Despite its critical role in neurodevelopment and brain function, vitamin D (vit-D) homeostasis, metabolism, and kinetics within the central nervous system remain largely undetermined. Thus, it is of critical importance to establish an accurate, highly sensitive, and reproducible method to quantitate vit-D in brain tissue. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method and for the first time, demonstrate detection of seven major vit-D metabolites in brain tissues of C57BL/6J wild-type mice, namely 1,25(OH)2D3, 3-epi-1,25(OH)2D3, 1,25(OH)2D2, 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, and 24,25(OH)2D2. Chromatographic separation was achieved on a pentaflurophenyl column with 3 mM ammonium formate water/methanol [A] and 3 mM ammonium formate methanol/isopropanol [B] mobile phase components. Detection was by positive ion electrospray tandem mass spectrometry with the EVOQ elite triple quadrupole mass spectrometer with an Advance ultra-high-performance liquid chromatograph and online extraction system. Calibration standards of each metabolite prepared in brain matrices were used to validate the detection range, precision, accuracy, and recovery. Isotopically labelled analogues, 1,25(OH)2D3-d3, 25(OH)D3-c5, and 24,25(OH)2D3-d6, served as the internal standards for the closest molecular-related metabolite in all measurements. Standards between 1 fg/mL and 10 ng/mL were injected with a resulting linear range between 0.001 and 1 ng, with an LLOD and LLOQ of 1 pg/mL and 12.5 pg/mL, respectively. The intra-/inter-day precision and accuracy for measuring brain vit-D metabolites ranged between 0.12-11.53% and 0.28-9.11%, respectively. Recovery in acetonitrile ranged between 99.09 and 106.92% for all metabolites. Collectively, the sensitivity and efficiency of our method supersedes previously reported protocols used to measure vit-D and to our knowledge, the first protocol to reveal the abundance of 25(OH)D2, 1,25(OH)D2, and 24,25(OH)2D2, in brain tissue of any species. This technique may be important in supporting the future advancement of pre-clinical research into the function of vit-D in neurophysiological and neuropsychiatric disorders, and neurodegeneration.
Branched fatty acid esters of hydroxy fatty acids (FAHFAs) represent trace lipids with significant natural biological functions. While exogenous FAHFAs have been extensively studied, research on FAHFAs in milk remains limited, constraining our grasp of their nutritional roles. This study introduces a non-targeted mass spectrometry approach combined with chemical networking of spectral fragmentation patterns to uncover FAHFAs. Through meticulous sample handling and comparisons of various data acquisition and processing modes, we validate the method's superiority, identifying twice as many FAHFAs compared to alternative techniques. This validated method was then applied to different milk samples, revealing 45 chemical signals associated with known and potential FAHFAs, alongside findings of 66 ceramide/hexosylceramide (Cer/HexCer), 48 phosphatidyl ethanolamine/lyso phosphatidyl ethanolamine (PE/LPE), 21 phosphatidylcholine/lysophosphatidylcholine (PC/LPC), 16 phosphatidylinositol (PI), 7 phosphatidylserine (PS), and 11 sphingomyelin (SM) compounds. This study expands our understanding of the FAHFA family in milk and provides a fast and convenient method for identifying FAHFAs.
Stable carbon isotope ratio measurements are used to investigate the provenance of vanillin. In this study, a variety of commercial vanillin samples and vanilla products were analyzed to provide a frame of reference for the variability of carbon isotope delta values in various vanillin samples, with the results ranging from -20.6 to -36.7‰ relative to the Vienna Peedee Belemnite (VPDB). We present information on the development of two synthetic vanillin reference materials, VANA-1 and VANB-1, prepared in 0.75 g units in glass vials, to be used for the calibration of carbon isotope delta measurements of vanillin and other easily combustible organic materials. Characterization of 40 vials each of VANA-1 and VANB-1 was performed by three laboratories over several measurement sequences. The certified carbon isotope delta values are -31.30 ± 0.06‰ (VANA-1) and -25.85 ± 0.05‰ (VANB-1). These uncertainties, for the 95% confidence level, include considerations for measurement uncertainty, coherence of the reference materials used for calibration, batch homogeneity, and stability during storage and transportation. The results are traceable to the VPDB through a set of nine reference materials (IAEA-CH-6, USGS65, IAEA-600, NBS22, USGS61, IAEA-603, IAEA-610, IAEA-611, and IAEA-612). For up to date certified values, users should refer to doi.org/10.4224/crm.2022.vana-1 and doi.org/10.4224/crm.2022.vanb-1.
This study aims to investigate the influence of copper(II) ions as a cofactor on the electrochemical performance of a biocomposite consisting of a mini protein mimicking uricase (mp20) and zeolitic immidazolate framework-8 (ZIF-8) for the detection of uric acid. A central composite design (CCD) was utilized to optimize the independent investigation, including pH, deposition potential, and deposition time, while the current response resulting from the electrocatalytic oxidation of uric acid was used as the response. The statistical analysis of variance (ANOVA) showed a good correlation between the experimental and predicted data, with a residual standard error percentage (RSE%) of less than 2% for predicting optimal conditions. The synergistic effect of the nanoporous ZIF-8 host, Cu(II)-activated mp20, and reduced graphene oxide (rGO) layer resulted in a highly sensitive biosensor with a limit of detection (LOD) of 0.21 μM and a reproducibility of the response (RSD = 0.63%). The Cu(II)-activated mp20@ZIF-8/rGO/SPCE was highly selective in the presence of common interferents, and the fabricated layer exhibited remarkable stability with signal changes below 4.15% after 60 days. The biosensor's reliable performance was confirmed through real sample analyses of human serum and urine, with comparable recovery values to conventional HPLC.
Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent inflammation of the sinonasal mucosa. CRSwNP treatments are associated with inconsistent efficacy and recurrence of symptoms. Dynorphin 1-17 (DYN 1-17) and its fragments have been shown to modulate the immune response in various inflammatory conditions. This study aimed to investigate the effect of different pH and degrees of inflammation on DYN 1-17 metabolism in human CRSwNP tissues. DYN 1-17 was incubated with grade 3 and grade 4 inflamed tissues of CRSwNP patients at pH 5.5 and pH 7.4 over a range of incubation periods. The resulting fragments were identified using an ultra-performance liquid chromatography (UPLC) system coupled to quadrupole-time of flight (QTOF) mass spectrometry based on their accurate mass. The rate of DYN 1-17 fragmentation was slower at pH 5.5 in comparison to pH 7.4. The extent and rate of metabolism of DYN 1-17 were much lower in grade 3 inflamed tissue (31-32 fragments) than in grade 4 (34-41 fragments). N-Terminal fragments (DYN 1-15, 1-11, 1-10, and 1-6) were metabolized slower at pH 5.5 as compared to pH 7.4. DYN 1-12, 1-8, 2-10, 4-10, 5-10, and 8-14 were only observed under the inflammatory pH while DYN 5-17 and 6-17 were only identified upon incubation with grade 4 CRSwNP tissues. DYN 1-17 metabolism was significantly affected by the pH level and the severity of the inflammation of CRSwNP tissues, indicating the potential roles of DYN 1-17 and its fragments in modulating the inflammatory response and their avenue as therapeutics in future studies.