Were diluted 5 fold with either homogenization buffer or fly homogenate. Subjected to different heat and pH treatment, these diluted NAD+ standards were subsequently assayed by enzyme cycling. The slopes between the rate of absorbance increase (Vmean) and the concentration of NAD+ standard are summarized in Table 1. When diluted in Drosophila homogenate, 30 min of 65uC incubation did not fully destroy all NAD+ as implied by a significant positive slope between Vmean and the amount of NAD+ added by standard 1676428 solution. Only with additional 0.01 M OH- present in Drosophila homogenate did this slope become not different from zero indicating all NAD+ was degraded. It appears fly homogenate is able to block the heat-related degradation of NAD+. The results are summarized in Table 1. However, we found fly homogenate does not appear to block the degradation of NADH, which is stable in 65uC with 0.01 M OH2 and quickly degraded in the same temperature in 0.01 M H+ as found by Lowry et al. [24] (Data not shown).Statistical MethodsMethods for estimating and testing the difference of redox ratio in different treatment group are included as Materials and Methods S1 unless otherwise described in the text.Results Typical Result for NADPx and NADx Assay, and the Optimization of ADH Based NADx AssayTypical standard curve and reaction kinetics for NADPx using PES/MTT assay are shown in Figure 2a and 2d. As more MTT is being reduced, the color of the assay mixture changes from yellow to green. However, over time, the reduced MTT forms precipitates which leads to a gradual decline of absorbance once it reached its peak OD570 of about 1.7 (Fig. 2d). A typical standard curve for NADx assay is shown in Figure 2b. Compared to the NADPx assay, the NADx assay is less linear and has a much smaller slope (compare y-axes of Fig. 2a and Fig. 2b). The reaction velocity decreases over time resulting in blended kinetics curves indicating either the enzyme activity or Autophagy ethanol is limiting (Fig. 2e, “Without hydrazine”). We found increasing the final concentration of enzyme, even up to 200 mg/ml, does not remedy the problem. The average reaction rate seems to be proportional to the concentration of ethanol (data not shown). As the oxidation of ethanol to acetaldehyde has a positive DG’0 of 47.2 kJ/mol, a relatively high concentration of ethanol is used in the reaction mixture in order to push the reaction forward. High ethanol concentration alone, although lowering the free energy change, does not improve the assay dramatically, as acetaldehyde is not removed and may eventually inhibit the reaction by blocking the enzyme catalytic site. We chose to couple the ethanol to acetaldehyde reaction with a reaction utilizing acetaldehyde to solve this problem. One possibility is 
to couple it to the acetaldehyde oxidation catalyzed by acetaldehyde dehydrogenase (ALDH), which has a DG’0 of 2215.12 kJ/mol. Another possibility is to couple it to Wolff?Kishner reduction, where acetaldehyde reacts with hydrazine eventually forming N2 and ethane. We found adding a final concentration of 0.02 hydrazine (supplied with 35 w/w hydrazine water solution, Sigma-Aldrich #309400) into reaction mixture inhibitor largely solved the problem (Fig. 3a). Vmean increases linearly with log[hydrazine] (Fig. 3a) but at concentrations higher than 0.02 the OD increase of the blank becomes large (Fig. 3b). Hydrazine increases Vmean of ADH reaction in an [NADx] dependent fashion (Fig. 2f) 
as well. In all, hydrazine.Were diluted 5 fold with either homogenization buffer or fly homogenate. Subjected to different heat and pH treatment, these diluted NAD+ standards were subsequently assayed by enzyme cycling. The slopes between the rate of absorbance increase (Vmean) and the concentration of NAD+ standard are summarized in Table 1. When diluted in Drosophila homogenate, 30 min of 65uC incubation did not fully destroy all NAD+ as implied by a significant positive slope between Vmean and the amount of NAD+ added by standard 1676428 solution. Only with additional 0.01 M OH- present in Drosophila homogenate did this slope become not different from zero indicating all NAD+ was degraded. It appears fly homogenate is able to block the heat-related degradation of NAD+. The results are summarized in Table 1. However, we found fly homogenate does not appear to block the degradation of NADH, which is stable in 65uC with 0.01 M OH2 and quickly degraded in the same temperature in 0.01 M H+ as found by Lowry et al. [24] (Data not shown).Statistical MethodsMethods for estimating and testing the difference of redox ratio in different treatment group are included as Materials and Methods S1 unless otherwise described in the text.Results Typical Result for NADPx and NADx Assay, and the Optimization of ADH Based NADx AssayTypical standard curve and reaction kinetics for NADPx using PES/MTT assay are shown in Figure 2a and 2d. As more MTT is being reduced, the color of the assay mixture changes from yellow to green. However, over time, the reduced MTT forms precipitates which leads to a gradual decline of absorbance once it reached its peak OD570 of about 1.7 (Fig. 2d). A typical standard curve for NADx assay is shown in Figure 2b. Compared to the NADPx assay, the NADx assay is less linear and has a much smaller slope (compare y-axes of Fig. 2a and Fig. 2b). The reaction velocity decreases over time resulting in blended kinetics curves indicating either the enzyme activity or ethanol is limiting (Fig. 2e, “Without hydrazine”). We found increasing the final concentration of enzyme, even up to 200 mg/ml, does not remedy the problem. The average reaction rate seems to be proportional to the concentration of ethanol (data not shown). As the oxidation of ethanol to acetaldehyde has a positive DG’0 of 47.2 kJ/mol, a relatively high concentration of ethanol is used in the reaction mixture in order to push the reaction forward. High ethanol concentration alone, although lowering the free energy change, does not improve the assay dramatically, as acetaldehyde is not removed and may eventually inhibit the reaction by blocking the enzyme catalytic site. We chose to couple the ethanol to acetaldehyde reaction with a reaction utilizing acetaldehyde to solve this problem. One possibility is to couple it to the acetaldehyde oxidation catalyzed by acetaldehyde dehydrogenase (ALDH), which has a DG’0 of 2215.12 kJ/mol. Another possibility is to couple it to Wolff?Kishner reduction, where acetaldehyde reacts with hydrazine eventually forming N2 and ethane. We found adding a final concentration of 0.02 hydrazine (supplied with 35 w/w hydrazine water solution, Sigma-Aldrich #309400) into reaction mixture largely solved the problem (Fig. 3a). Vmean increases linearly with log[hydrazine] (Fig. 3a) but at concentrations higher than 0.02 the OD increase of the blank becomes large (Fig. 3b). Hydrazine increases Vmean of ADH reaction in an [NADx] dependent fashion (Fig. 2f) as well. In all, hydrazine.
