Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.
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.
Hamase
K
K
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
Inoue
T
T
Morikawa
A
A
Konno
R
R
Zaitsu
K
K
D000818:Animals; D001921:Brain / Q000201:enzymology*; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D003605:D-Amino-Acid Oxidase / Q000172:deficiency*; D007930:Leucine / Q000032:analysis*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008817:Mice, Mutant Strains; D011392:Proline / Q000032:analysis*
D011392:Proline; D003605:D-Amino-Acid Oxidase; D007930:Leucine