Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction

Abstract

The production of ATP is vital for muscle contraction, chemiosmotic homeostasis, and normal cellular function. Many studies have measured ATP content or qualitative changes in ATP production, but few have quantified ATP production in vivo in isolated mitochondria. Because of the importance of understanding the energy capacity of mitochondria in biology, physiology, cellular dysfunction, and ultimately, disease pathologies and normal aging, we modified a commercially available bioluminescent ATP determination assay for quantitatively measuring ATP content and rate of ATP production in isolated mitochondria. The bioluminescence assay is based on the reaction of ATP with recombinant firefly luciferase and its substrate luciferin. The stabilities of the reaction mixture as well as relevant ATP standards were quantified. The luminescent signals of the reaction mixture and a 0.5 microM ATP standard decreased linearly at rates of 2.16 and 1.39% decay/min, respectively. For a 25 microM ATP standard, the luminescent signal underwent a logarithmic decay, due to intrinsic deviations from the Beer-Lambert law. Moreover, to test the functionality of isolated mitochondria, they were incubated with 1 and 5 mM oligomycin, an inhibitor of oxidative phosphorylation. The rate of ATP production in the mitochondria declined by 34 and 83%, respectively. Due to the sensitivity and stability of the assay and methodology, we were able to quantitatively measure in vivo the effects of age and caloric restriction on the ATP content and production in isolated mitochondria from the brain and liver of young and old Fischer-344 rats. In both tissues, neither age nor caloric restriction had any significant effect on the ATP content or the rate of ATP production. This study introduces a highly sensitive, reproducible, and quick methodology for measuring ATP in isolated mitochondria.

Deficiency in Apoptosis-Inducing Factor Recapitulates Chronic Kidney Disease via Aberrant Mitochondrial Homeostasis

“…Mitochondrial ATP was measured using a Molecular Probes bioluminescence ATP determination kit (Life Technologies, Melbourne, VIC, Australia), as described previously (19,20) with the following modifications. A 10-point standard curve was prepared, ranging from 50,000 to 125 nmol/L.…”

Normal brain ageing: models and mechanisms

“…Whether these considerations are applicable to the metabolic state of the aged neurons is not yet clear, and only few direct data are available. Recent direct luminometric measurements of the ATP content and rate of ATP production in resting conditions using mitochondria acutely obtained from Fischer 344 rats showed no difference between adult (12 months) and old (24 months) animals (Drew & Leeuwenburgh 2003). In measuring ATP from whole brain slices (Xiong & Toescu unpublished results) we observed little difference in ATP content between young and old cerebellar slices, but following neuronal activity (45 min after a 5 min pulse of 75 mM KCl) the ATP content in the old slices (from 20 to 23 months old animals) was about 50% less than in the younger slices (12 months old), indicating a decreased functional reserve.…”

Evaluation of sex differences on mitochondrial bioenergetics and apoptosis in mice

“…Mitochondria isolated from heart and skeletal muscle were used immediately after isolation to determine mitochondrial ATP content (Drew and Leeuwenburgh, 2003). In order to determine ATP content, freshly isolated mitochondria were added to a cuvette plus 1.25 mM pyruvate, 1.25 mM malate, and a luciferin-luciferase ATP monitoring reagent (ATP Determination Kit A-22066, Molecular Probes, Eugene, OR).…”