2-(4-Thiazolylmethyl)-D-proline hydrochloride

In mammals, metabolism of free d-glutamate is regulated by d-glutamate cyclase (DGLUCY), which reversibly converts d-glutamate to 5-oxo-d-proline and H2O. Metabolism of these d-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-d-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-d-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet-visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of d-glutamate and 5-oxo-d-proline, and cardiac function.

Background: Pyridoxal 5'-phosphate (PLP) plays a crucial role as a cofactor in amino acid metabolism. There is a prevalence of moderate vitamin B-6 deficiency in the population that may be exacerbated through the ingestion of 1-amino d-proline (1ADP), a vitamin B-6 antagonist found in flaxseed. Objective: Given prior evidence of the impact of synthetic 1ADP on indexes of pyridoxine metabolism, the current study was designed to investigate the effects of 1ADP derived from flaxseed on amino acid metabolism in moderately vitamin B-6-deficient rats. Methods: Male weanling rats (n = 8/treatment) consumed a semipurified diet containing either 7 mg pyridoxine hydrochloride/kg diet [optimum vitamin B-6 (OB)] or 0.7 mg pyridoxine hydrochloride/kg diet [moderately vitamin B-6 deficient (MB)], each with 0 or 10 mg vitamin B-6 antagonist/kg diet, in either a synthetic form (1ADP) or as a flaxseed extract (FE), for 5 wk. At the end of the experiment, plasma vitamin B-6 and amino acid concentrations and the activities of hepatic PLP-dependent enzymes were analyzed. Results: Compared with the MB control group, plasma PLP concentrations were 26% and 69% lower, respectively, in the MB+FE and MB+1ADP rats (P ≤ 0.001). In the MB+FE group, the plasma cystathionine concentration was 100% greater and the plasma α-aminobutyric acid and glutamic acid concentrations were 59% and 30% lower, respectively, than in the MB control group. Both synthetic 1ADP and FE significantly (P < 0.001) inhibited the in vitro hepatic activities of 2 PLP-dependent enzymes, cystathionine β-synthase (up to 44%) and cystathionine γ-lyase (up to 60%), irrespective of vitamin B-6 concentrations. Because of vitamin B-6 antagonist exposure, observed perturbations in plasma biomarkers and hepatic enzyme activities were not evident or of lesser magnitude in rats consuming adequate vitamin B-6. Conclusion: The current data from a rat model provide evidence that a vitamin B-6 antagonist now prevalent in the human food supply may pose challenges to individuals of moderate vitamin B-6 status.