Phospholipids in the central nervous system are enriched in n-3 and n-6 polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and arachidonic acid (ARA). These PUFA can undergo enzymatic reactions to produce lipid mediators, as well as reaction with oxygen free radicals to produce 4-hydroxyhexenal (4-HHE) from DHA and 4-hydroxynonenal (4-HNE) from ARA. Recent studies demonstrated pleiotropic properties of these peroxidation products through interaction with oxidative and anti-oxidant response pathways. In this study, BV-2 microglial cells were used to investigate ability for DHA, 4-HHE, and 4-HNE to stimulate the anti-oxidant stress responses involving the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway and synthesis of heme oxygenase (HO-1), as well as to mitigate lipopolysaccharide (LPS)-induced nitric oxide (NO), reactive oxygen species (ROS), and cytosolic phospholipase A2 (cPLA2). In addition, LC-MS/MS analysis was carried out to examine effects of exogenous DHA and LPS stimulation on endogenous 4-HHE and 4-HNE levels in BV-2 microglial cells. Effects of DHA, 4-HHE, and 4-HNE on LPS-induced NO production was determined using the Griess reagent. LPS-induced ROS production was measured using CM-H2DCFDA. Western blots were used to analyze expression of p-cPLA2, Nrf2, and HO-1. Cell viability and cytotoxicity were measured using the WST-1 assay, and cell protein concentrations were measured using the BCA protein assay kit. An ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to determine levels of free 4-HHE and 4-HNE in cells. DHA (12.5–100 μM), 4-HHE (1.25–10 μM), and 4-HNE (1.25–10 μM) dose dependently suppressed LPS-induced production of NO, ROS, and as p-cPLA2 in BV-2 microglial cells. With the same concentrations, these compounds could enhance Nrf2 and HO-1 expression in these cells. Based on the estimated IC50 values, 4-HHE and 4-HNE were five- to tenfold more potent than DHA in inhibiting LPS-induced NO, ROS, and p-cPLA2. LC-MS/MS analysis indicated ability for DHA (10–50 μM) to increase levels of 4-HHE and attenuate levels of 4-HNE in BV-2 microglial cells. Stimulation of cells with LPS caused an increase in 4-HNE which could be abrogated by cPLA2 inhibitor. In contrast, bromoenol lactone (BEL), a specific inhibitor for the Ca2+-independent phospholipase A2 (iPLA2), could only partially suppress levels of 4-HHE induced by DHA or DHA + LPS. This study demonstrated the ability of DHA and its lipid peroxidation products, namely, 4-HHE and 4-HNE at 1.25–10 μM, to enhance Nrf2/HO-1 and mitigate LPS-induced NO, ROS, and p-cPLA2 in BV-2 microglial cells. In addition, LC-MS/MS analysis of the levels of 4-HHE and 4-HNE in microglial cells demonstrates that increases in production of 4-HHE from DHA and 4-HNE from LPS are mediated by different mechanisms.

Cyanogenic glycosides (CNGs) are naturally occurring plant molecules (nitrogenous plant secondary metabolites) which consist of an aglycone and a sugar moiety. Hydrogen cyanide (HCN) is released from these compounds following enzymatic hydrolysis causing potential toxicity issues. The presence of CNGs in American elderberry (AE) fruit, Sambucus nigra (subsp. canadensis), is uncertain. A sensitive, reproducible and robust LC-MS/MS method was developed and optimized for accurate identification and quantification of the intact glycoside. A complimentary picrate paper test method was modified to determine the total cyanogenic potential (TCP). TCP analysis was performed using a camera-phone and UV-Vis spectrophotometry. A method validation was conducted and the developed methods were successfully applied to the assessment of TCP and quantification of intact CNGs in different tissues of AE samples. Results showed no quantifiable trace of CNGs in commercial AE juice. Levels of CNGs found in various fruit tissues of AE cultivars studied ranged from between 0.12 and 6.38 µg/g. In pressed juice samples, the concentration range measured was 0.29–2.36 µg/mL and in seeds the levels were 0.12–2.38 µg/g. TCP was highest in the stems and green berries. Concentration levels in all tissues were generally low and at a level that poses no threat to consumers of fresh and processed AE products.

