Department of Chemistry, Faculty of Science, University of South Bohemia in České Budějovice, České Budějovice, Czech Republic.
Prague Zoo, Prague, Czech Republic.
PLoS One. 2022 Jul 14;17(7):e0271444. doi: 10.1371/journal.pone.0271444. eCollection 2022.
Obligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, suggesting that levels of B vitamins may vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood can provide valuable informative background and understanding of general principles of insect symbiosis. In this work, a chromatographic separation of eight B vitamins (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folic acid, and cyanocobalamine), four B vitamin derivatives (niacinamide, pyridoxal-5-phosphate, 4-pyridoxic acid, and tetrahydrofolic acid), and 3 stable isotope labelled internal standards was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Except for vitamin B9 (tetrahydrofolic acid), the instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 μg/L, correlation coefficients from 0.9997 to 1.0000, and QC coefficients from 0.53 to 3.2%. Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. The best results were obtained for zinc sulphate in methanol, but only nine analytes were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120%, precision from 0.5 to 13%, and process efficiency from 65 to 108%. The content of B vitamins in whole blood samples from human and various vertebrates is presented as an application example of this newly developed method.
专性共生细菌与专门以脊椎动物血液为食的昆虫有关，据推测，这些共生细菌可以补充其饮食中缺乏的 B 族维生素。最近的基因组分析显示，不同共生体的生物合成能力存在很大差异，这表明不同脊椎动物宿主的 B 族维生素水平可能有所不同。然而，尚未对各种脊椎动物血液中的 B 族维生素含量进行严格测定。一种针对血液中 B 族维生素复合物的可靠分析方法可以为昆虫共生的一般原理提供有价值的信息背景和理解。在这项工作中，开发了一种色谱分离法，可分离八种 B 族维生素（硫胺素、核黄素、烟酸、泛酸、吡哆醇、生物素、叶酸和氰钴胺素）、四种 B 族维生素衍生物（烟酰胺、吡哆醛-5-磷酸、4-吡啶酮酸和四氢叶酸）以及 3 种稳定同位素标记的内标。使用双重压力线性离子阱质谱仪在 FullScan MS/MS 和 SIM 模式下进行检测。除了维生素 B9（四氢叶酸）之外，所有分析物的仪器定量限均在 0.42 至 5.0 μg/L 之间，相关系数在 0.9997 至 1.0000 之间，QC 系数在 0.53 至 3.2%之间。全血样品制备步骤的优化特别侧重于评估两种类型的蛋白质沉淀剂：三氯乙酸和甲醇中的硫酸锌。甲醇中的硫酸锌效果最佳，但只有九种分析物成功得到验证。使用这种蛋白质沉淀剂的程序的准确性在 89%至 120%之间，精密度在 0.5%至 13%之间，处理效率在 65%至 108%之间。作为这种新方法的应用实例，呈现了来自人类和各种脊椎动物的全血样本中 B 族维生素的含量。
