Determination of Free [formula omitted]-Proline and [formula omitted]-Leucine in the Brains of Mutant Mice Lacking [formula omitted]-Amino Acid Oxidase Activity

Abstract

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This d-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for d-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free d-proline in eight brain regions and sera of mutant ddY/DAO− mice, lacking d-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free d-leucine was also investigated. The amount and distribution of d-proline in the brains of ddY/DAO+ mice and ddY/DAO− mice are almost the same, and relatively high amounts of d-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of d-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of d-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO− mice, d-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of l-proline and l-leucine are not significantly different between the two strains. In the serum, the amounts of both free d-proline and d-leucine are significantly higher in the ddY/DAO− mice than those obtained in ddY/DAO+ mice.