*4.4. High-Performance Liquid Chromatography (HPLC) Analysis*

Blood samples were obtained by cardiac puncture in gerbils for immunohistochemical staining described in Section 2.3. PLP and homocysteine levels were measured in the serum as described before [20]. Briefly, serum was injected onto a C18 reverse-phase column (250 mm × 4.6 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA) in an HPLC system (Agilent 1100 series, Agilent Technologies, Santa Clara, CA, USA) equipped with an electrochemical detector.

#### *4.5. Malondialdehyde Assay*

To elucidate the effects of pyridoxine deficiency on lipid peroxidation induced by ischemia, transient forebrain ischemia was induced with occlusion of common carotid arteries, and the animals (*n* = 5 per group) were sacrificed at 3, 12, and 24 h after ischemia/reperfusion on the 56th day of diet feeding. Both hippocampi were quickly removed, and MDA levels were measured with a Bioxytech MDA-586 kit (Oxis Research, Portland, OR, USA) as described in the previous study [59]. MDA levels were normalized to the protein concentration and the assay was triplicated.
