Vitamin B6, or more specifically the active form pyridoxal 5ʹ-phosphate (PLP), plays a crucial role as a cofactor for numerous enzymes linked to carbohydrate, fatty acid, and amino acid metabolism. There is a high prevalence of moderate vitamin B6 deficiency in the population that may be further exacerbated through the ingestion of vitamin B6 antagonists present in the food supply. For example, flaxseed contains the anti-pyridoxine factor 1-amino D-proline (1ADP) in the form of a dipeptide called linatine. In order to address these issues, the current study was designed to: 1) characterize and quantify the total amount of anti-pyridoxine factors present in flaxseed through the use of UPLC/ESI-MS analysis, 2) investigate the in vivo effects of synthetic and flaxseed-derived 1ADP on amino acid metabolism using a rat model of moderate B6 deficiency, and 3) identify novel biomarkers of vitamin B6 inadequacy using a LC-Qtof-MS based non-targeted metabolomics approach. The total anti-pyridoxine content, measured as 1ADP equivalents, in the flaxseed extract was found to be 177-437 μg/g of whole flaxseed, depending on the variety tested. Plasma biochemical analyses revealed that B6 vitamers, particularly PLP concentrations were reduced (P≤0.001), due to 1ADP ingestion (10 mg/kg diet) irrespective of the sources. Oral ingestion of flaxseed-derived 1ADP in moderately vitamin B6-deficient rats increased plasma cystathionine (P≤0.001), and decreased plasma α-aminobutyric acid (P≤0.001) and glutamic acid (P=0.017) concentrations compared to the controls. However, the ingestion of synthetic 1ADP elicited greater perturbations in amino acid profile compared to the flaxseed-derived 1ADP, which was predominantly in the form of the dipeptide linatine. Additionally, oral ingestion of the synthetic as well as the flaxseed-derived 1ADP significantly (P≤0.05) inhibited the activities of hepatic PLP-dependent enzymes involved in transsulphuration reactions of methionine metabolism. The use of a non-targeted metabolomics approach identified ten potential lipophilic markers of vitamin B6-insufficiency: glycocholic acid, glycoursodeoxycholic acid, murocholic acid, N-docosahexaenoyl GABA, N-arachidonoyl GABA, lumula, nandrolone, orthothymotinic acid, cystamine and 3-methyleneoxindole. These data serve to highlight potential deleterious effects of anti-pyridoxine factors linked to flaxseed in a population at risk for moderate vitamin B6 deficiency. / October 2015