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A hydrazine coupled cycling assay validates the decrease in redox ratio under starvation in Drosophila.

Chen-Tseh Zhu1, David M Rand

  • 1Department of Ecology and Evolution Biology, Brown University, Providence, Rhode Island, USA. ctzhu@brown.edu

Plos One
|October 20, 2012
PubMed
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Researchers developed a new assay to measure the NAD+/NADH redox ratio in fruit flies (Drosophila melanogaster). This method accurately quantifies pyridine nucleotides and shows a significant decrease in the redox ratio during starvation.

Area of Science:

  • Biochemistry
  • Metabolomics
  • Model Organisms

Background:

  • Pyridine nucleotides, NAD+/NADH and NADP+/NADPH, are crucial for cellular redox homeostasis.
  • Measuring the NAD+/NADH redox ratio is vital for understanding metabolic states.
  • Existing methods for measuring pyridine nucleotides can be limited in sensitivity and stability.

Purpose of the Study:

  • To adapt and validate an enzymatic recycling assay for direct measurement of the NAD+/NADH redox ratio in Drosophila melanogaster.
  • To assess the suitability of this assay for quantifying NADP+ and NADPH.
  • To investigate changes in the NAD+/NADH redox ratio in Drosophila melanogaster under starvation conditions.

Main Methods:

  • Adaptation of a common enzymatic recycling assay for pyridine nucleotide measurement.

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  • Inclusion of a coupling reaction to remove acetaldehyde byproduct from alcohol dehydrogenase (ADH) reaction to improve assay linearity.
  • Simultaneous determination of NAD+ and NADH levels with minimal enzymatic degradation and enhanced sensitivity.

    • Main Results:

    • The adapted assay directly measures the NAD+/NADH redox ratio in Drosophila melanogaster.
    • The method is also effective for quantifying NADP+ and NADPH.
    • A substantial decrease in the NAD+/NADH redox ratio was observed in Drosophila melanogaster during starvation.

    Conclusions:

    • The developed enzymatic assay provides a sensitive and stable method for determining the NAD+/NADH redox ratio and other pyridine nucleotides in Drosophila melanogaster.
    • This assay is valuable for studying metabolic changes, particularly the impact of starvation on redox homeostasis in vivo.
    • The findings highlight the significant metabolic disruption caused by starvation in fruit flies, as indicated by the reduced NAD+/NADH ratio.