A Hydrazine Coupled Cycling Assay Validates the Decrease in Redox Ratio under Starvation in Drosophila

A commonly used enzymatic recycling assay for pyridine nucleotides has been adapted to directly measure the NAD+/NADH redox ratio in Drosophila melanogaster. This method is also suitable for quantification of NADP+ and NADPH. The addition of a coupling reaction removing acetaldehyde produced from the alcohol dehydrogenase (ADH) reaction was shown to improve the linearity of NAD(H) assay. The advantages of this assay method are that it allows the determination of both NAD+ and NADH simultaneously while keeping enzymatic degradation of pyridine nucleotides minimal and also achieving better sensitivity. This method was used to determine the redox ratio of D. melanogaster and validated substantial decrease of redox ratio during starvation.

本研究将一种常用于吡啶核苷酸（pyridine nucleotides）检测的酶循环测定法进行改造，使其可直接测定黑腹果蝇（Drosophila melanogaster）体内的NAD+/NADH氧化还原比率。该方法同样适用于NADP+与NADPH的定量检测。通过添加可清除乙醇脱氢酶（alcohol dehydrogenase, ADH）反应生成的乙醛的偶联反应，可提升NAD(H)测定的线性性能。该测定法的优势在于可同时检测NAD+与NADH，同时将吡啶核苷酸的酶促降解降至最低，且具备更优异的检测灵敏度。本研究采用该方法测定黑腹果蝇的氧化还原比率，并验证了饥饿状态下其氧化还原比率会出现显著下降。