LC-MS/MS method development for quantitative analysis of cyanogenic glycosides in elderberry and lipid peroxidation products in mice
Authors:   , Appenteng Michael Kwame 
Published: August 2021

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 0.12-6.38 [micro-gram]/g. In pressed juice samples, the concentration range measured was 0.29-2.36 [micro-gram]/mL and in seeds the amounts was 0.12-2.38 [micro-gram]/g. TCP was highest in the stems and green berries. CNG levels in all tissues were generally low and at a level that poses no threat to consumers of fresh and processed AE products. The abundance of docosahexaenoic acid (DHA) in phospholipids in the brain and retina has generated interest to search for its role in mediating neurological functions. Besides the source of many oxylipins with pro-resolving properties, DHA also undergoes peroxidation, producing 4-hydroxyhexenal (4-HHE), although its function remains elusive. Despite wide dietary consumption, whether supplementation of DHA may alter the peroxidation products and their relationship to phospholipid species in brain and other body organs have not been explored sufficiently. In this study, adult mice were administered a control or DHA-enriched diet for three weeks, and phospholipid species and peroxidation products were examined in brain, heart and plasma. Results demonstrated that this dietary regimen increased (n-3) and decreased (n-6) species to different extent in all major phospholipid classes (PC, dPE, PE-pl, PI and PS) examined. Besides changes in phospholipid species, DHA-enriched diet also showed substantial increases in 4-HHE in brain, heart and plasma. Among different brain regions, the hippocampus responded to the DHA-enriched diet showing significant increase in 4-HHE. Considering the pro- and anti-inflammatory pathways mediated by the (n-6) and (n-3) polyunsaturated fatty acids, unveiling the ability for DHA-enriched diet to alter phospholipid species and lipid peroxidation products in the brain and in different body organs may be an important step forward towards understanding the mechanism(s) for this (n-3) fatty acid on health and diseases. Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted social communication and repetitive behaviors. Prenatal stress is critical in neurodevelopment and increases risk for ASD, particularly in those with greater genetic susceptibility to stress. Docosahexaenoic acid (DHA) is one of the most abundant [omega]-3 fatty acids in mammalian brain, and dietary [omega]-3 fatty acid affects the development and maintenance of brain structure. We investigated whether prenatal supplementation of DHA alleviates autistic-like behaviors in a gene/stress mouse model and how it alters lipid peroxidation activity in the brain. Pregnant heterozygous serotonin transporter knockout (SERT-KO) and wild-type (WT) dams were placed in either non-stressed control conditions or chronic variable stress conditions and fed either a control diet or a DHA-rich (1 [percent] by wt) diet. Offspring of each group were assessed for anxiety and autism-associated behavior at post-natal day 60, including an open field test, elevatedplus maze test, repetitive behavior, and the 3-chamber social approach test. Our LC-MS-based method was used to follow changes in peroxidation product concentrations in mouse plasma, heart, and cerebral cortex.