Phospholipids are major components in the lipid bilayer of cell membranes. These molecules are comprised of two acyl or alkyl groups and different phospho-base groups linked to the glycerol backbone. Over the years, substantial interest has focused on metabolism of phospholipids by phospholipases and the role of their metabolic products in mediating cell functions. The high levels of polyunsaturated fatty acids (PUFA) in the central nervous system (CNS) have led to studies centered on phospholipases A2 (PLA2s), enzymes responsible for cleaving the acyl groups at the sn-2 position of the phospholipids and resulting in production of PUFA and lysophospholipids. Among the many subtypes of PLA2s, studies have centered on three major types of PLA2s, namely, the calcium-dependent cytosolic cPLA2, the calcium-independent iPLA2 and the secretory sPLA2. These PLA2s are different in their molecular structures, cellular localization and, thus, production of lipid mediators with diverse functions. In the past, studies on specific role of PLA2 on cells in the CNS are limited, partly because of the complex cellular make-up of the nervous tissue. However, understanding of the molecular actions of these PLA2s have improved with recent advances in techniques for separation and isolation of specific cell types in the brain tissue as well as development of sensitive molecular tools for analyses of proteins and lipids. A major goal here is to summarize recent studies on the characteristics and dynamic roles of the three major types of PLA2s and their oxidative products towards brain health and neurological disorders.

The presence of phenolic compounds in honey can serve as potential authenticity markers for honey’s botanical or geographical origins. The composition and properties of honey can vary greatly depending on the floral and geographical origins. This study focuses on identifying the specific markers that can distinguish honey based on their geographical areas in the United States. The main approach presented in this study to identify the geographic origins of honey involves chemometric methods combined with phenolic compound fingerprinting. Sample clean-up and phenolic compound extraction was carried out using solid phase extraction (SPE). Reversed phase liquid chromatography in combination with tandem mass spectrometry were utilized for the separation of the compounds. The honey physicochemical qualities were predominantly determined via spectrophotometric methods. Multivariate statistical tools such as principal component analysis (PCA), analysis of variance (ANOVA), and partial-least squares discriminant analysis (PLS-DA) were employed as both classification and feature selection tools. Overall, the present study was able to identify the presence of 12 potential markers to differentiate the honey’s geographical origins. The total phenolic content ranged from 81.6 to 105.7 mg GAE/100 g corresponding to honey from Colorado and Washington, respectively (GAE: gallic acid equivalents). The regression analysis shows a tendency for the total phenolic content of honey to increase as the color of honey increases. The most important result obtained in this study is the demonstration that the geographical origin of honey plays a critical role in predicting the physical properties and phenolic composition of honey.

Background: Nitric oxide (NO) is a signaling molecule regulating numerous cellular functions in development and disease. In the brain, neuronal injury or neuroinflammation can lead to microglial activation, which induces NO production. NO can react with critical cysteine thiols of target proteins forming S-nitroso-proteins. This modification, known as S-nitrosylation, is an evolutionarily conserved redox-based post-translational modification (PTM) of specific proteins analogous to phosphorylation. In this study, we describe a protocol for analyzing S-nitrosylation of proteins using a gel-based proteomic approach and use it to investigate the modes of action of a botanical compound found in green tea, epigallocatechin-3-gallate (EGCG), on protein S-nitrosylation after microglial activation. Methods/Results: To globally and quantitatively analyze NO-induced protein S-nitrosylation, the sensitive gel-based proteomic method, termed NitroDIGE, was developed by combining two-dimensional differential in-gel electrophoresis (2-D DIGE) with the modified biotin switch technique (BST) using fluorescence-tagged CyDye™ thiol reactive agents to label S-nitrosothiols. The NitroDIGE method showed high specificity and sensitivity in detecting S-nitrosylated proteins (SNO-proteins). Using this approach, we identified a subset of SNO-proteins ex vivo by exposing immortalized murine BV-2 microglial cells to a physiological NO donor, or in vivo by exposing BV-2 cells to endotoxin lipopolysaccharides (LPS) to induce a proinflammatory response. Moreover, EGCG was shown to attenuate S-nitrosylation of proteins after LPS-induced activation of microglial cells primarily by modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response. Conclusions: These results demonstrate that NitroDIGE is an effective proteomic strategy for “top-down” quantitative analysis of protein S-nitrosylation in multi-group samples in response to nitrosative stress due to excessive generation of NO in cells. Using this approach, we have revealed the ability of EGCG to down-regulate protein S-nitrosylation in LPS-stimulated BV-2 microglial cells, consistent with its known antioxidant effects.