Dynamics of Organic Acids during the Droplet-Vitrification Cryopreservation Procedure Can Be a Signature of Oxidative Stress in Pogostemon yatabeanus.

Cryopreservation in liquid nitrogen (LN, -196 °C) is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. In previous studies, a systematic approach was used to develop a droplet-vitrification (DV) cryopreservation protocol for Postemon yatabeanus shoot tips that includes preculture with 10% sucrose, osmoprotection with C4-35%, cryoprotection with A3-80% vitrification solution, and a three-step regrowth starting with the ammonium-free medium. The tricarboxylic acid (TCA) cycle is a crucial component of plant cell metabolism as it is involved in redox state regulation and energy provision. We hypothesized that organic acids (OAs) associated with the TCA and its side reactions indirectly indicate metabolism intensity and oxidative stress development in shoot tips under the cryopreservation procedure. In this study, the contents of 14 OAs were analyzed using gas chromatography-tandem mass spectrometry (GC-MS/MS) in P. yatabeanus shoot tips in a series of treatments including individual steps of the DV procedure, additional stress imposed by non-optimum protocol conditions (no preculture, no osmoprotection, various vitrification solution composition, using vials instead of aluminum foils, etc.) and regrowth on different media with or without ammonium or growth regulators. The possible relation of OA content with the total cryoprotectant (CPA) concentration and shoot tips regeneration percentage was also explored. Regeneration of cryopreserved shoot tips reduced in descending order as follows: standard protocol condition (91%) > non-optimum vitrification solution (ca. 68%) > non-optimum preculture (60-62%) > regrowth medium (40-64%) > no osmoprotection, cryopreservation in vials (28-30%). Five OAs (glycolic, malic, citric, malonic, and lactic) were the most abundant in the fresh (control) shoot tips. The dynamic pattern of OAs during the DV procedure highly correlated (r = 0.951) with the total CPA concentration employed: it gradually increased through the preculture, osmoprotection, and cryoprotection, peaked at cooling/rewarming (6.38-fold above control level), and returned to the fresh control level after 5 days of regrowth (0.89-fold). The contents of four OAs (2-hydroxybutyric, 3-hydroxypropionic, lactic, and glycolic) showed the most significant (10-209-fold) increase at the cooling/rewarming step. Lactic and glycolic acids were the major OAs at cooling/rewarming, accounting for 81% of the total OAs content. The OAs were categorized into three groups based on their dynamics during the cryopreservation protocol, and these groups were differently affected by protocol step modifications. However, there was no straightforward relationship between the dynamics of OAs and shoot tip regeneration. The results suggest that active modulation of OAs metabolism may help shoot tips to cope with osmotic stress and the chemical cytotoxicity\ of CPAs. Further intensive studies are needed to investigate the effect of cryopreservation on cell primarily metabolism and identify oxidative stress-related biomarkers in plant materials.

Chiral LC-MS/MS method for the discrimination of triiodothyronine enantiomers on a crown ether-based chiral stationary phase.

Considering the greater pharmaceutical and clinical interest of triiodothyronine (T3 ) thyroid hormone, an effective D/L-T3 enantiomer separation was performed on a crown ether-based chiral stationary phase by LC-MS/MS. In optimal analytical condition and selected reaction monitoring mode, the two enantiomers of T3 were baseline separated within 10 min. The limit of detection and limit of quantitation were found to be 0.05 and 0.10 ng/µl; 0.20 and 0.50 ng/µl for D- and L-T3 , respectively. During validation, this method proved to be feasible, accurate as well as enantioselective and sensitive for the resolution of T3 enantiomers. For commercial D- and L-T3 chemicals, the enantiomeric impurities as the other enantiomer were 0.11% and 4.61%. On the other hand, the impurity as D-T3 for commercial pharmaceutical products (liothyronine sodium tablets, two suppliers) was 0.68% and 6.57%.

Alcohol perturbed locomotor behavior, metabolism, and pharmacokinetics of gamma-hydroxybutyric acid in rats.

Gamma-hydroxybutyric acid (GHB), both a metabolic precursor and product of gamma-aminobutyric acid (GABA), is a central nervous system depressant used for the treatment of narcolepsy-associated cataplexy and alcohol withdrawal. However, administration of GHB with alcohol (ethanol) is a major cause of hospitalizations related to GHB intoxication. In this study, we investigated locomotor behavior as well as metabolic and pharmacokinetic interactions following co-administration of GHB and ethanol in rats. The locomotor behavior of rats was evaluated following the intraperitoneal administration of GHB (sodium salt, 500 mg/kg) and/or ethanol (2 g/kg). Further, time-course urinary metabolic profiling of GHB and its biomarker metabolites glutamic acid, GABA, succinic acid, 2,4-dihydroxybutyric acid (OH-BA), 3,4-OH-BA, and glycolic acid as well as pharmacokinetic analysis were performed. GHB/ethanol co-administration significantly reduced locomotor activity, compared to the individual administration of GHB or ethanol. The urinary and plasma concentrations of GHB and other target compounds, except for 2,4-OH-BA, were significantly higher in the GHB/ethanol co-administration group than the group administered only GHB. The pharmacokinetic analysis results showed that the co-administration of GHB and ethanol significantly increased the half-life of GHB while the total clearance decreased. Moreover, a comparison of the metabolite-to-parent drug area under the curve ratios demonstrated that the metabolic pathways of GHB, such α- and ß-oxidation, were inhibited by ethanol. Consequently, the co-administration of GHB and ethanol aggravated the metabolism and elimination of GHB and enhanced its sedative effect. These findings will contribute to clinical interpretation of GHB intoxication.