Targeted UHPLC-ESI-MS/MS Analysis of Selected Neurotransmitters, Tryptophan and Its Metabolite Kynurenine in Tau Transgenic Rat Brain Tissue: A Pivotal Study

Neurotransmitters (NT) are widely distributed in the central nervous system. These molecules are important for many physiological processes and the function of the immune system. Imbalance of NT are linked to numerous neurological disorders and diseases, including tauopathies. Here, a targeted approach based on on-line combination of ultra-high performance liquid chromatography with tandem mass spectrometry was validated and applied to the quantitative analysis of nine NT (acetylcholine, choline, aspartic acid, asparagine, glutamic acid, glutamine, pyroglutamate, γ-aminobutyric acid, N-acetyl-L-aspartic acid), tryptophan and its metabolite kynurenine in brain tissue samples of a rat model for tauopathy. The applied analytical method was characterized by excellent validation parameters for all analytes, such as limits of detection in the range of 0.01–1.70 µg/mL, regression coefficients of the calibration curves ≥ 0.9946, intra-day and inter-day precision expressed as coefficient of variation in the range of 0.6–11.9% and 0.6–14.4%, and accuracy in the range of 87.6–107.1% and 87.2–119.6%. Our analytical approach led to the identification of increased levels of choline and γ-aminobutyric acid in pons, and elevated concentration levels of pyroglutamate in medulla oblongata. These findings indicate that NT could play a valuable role in the study and clarification of neuroinflammation and neurodegenerative diseases.

Optimization of Gamma Aminobutyric Acid Production Using High Pressure Processing (HPP) by Lactobacillus brevis PML1

In the present research, the production potential of gamma aminobutyric acid (GABA) using Lactobacillus brevis PML1 was investigated. In addition, the microorganism viability was examined in MAN, ROGOSA, and SHARPE (MRS) after undergoing high hydrostatic pressure at 100, 200, and 300 MPa for 5, 10, and 15 min. Response surface methodology (RSM) was applied to optimize the production conditions of GABA as well as the bacteria viability. Analysis of variance (ANOVA) indicated that both the independent variables (pressure and time) significantly influenced the dependent ones (GABA and bacteria viability) ( P < 0.05 ). The optimum extraction conditions to maximize the production of GABA included the pressure of 300 MPa and the time of 15 min. The amount of the compound was quantified using thin-layer chromatography (TLC) and spectrophotometry. For the process optimization, a central composite design (CCD) was created using Design Expert with 5 replications at the center point, whereby the highest content of GABA was obtained to be 397.73 ppm which was confirmed by high performance liquid chromatography (HPLC). Moreover, scanning electron microscopy (SEM) was utilized to observe the morphological changes in the microorganism. The results revealed that not only did have Lactobacillus brevis PML1 the potential for the production of GABA under conventional conditions (control sample) but also the content of this bioactive compound could be elevated by optimizing the production parameters.

Involvement of TauT/SLC6A6 in Taurine Transport at the Blood–Testis Barrier

Taurine transport was investigated at the blood–testis barrier (BTB) formed by Sertoli cells. An integration plot analysis of mice showed the apparent influx permeability clearance of [3H]taurine (27.7 μL/(min·g testis)), which was much higher than that of a non-permeable paracellular marker, suggesting blood-to-testis transport of taurine, which may involve a facilitative taurine transport system at the BTB. A mouse Sertoli cell line, TM4 cells, showed temperature- and concentration-dependent [3H]taurine uptake with a Km of 13.5 μM, suggesting that the influx transport of taurine at the BTB involves a carrier-mediated process. [3H]Taurine uptake by TM4 cells was significantly reduced by the substrates of taurine transporter (TauT/SLC6A6), such as β-alanine, hypotaurine, γ-aminobutyric acid (GABA), and guanidinoacetic acid (GAA), with no significant effect shown by L-alanine, probenecid, and L-leucine. In addition, the concentration-dependent inhibition of [3H]taurine uptake revealed an IC50 of 378 μM for GABA. Protein expression of TauT in the testis, seminiferous tubules, and TM4 cells was confirmed by Western blot analysis and immunohistochemistry by means of anti-TauT antibodies, and knockdown of TauT showed significantly decreased [3H]taurine uptake by TM4 cells. These results suggest the involvement of TauT in the transport of taurine at the BTB.

Embryonic Thermal Manipulation and in ovo Gamma-Aminobutyric Acid Supplementation Regulating the Chick Weight and Stress-Related Genes at Hatch

Chickens are exposed to numerous types of stress from hatching to shipping, influencing poultry production. Embryonic manipulation may develop resistance against several stressors. This study investigates the effects of thermoneutral temperature (T0; 37.8°C) with no injection (N0) (T0N0), T0 with 0.6 ml of 10% in ovo gamma-aminobutyric acid (GABA) supplementation (N1) at 17.5th embryonic day (ED) (T0N1), thermal manipulation (T1) at 39.6°C from the 10th to 18th ED (6 h/day) with N0 (T1N0), and T1 with N1 (T1N1) on hatchability parameters and hepatic expression of stress-related genes in day-old Arbor Acres chicks. The parameters determined were hatchability, body weight (BW), organ weight, hepatic malondialdehyde (MDA), and antioxidant-related gene expression. Percent hatchability was calculated on a fertile egg basis. Growth performance was analyzed using each chick as an experimental unit. Eight birds per group were used for organ weight. Two-way ANOVA was used taking temperature and GABA as the main effect for growth performance and gene expression studies. Analysis was performed using an IBM SPSS statistics software package 25.0 (IBM software, Chicago, IL, USA). Hatchability was similar in all the groups and was slightly lower in the T1N1. Higher BW was recorded in both T1 and N1. Intestinal weight and MDA were higher in T0N1 against T0N0 and T1N1, respectively. The expression of HSP70, HSP90, NOX1, and NOX4 genes was higher and SOD and CAT genes were lower in the T1 group. The present results show that T1 and N1 independently improve the BW of broiler chicks at hatch, but T1 strongly regulates stress-related gene expression and suggests that both T1 and N1 during incubation can improve performance and alleviate stress after hatch.

Pengaruh Waktu Perendaman Beras terhadap Profil Gelatinisasi dan Komponen Bioaktif Tepung Beras Pecah Kulit Berkecambah

Tepung beras pecah kulit berkecambah dapat dimanfaatkan sebagai bahan pangan fungsional karena mengandung senyawa γ-aminobutyric acid (GABA) dan komponen bioaktif lainnya. Penelitian ini bertujuan untuk menganalisis pengaruh waktu perendaman terhadap profil pasting dan komponen bioaktif tepung beras pecah kulit berkecambah. Perkecambahan dilakukan dengan merendam beras pecah kulit selama 120 jam dengan pengambilan sampel setiap 24 jam. Tepung beras pecah kulit berkecambah mengalami penurunan viskositas puncak, breakdown, dan setback seiring dengan lama waktu perendaman. Kandungan GABA mengalami peningkatan dan mencapai nilai tertinggi setelah perendaman 72 jam. Kandungan total fenol, kapasitas antioksidan, dan γ-orizanol mengalami penurunan seiring dengan lamanya waktu perendaman. Sementara itu, hasil analisis komposisi asam lemak tepung beras pecah kulit berkecambah pada perlakuan perendaman 120 jam menunjukkan adanya dominasi asam lemak tidak jenuh. Berdasarkan hasil penelitian ini, tepung beras kecambah dengan perendaman 72 jam dapat dipilih sebagai perlakuan terbaik karena memiliki akumulasi GABA paling tinggi.