High sensitivity can be achieved. Furthermore, an enzymatic assay is highly versatile and specific. By changing the dehydrogenase reaction this assay can be adopted for detecting different redox coenzymes and in this report both NADx and NADPx assay are shown. By replacing PES/MTT with PMS/resazurin, it can be turned into a sensitive fluorescence assay as well. Because the reaction depends on dehydrogenase, specificity can be granted by carefully selecting dehydrogenase specific to only NAD+ or NADP+ (for this reason, enzymes such as mammalian glutamate dehydrogenase EC 1.4.1.2 should be avoided as it is capable of using both NAD+ and NADP+ as substrate). In rats, Williamson et al. [27] reported that during a shift from well-fed to starved, the free concentration ratio [NAD+]/[NADH] changes as follows: from 725 to 528 in cytosol and from 8 to 5 in mitochondria. They also showed that the ratio of total NAD+/ NADH was 7.2 in cytoplasm and 2.2 in mitochondria 22948146 hence most of NADH are in mitochondria and protein bound [27]. The result regarding the total amounts of pyridine nucleotides was obtained using a method contributed by Glock and McLean [37], in which in order to measure total amount of pyridine nucleotides, sample was divided in two parts and extracted separately. One extraction is made in acid for assaying NAD+ and the other in alkaloid for NADH assay. Using the enzyme cycling method, our finding of total NAD+/NADH being around 8 and halved with starvation Anlotinib biological activity MedChemExpress Docosahexaenoyl ethanolamide agrees with 25837696 these findings. NADPx predominantly exists in reduced form; we found the ratio of oxidized over reduced form to be around 0.2 for well-fed and decreased with starvation. The concentrations of PMS and resazurin in this study are carefully chosen based on the study of reaction mechanism by Candeias et al. [38]. It was shown in that report that when the concentration of PMS exceeds 1000 mM, it can form secondary products. Note that the low concentration of dye will certainly limit the upper detection range. This assay depends on the pH-dependent instability of pyridine nucleotides to distinguish between NAD(P)+ from NAD(P)H. In contrast to what has been reported before, we found 30 min of 65uC heat treatment alone cannot fully degrade NAD+ in fruit fly whole body homogenate (Table 1). As most of the NADx assays are optimized and tested for cell lines and mammalian tissue, we suspect this to be due to some unknown insect-specific metabolites which are capable of blocking NAD+ degradation by heat in neutral pH. It has also been suggested that two extractions be made from the same biological sample, one in acid and the other in alkali, and NAD+ and NADH can be assayed separately [19,21,26]. However, it has long been known as well that even high concentrations of OH2 or H+ cannot destroy the activity of pyridine nucleotide-consuming enzymes [26]. Considering these factors, we opt to prepare a common protein free homogenate and treat with OH2 or H+ after rather than before extraction. It is important to minimize enzymatic degradation and to effectively dissociate protein bound NADH when aiming to measure its total amount. Both of these can be accomplished using the phenol chloroform extraction invented by Chomczynski and Sacchi [28]. After extraction, hydrophobic molecules, including proteins, are 
removed in chloroform phase while hydrophilic molecules remain in aqueous phase. Residual phenol chloroformcarry-over did not seem to have a strong negative impact on the assay at le.High sensitivity can be achieved. Furthermore, an enzymatic assay is highly versatile and specific. By changing the dehydrogenase reaction this assay can be adopted for detecting different redox coenzymes and in this report both NADx and NADPx assay are shown. By replacing PES/MTT with PMS/resazurin, it can be turned into a sensitive fluorescence assay as well. Because the reaction depends on dehydrogenase, specificity can be granted by carefully selecting dehydrogenase specific to only NAD+ or NADP+ (for this reason, enzymes such as mammalian glutamate dehydrogenase EC 1.4.1.2 should be avoided as it is capable of using both NAD+ and NADP+ as substrate). In rats, Williamson et al. [27] reported that during a shift from well-fed to starved, the free concentration ratio [NAD+]/[NADH] changes as follows: from 725 to 528 in cytosol and from 8 to 5 in mitochondria. They also showed that the ratio of total NAD+/ NADH was 7.2 in cytoplasm and 2.2 in mitochondria 22948146 hence most of NADH are in mitochondria and protein bound [27]. The result regarding the total amounts of pyridine nucleotides was obtained using a method contributed by Glock and McLean [37], in 
which in order to measure total amount of pyridine nucleotides, sample was divided in two parts and extracted separately. One extraction is made in acid for assaying NAD+ and the other in alkaloid for NADH assay. Using the enzyme cycling method, our finding of total NAD+/NADH being around 8 and halved with starvation agrees with 25837696 these findings. NADPx predominantly exists in reduced form; we found the ratio of oxidized over reduced form to be around 0.2 for well-fed and decreased with starvation. The concentrations of PMS and resazurin in this study are carefully chosen based on the study of reaction mechanism by Candeias et al. [38]. It was shown in that report that when the concentration of PMS exceeds 1000 mM, it can form secondary products. Note that the low concentration of dye will certainly limit the upper detection range. This assay depends on the pH-dependent instability of pyridine nucleotides to distinguish between NAD(P)+ from NAD(P)H. In contrast to what has been reported before, we found 30 min of 65uC heat treatment alone cannot fully degrade NAD+ in fruit fly whole body homogenate (Table 1). As most of the NADx assays are optimized and tested for cell lines and mammalian tissue, we suspect this to be due to some unknown insect-specific metabolites which are capable of blocking NAD+ degradation by heat in neutral pH. It has also been suggested that two extractions be made from the same biological sample, one in acid and the other in alkali, and NAD+ and NADH can be assayed separately [19,21,26]. However, it has long been known as well that even high concentrations of OH2 or H+ cannot destroy the activity of pyridine nucleotide-consuming enzymes [26]. Considering these factors, we opt to prepare a common protein free homogenate and treat with OH2 or H+ after rather than before extraction. It is important to minimize enzymatic degradation and to effectively dissociate protein bound NADH when aiming to measure its total amount. Both of these can be accomplished using the phenol chloroform extraction invented by Chomczynski and Sacchi [28]. After extraction, hydrophobic molecules, including proteins, are removed in chloroform phase while hydrophilic molecules remain in aqueous phase. Residual phenol chloroformcarry-over did not seem to have a strong negative impact on the assay at le.
