Evaluation of D-amino acid levels in rat by gas chromatography-selected ion monitoring mass spectrometry: no evidence for subacute toxicity of orally fed D-proline and D-aspartic acid.

Abstract

Sprague-Dawley rats received deionized water (controls) during 28 days or drinking water with added D-proline, L-proline, D-aspartic acid or L-aspartic acid corresponding to a mean daily load of approximately 50 mg amino acid enantiomer kg-1 body weight. Parameters indicating the physiological status (food intake and body weight, glutamic-oxalic-transaminase, glutamic-pyruvic-transaminase, alkaline phosphatase, urea and creatinine in serum, and creatine and osmomolality of urine) were determined. After 28 days the weights of the supposed target organs of toxicity (kidney, liver, brain, thymus) were determined and organs were inspected for macroscopic and microscopic alterations. No pathological changes in the organs were observed and no signs of subacute toxicity (liver, kidney) were found. In serum, homogenates of liver, kidney and brain, and in part, in urine, the amounts of D-amino acids (D-AAs) were quantitatively determined using chiral phase capillary gas chromatography-selected ion monitoring mass spectrometry. Significant levels of certain D-AAs (Ala, Pro, Ser, Asx, Glx, Orn and Lys) were already detectable in kidney and liver homogenates and serum of controls. In brain homogenates the highest amounts among the D-AAs were found for D-Ser (up to 382 nmol g-1), moderate amounts for D-Ala, D-Asx and D-Glx, and, in a few cases, trace amounts for D-Orn and D-Lys (1-2 nmol g-1). D-Pro was not detected either in the brains of controls or in the brains of animals loaded with D-Pro. Feeding with D-Pro resulted in a 20-30 fold increased renal excretion of D-Pro at the end of the experiment. Continuous feeding with D-Asp did not increase renal excretion of this enantiomer, but in the serum, higher amounts (0.8-4.0 mumol-1) were determined in comparison to the control group (0.3-0.9 mumol-1). Feeding with D-Pro led to an increase of this enantiomer in serum (1.3-10.5 mumol-1). Feeding with D-Asp did not increase its amounts in brain homogenates (38 and 43 nmol g-1) in comparison to controls.