Fig 1.
Chemical structure of N-acetyl-leucine.
(a) Stereochemistry of the enantiomers. (b) Amide resonance structures showing similarity to an imine. Extending from the tetrahedral chiral carbon is a solid wedge to indicate a bond projecting above the plane of the paper and a hashed wedge to indicate a bond projecting below the plane of the paper.
Fig 2.
Schematic outlining the experimental procedure.
Male mice were orally administered N-acetyl-leucine as either the racemate (50% each enantiomer) or purified L-enantiomer (2.6% D-enantiomer and 97.4% L-enantiomer). At specific times (0.25 to 8 h) after administration, blood was taken, plasma was separated and quantified by chiral liquid chromatography/mass spectrometry. Plots of the plasma concentration of each enantiomer over time were used to visualize pharmacokinetics and a noncompartmental model was used to calculate the pharmacokinetic parameters Cmax (maximum peak concentration), Tmax (time to reach Cmax), ke (first order elimination rate constant), T1/2 (half-life) and AUC (area under the curve). Samples of brain and skeletal muscle were also taken at specific times and used to determine compound distribution and to search for metabolites with high-resolution mass spectrometry.
Fig 3.
Graphs of plasma concentration of enantiomers versus time after administration of racemic N-acetyl-DL-leucine or purified N-acetyl-L-leucine.
Data are presented as (a,c) linear-linear plots or (b,d) semilog plots. Values are the mean ± standard error of the mean with n = 3 (mice).
Fig 4.
Replots of the data to facilitate direct comparison of the plasma concentration of N-acetyl-leucine enantiomers after oral administration of racemic N-acetyl-DL-leucine or purified N-acetyl-L-leucine.
Data are presented as (a,c) linear-linear plots or (b,d) semilog plots. Values are the mean ± standard error of the mean with n = 3 (mice).
Fig 5.
Bar charts showing the pharmacokinetic parameters for the enantiomers of N-acetyl-L-leucine after administration of racemic N-acetyl-DL-leucine (denoted as DL) or N-acetyl-L-leucine (denoted as L).
(a-g) Conventional pharmacokinetic parameters calculated from the plasma concentration of drug. (h-j) Parameters derived from the conventional pharmacokinetic parameters to detect and highlight the effects of pharmacokinetic differences between the enantiomers. Values are the mean ± standard error of the mean with n = 3 (mice). Means were statistically analysed by either (a-g) pre-planned t tests; (h and i) a one-sample t test comparing the measured value with the expected value: 1 when administered as DL and 36 when administered as purified L; and (j) a one-sample t test comparing the measured value with the expected value of 2. The means compared are indicated by the horizontal lines on the charts, and exact p values are provided for the comparisons.
Table 1.
The calculated pharmacokinetic parameters for N-Acetyl-D-Leucine and N-Acetyl-L-Leucine plasma after oral administration of N-Acetyl-DL-Leucine or N-Acetyl-L-Leucine at a nominal dose of 100 mg/kg.
Fig 6.
Graphs of the concentration of enantiomers in tissue versus time after administration of racemic N-acetyl-DL-leucine or purified N-acetyl-L-leucine.
Data are for (a,b) muscle and (c,d) brain and presented as linear-linear plots. Values are the mean ± standard error of the mean with n = 3 (mice).