唾液短链脂肪酸和羟基酸的变化(例如,乳酸,和3-羟基丁酸)水平已被建议反映人类肠道微生物群的生态失调,这代表了与心力衰竭(HF)疾病发作有关的另一个因素。这些代谢物的物理化学性质与生物基质的复杂组成相结合意味着样品预处理程序几乎是不可避免的。这项工作描述了一个可靠的,简单,和不含有机溶剂的方案,用于测定从心力衰竭收集的刺激唾液样本中的短链脂肪酸和羟基酸,肥胖,和高血压患者。该程序基于原位五氟苄基溴(PFB-Br)衍生化和HiSorb吸附萃取以及热解吸和气相色谱-串联质谱法。HiSorb提取装置与水性基质完全兼容,从而节省与有机溶剂萃取方法相关的时间和材料。中心复合面为中心的实验设计用于优化PFB-Br与目标分析物之间的摩尔比,衍生化温度,反应时间为100,60℃,180分钟,分别。使用少量唾液(20μL)达到0.1-100μM的检测限。使用乙酸钠-1-13C作为内标提高了该方法的日内和日间精度,范围为10%至23%。优化的方案被成功地应用于我们认为是第一次评估四组患者唾液样本中短链脂肪酸和羟基酸的唾液水平:i)入院的患者患有急性HF症状,ii)患有慢性HF症状的患者,iii)没有HF症状但肥胖的患者,和iv)没有HF症状但有高血压的患者。第一组患者入院时唾液乙酸和乳酸水平明显较高,己酸和庚酸的含量最低。此外,显著高水平的乙酸,丙酸,与其他患者相比,在HF中观察到的丁酸和丁酸表明口腔细菌与肠道菌群失调之间存在潜在联系。
Variations in salivary short-chain fatty acids and hydroxy acids (e.g., lactic acid, and 3-hydroxybutyric acid) levels have been suggested to reflect the dysbiosis of human gut microbiota, which represents an additional factor involved in the onset of heart failure (HF) disease. The physical-chemical properties of these metabolites combined with the complex composition of biological matrices mean that sample pre-treatment procedures are almost unavoidable. This work describes a reliable, simple, and organic solvent free protocol for determining short-chain fatty acids and hydroxy acids in stimulated saliva samples collected from heart failure, obese, and hypertensive patients. The procedure is based on in-situ pentafluorobenzyl bromide (PFB-Br) derivatization and HiSorb sorptive extraction coupled to thermal desorption and gas chromatography-tandem mass spectrometry. The HiSorb extraction device is completely compatible with aqueous matrices, thus saving on time and materials associated with organic solvent-extraction methods. A Central Composite Face-Centred experimental design was used for the optimization of the molar ratio between PFB-Br and target analytes, the derivatization temperature, and the reaction time which were 100, 60 °C, and 180 min, respectively. Detection limits in the range 0.1-100 µM were reached using a small amount of saliva (20 µL). The use of sodium acetate-1-13C as an internal standard improved the intra- and inter-day precision of the method which ranged from 10 to 23%. The optimized protocol was successfully applied for what we believe is the first time to evaluate the salivary levels of short chain fatty acids and hydroxy acids in saliva samples of four groups of patients: i) patients admitted to hospital with acute HF symptoms, ii) patients with chronic HF symptoms, iii) patients without HF symptoms but with obesity, and iv) patients without HF symptoms but with hypertension. The first group of patients showed significantly higher levels of salivary acetic acid and lactic acid at hospital admission as well as the lowest values of hexanoic acid and heptanoic acid. Moreover, the significant high levels of acetic acid, propionic acid, and butyric acid observed in HF respect to the other patients suggest the potential link between oral bacteria and gut dysbiosis.