sample is sufficient) 4, 6
• Fibroblasts: Can show differences between nuclear DNA and mitochondrial DNA-related disorders 7
• Blood samples: Limited utility but can be used for initial screening

Clinical Application Algorithm

1. Initial screening:

   • Lactate/pyruvate ratio
   • Coenzyme Q10 levels
   • Basic metabolic panel

2. Confirmatory testing:

   • Muscle biopsy for OCR and ATP production measurement
   • Respiratory chain complex activity assays

3. Advanced diagnostics:

   • Neuroimaging with magnetic resonance spectroscopy when metabolic etiology is suspected 1
   • Genetic testing guided by biochemical findings 6

Pitfalls and Caveats

• Fluorescent probes can produce false positives due to photo-toxicity, auto-oxidation, or pH sensitivity 1
• Isolation of mitochondria is delicate and time-consuming; quality affects experimental results 3
• Excessive uncoupling of mitochondria can impair oxygen utilization despite increased oxygen consumption 1
• pH sensitivity of some probes requires simultaneous use of pH sensors 3
• Interpretation of results requires consideration of multiple parameters, as single measurements may be insufficient for diagnosis 6

By systematically applying these methods, clinicians can effectively evaluate mitochondrial function to diagnose disorders and monitor therapeutic interventions.