This is the first study to apply the PITC method to detect L-theanine in biological samples and report the effect of L-theanine on pharmacokinetic parameters and serum amino acid composition in mice.
Among the several amino acid analyses, the separation and quantification of glutamic acid using the PITC method has been reported to be superior to the O-phthaldehyde (OPA) labeling method (Fürst et al. 1990). Further, Thippeswamy et al. (2006) have reported that the L-theanine peak could be analyzed using the PITC method without interference from Asp, Glu, Ser, Gly, His, Arg, Thr, Ala, Pro, Tyr, Val, Met, Cys, Ile, Leu, Phe, or Lys. Additionally, Zhu et al. (2016) and Thippeswamy et al. (2006) have reported that L-theanine in pu-erh and green tea could be analyzed using the PITC method and that other amino acids in tea leaf samples could be detected simultaneously; however, these two reports were not on biological samples such as blood. Terashima et al. (1999) quantified L-theanine concentrations in rat blood; however, they used an automated amino acid analyzer with ninhydrin, and chromatograms were not shown and quantitation was not discussed. Therefore, we adopted the PITC method to analyze L-theanine, including Glu, a degradation product of L-theanine. Furthermore, this study revealed that β-Hyp, Tau, GABA, Trp, and Orn, which have not been previously examined, did not interfere with the analysis of L-theanine. In this study, we isolated L-theanine without interference from the serum components and other 18 amino acids (Asp, Glu, Ser, Gly, His, Cit, Thr, GABA, Ala, Pro, Tyr, Val, Met, Cys, Ile, Leu, Phe, and Lys) that could be quantified simultaneously. Therefore, the PITC method is an excellent analytical method that can separate and quantify L-theanine in biological samples to the limit of quantification of 0.44 µg/mL, suggesting its application for quantifying L-theanine in the brain and other tissues.
The Cmax and AUC results of L-theanine oral administration to mice indicated that the blood concentration of L-theanine showed dose-dependent linearity. The average T1/2 of L-theanine, when administered orally to mice, was 42.16 ± 7.23 min. However, L-theanine is less likely to be eliminated than Arg, compared with the reported T1/2 of Arg in rats, which was 10.1 min (Campistron et al. 1982). Further, our results suggest that although L-theanine is rapidly absorbed, its elimination may be delayed at higher doses, such as 1000 mg/kg. A study on the pharmacokinetics of L-theanine in rats by Terashima et al. (1999) reported that at a dose of 4000 mg/kg administered intragastrically, the Tmax was reached 1 h post administration and the blood concentration of L-theanine decreased to approximately 15% or less of Cmax 16 h after administration. However, they collected blood at 0, 1, 2, 5, 8, 16, and 24 h, and considering that a very high dose of L-theanine (4000 mg/kg) was administered, the Tmax may have been achieved earlier, resulting in the delayed disappearance of L-theanine. For sleep-improving effects, L-theanine supplements are often taken before sleep, and Kobayashi and Nagato (1998) reported that L-theanine takes approximately 30 min to produce its effect. Therefore, it is necessary to observe the changes in the blood concentration of L-theanine within 1 h after administration.
Scheid et al. (2012) suggested that L-theanine is distributed not only in plasma but also in other tissues, and the volume of distribution in mice in the present study suggested the same. Considering the body weight of the mice used in this study, it was suggested that although L-theanine is distributed in tissues, but its transferability is low.
Further, we have clarified the previously unknown bioavailability and clearance of L-theanine. Although the bioavailability of theanine in mice may differ from that in humans, it will be useful for future studies to elucidate the effects of theanine on brain function.
In the present study, mice survived well after tail vein administration at a dose of 1000 mg/kg, suggesting that the biological effects of L-theanine ingestion are mild and may not have a significant effect on serum amino acid composition. Terashima et al. (1999) reported that when L-theanine was administered to rats, the Trp concentration tended to decrease 24 h after administration, but other amino acid compositions in the blood did not change significantly within 24 h. In this study, considering that investigating the immediate effect of L-theanine after administration is also important, we observed the effect of amino acid composition for 2 h after administration. Our results showed that the serum concentrations of Phe, Thr, Tyr, Met, Ala, Val, Leu, Ile, Lys, His, Glu, Gln, and Asp were unchanged 2 h after L-theanine administration, consistent with the results of Terashima et al. (1999). Notably, Trp was not compared as it could not be separated or quantified using the PITC method. We observed an increase in the Gly peak area 30 min after the oral administration of L-theanine at doses of 400 and 1000 mg/kg. This increase in peak area was significantly greater at the 1000 mg/kg dose than at the 400 mg/kg dose. However, this increase in the peak area was not sharp and could not be attributed to Gly alone. Based on the retention time of the peak, the possibility of ethylamine and ammonia, which are metabolites of theanine, was not considered. According to Tsuge et al. (2003), L-theanine may be converted to y-glutamylglycine in the presence of glycylglycine. Yamada et al. (2005) also reported that the administration of L-theanine to rats may induce inhibitory neurotransmission via glycine receptors by releasing Gly from brain neurons. However, we were unable to determine whether the increase in this peak was owing to an increase in Gly alone. Given that Gly, similar to L-theanine, is used as a dietary supplement for its anxiolytic effects and to improve sleep, determining the effects of L-theanine administration on Gly levels is important and needs further research.