定量检测动物和人体小体积血样中的循环短链脂肪酸。

Quantitation of circulating short-chain fatty acids in small volume blood samples from animals and humans.

机构信息

Chalmers University of Technology, Department of Life Sciences, Division of Food and Nutrition Science, Gothenburg, Sweden.

出版信息

Talanta. 2024 May 15;272:125743. doi: 10.1016/j.talanta.2024.125743. Epub 2024 Feb 3.

DOI:10.1016/j.talanta.2024.125743

PMID:38382298

Abstract

BACKGROUND

The role of gut microbiota in human health has been intensively studied and more recently shifted from emphasis on composition towards function. Function is partly mediated through formed metabolites. Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate as well as their branched analogues represent major products from gut fermentation of dietary fibre and proteins, respectively. Robust and high-throughput analysis of SCFAs in small volume blood samples have proven difficult. Major obstacles come from the ubiquitous presence of SCFAs that leads to contaminations and unstable analytical results because of the high volatility of these small molecules. Comprehensive and comparable data on the variation of SCFAs in blood samples from different blood matrices and mammal species including humans is lacking. Therefore, our aim was to develop and evaluate a stable and robust method for quantitation of 8 SCFAs and related fermentation products in small volume blood plasma samples and to investigate their variation in humans and different animal species.

RESULTS

Derivatization was a successful approach for measurement of SCFAs in biological samples but quenching of the derivatization reaction was crucial to obtain long-term stability of the derivatized analytes. In total 9 compounds (including succinic acid) were separated in 5 min. The method was linear over the range 0.6-3200 nM formic (FA), acetic (AA), 0.3-1600 nM propionic (PA), and 0.16-800 nM for butyric (BA)-, isobutyric (IBA)-, valeric (VA)-, isovaleric (IVA)-, succinic (SA) and caproic acid (CA). The precision ranged ≤12 % within days and ≤28 % between days (except for CA and VA) in three different plasma quality control (QC) samples (29 batches analyzed over 3 months). The extraction recovery was on average 94 % for the different SCFAs. Typical interquartile range (IQR) concentrations (μM) of SCFAs in human plasma samples were 168 μM (FA), 64 μM (AA), 2.2 μM (PA), 0.54 μM (BA), 0.66 μM (IBA), 0.18 μM (VA), 0.40 μM (IVA), and 0.34 μM (CA). In total, 55 samples per batch/day were successfully analyzed and in total 5380 human plasma samples measured over a 3-year timespan.

SIGNIFICANCE

The developed UHPLC-MS based method was suitable for measuring SCFAs in small blood volume samples and enabled robust quantitative data.

摘要

背景

肠道微生物群在人类健康中的作用已被深入研究，最近的研究重点已从组成转向功能。功能部分通过形成代谢物来介导。短链脂肪酸（SCFAs），如乙酸盐、丙酸盐和丁酸盐，以及它们的支链类似物，分别代表膳食纤维和蛋白质肠道发酵的主要产物。在小体积的血液样本中，对 SCFAs 进行稳健且高通量的分析一直具有挑战性。主要障碍来自无处不在的 SCFAs，这会导致污染和由于这些小分子的高挥发性而导致不稳定的分析结果。关于不同血液基质和哺乳动物物种（包括人类）血液样本中 SCFAs 变化的综合和可比数据仍然缺乏。因此，我们的目标是开发和评估一种稳定且强大的方法，用于定量分析小体积血浆样本中 8 种 SCFAs 和相关发酵产物，并研究它们在人类和不同动物物种中的变化。

结果

衍生化是一种成功的测量生物样本中 SCFAs 的方法，但衍生化反应的淬灭对于获得衍生化分析物的长期稳定性至关重要。总共分离出 9 种化合物（包括琥珀酸），在 5 分钟内完成。该方法在 0.6-3200 nM 甲酸（FA）、乙酸（AA）、0.3-1600 nM 丙酸（PA）和 0.16-800 nM 丁酸（BA）、异丁酸（IBA）、戊酸（VA）、异戊酸（IVA）、琥珀酸（SA）和己酸（CA）的范围内呈线性。在三个不同的血浆质量控制（QC）样本（3 个月内分析了 29 批）中，日内精度≤12%，日间精度≤28%（除 CA 和 VA 外）。不同 SCFAs 的提取回收率平均为 94%。人血浆样本中 SCFAs 的典型四分位间距（IQR）浓度（μM）为 168 μM（FA）、64 μM（AA）、2.2 μM（PA）、0.54 μM（BA）、0.66 μM（IBA）、0.18 μM（VA）、0.40 μM（IVA）和 0.34 μM（CA）。每个批次/天成功分析了 55 个样本，在 3 年的时间内共测量了 5380 个人血浆样本。