Figure 1.
A representative scheme of a cycling assay for pyridine nucleotides.
In this case, the oxidation of ethanol to acetaldehyde catalyzed by ADH is used to assay NADx. The redox indicator is a MTT/PES coupled reaction. Acetaldehyde is removed by reacting with hydrazine in a Wolff-Kishner reduction (see detail in text).
Figure 2.
Comparison of reaction kinetics of NADPx, NADx assay with and without hydrazine.
A: Standard curve of NADPx assay. B: Standard curve of NADx assay without hydrazine. C: Standard curve of NADx assay with hydrazine. D: reaction kinetic of NADPx assay. E: NADx assay kinetic, showing additional hydrazine increases Vmean. F: the increase of Vmean by addition of hydrazine is dependent on the concentration of NAD+. All assays were performed in duplicates and hydrazine concentration is 0.02%.
Figure 3.
The optimal concentration of hydrazine is around 0.02%.
A: In a reaction containing 25 µM NAD+, Vmean increases with hydrazine concentration log-linearly up to 0.02%. B: The rate of absorbance increase in no NAD+ blank control is affected by hydrazine concentration higher than 0.02%.
Table 1.
Slopes between the rate of absorbance increase (Vmean) and the concentration of NAD+ (10−4·OD·min−1·µM−1) in either homogenate or homogenization buffer.
Figure 4.
Comparison of three different extraction methods.
Three sets of 15 D. melanogaster adult males were subjected to different treatments. Homogenized in buffer with additional 6 M guanidine-HCl or: homogenized and treated with equal volume phenol-chloroform or chloroform only. The samples were then assayed for NADx in duplicates.
Table 2.
Phenol-Chloroform extraction is efficient for extracting NADH.
Table 3.
The level of NADx and NADPx of fed and starved flies.
Figure 5.
Changes in the concentrations of protein and metabolites following starvation.
Comparisons are between flies held on food (‘Fed’) and on 2% agar (starved, ‘Stv’ on x-axes). Unit: µmol/g protein For NADH, NAD+, NADPH and NADP+. mg/g protein for TAG, glycogen and glucose. µg/ml for protein. n = 8 and measured in duplicates. The between group differences are tested by 2 sample t-test.