Development of selective methods for the determination of small amounts of D-amino acids in mammals

Sensitive and selective methods for the determination of small amounts of D-amino acids in mammalian tissues have been established using a two-step HPLC system with a reversed-phase column and a chiral column. A chiral derivatization method using o-phthalaldehyde and N-tertbutyloxycarbonyl-L-cysteine has also been reported for the simultaneous reversed-phase HPLC separation of D-aspartic acid, D-serine and D-alanine. Using these methods, the distribution and alteration of the amounts of D-alanine, D-leucine and D-proline in mammals have been clarified for the first time, and compared with those of D-aspartic acid and D-serine, which have already been reported for several mammalian tissues. The amounts of D-alanine, D-leucine and D-proline are much smaller than those of D-aspartic acid and D-serine, and the values are less than 1% of that of the L-form in most tissues. The amounts of D-alanine have been determined in 22 tissues of rats including the central nervous system and the periphery, and the highest value of 86.4 nmol/g wet tissue has been observed in the anterior pituitary gland. The amounts of D-alanine in this tissue increased until 6 weeks of age, and gradually decreased thereafter. The circadian changes of the D-alanine amounts in the anterior pituitary gland have also been revealed to show higher amounts during the light period. The distribution and alteration of the amounts of D-leucine and D-proline are totally different from those of D-alanine, indicating that these D-amino acids are strictly recognized in mammalian tissues.