2.4. NAD+ Measurement

NAD⁺ level in hippocampus tissue was determined using the colorimetric method by NAD⁺/NADH assay kit described by the manufacturer (Abcam, Cat #65348, UK) [43]. Hippocampal tissues were homogenized in ice-cold and centrifuged for 5 minutes at 4°C at 16000 g. The supernatant was filtered via a microspin column with a 10 kDa cutoff to separate the NADH-consuming enzymes. In a 96-well microplate, the heated (NADH) and unheated (total NAD(H) samples were mixed separately for five minutes using NAD⁺/NADH cycling assay mix. After two hours, the color was developed with NADH developer solution, and the absorbance was measured at 450 nm using a microplate reader. An aliquot of homogenized material was utilized to assess the concentration before ultrafiltration. Before ultrafiltration, an aliquot of homogenized material was used to test the protein concentration using the conventional bio-rad procedure of measuring the absorbency at 595 nm. Based on conventional NADH measurements, the amount of NAD⁺ was represented in pmol per mg protein.