Quantification of Plasma or Serum Short-Chain Fatty Acids: Choosing the Correct Blood Tube Lise Deroover 1 , Eef Boets 1 , Yaxin Tie 1 , Greet Vandermeulen 1 and Kristin Verbeke *1,2 1 Translational Research in Gastrointestinal Disorders, KU Leuven, Leuven, Belgium 2 Leuven Food Science and Nutrition Research Centre, KU Leuven, Leuven, Belgium Journal of Nutritional Health & Food Science Open Access Research Article Abstract Background: Short-Chain Fatty Acids (SCFA; acetate, propionate and butyrate) are more and more recognised as mediators of local gut and systemic health. Quantification of SCFA in plasma and serum is challenging due to their low concentrations in human blood and the ubiquitous nature of acetate, requiring careful standardisation of the sample preparation procedure. Also the choice of the blood tube might affect the resulting concentrations. Methods: SCFA concentrations were measured in blood samples (10 mL), collected from 10 healthy subjects in 7 different blood tubes. Control samples included milliQ (MQ) water and standard SCFA solutions. After preconcentration and clean-up of the samples using a hollow fiber liquid membrane extraction, SCFA concentrations were measured using Gas Chromatography (GC) coupled to Flame Ionisation Detection (FID). Results: Acetate concentrations were significantly higher (ANOVA, p<0.01) when blood was collected in an EDTA K2 tube, where as propionate and/or butyrate levels were significantly higher in plasma prepared in a PST tube and a Barricor tube and serum prepared in a SST tube (ANOVA, p<0.01 for all three tubes). Similar profiles of contamination were observed when analysing standard SCFA solutions that had been centrifuged in the different blood tubes. Lowest levels of contamination were observed when using red top glass serum tubes. Conclusions: A red top glass serum tube is the preferred tube to collect blood for the quantification of SCFA. When plasma is preferred over serum, a lithium heparin tube is the most appropriate test tube. Keywords: plasma/serum short-chain fatty acids; blood tube; polyacrylamide gel; gas chromatography; flame ionisation detection. Received: July 12, 2017; Accepted: September 12, 2017; Published: November 11, 2017 *Corresponding author: Kristin Verbeke, Translational Research in Gastrointestinal Disorders, KU Leuven, Herestraat 49 Box 701, Leuven 3000, Belgium, Tel: +32 16 33 01 50; Fax: +32 16 33 07 23; E-mail: kristin.verbeke@kuleuven.be Symbiosis www.symbiosisonline.org www.symbiosisonlinepublishing.com Symbiosis Group * Corresponding author email: kristin.verbeke@kuleuven.be Introduction Short-Chain Fatty Acids (SCFA; acetate, propionate and butyrate) are mainly produced by bacterial fermentation of undigested carbohydrates in the human colon [1]. They are considered as signaling molecules that provide a link between the diet, the microbiota and the host and mediate health benefits locally in the gut and on the systemic level [2,3]. Colonic-derived SCFA have been suggested to be involved in the prevention and treatment of metabolic syndrome, obesity, inflammatory bowel diseases and colon cancer [4-8]. However, these beneficial effects have mainly been demonstrated in in vitro experiments and animal models and need to be confirmed in human studies. The molecular mechanisms by which SCFA induce these effects are not completely understood and are the subject of many research. The fact that SCFA, in particular butyrate and to a lesser extent propionate inhibit histone deacetylases and in this way impact on the expression of genes with functions including cell proliferation, inflammation and apoptosis, may explain at least part of their effect [9]. In addition, SCFA are endogenous ligands for the G-protein coupled receptors 43 and 41 that have been found in the intestinal tract but also in adipose tissue, on immune cells, spleen and muscle cells. Activation of these receptors may be involved is the regulation of lipid and glucose metabolism [10]. Accurate quantification of SCFA in plasma and serum is essential to understand their role in mediating systemic effects. Colonic luminal SCFA concentrations, as well as fecal SCFA concentrations, are in the millimolar range and are relatively easy to quantify using gas chromatography after extraction into an organic solvent [11,12]. However, colonic SCFA are largely absorbed into the colonocytes where a part of the SCFA is used as an energy source and is immediately oxidized. The remaining SCFA are transported to the liver via the portal circulation where another (unknown) fraction is metabolized. Only the SCFA that pass the liver and escape splanchnic extraction end up in the peripheral circulation. As a result SCFA concentrations in plasma and serum are considerably lower than in the lumen of the colon and range between 50-100 µmol/L for acetate and 0.5-10 µmol/L for propionate and butyrate [13,14]. Because of these low concentrations, accurate analytical techniques are required. The precision of the measurements is further complicated by the ubiquitous nature of acetate, easily resulting in contaminations, which requires careful standardization of each step in the sample preparation [15]. In addition, the choice of blood collection tube may be important, as different blood tubes contain different additives, which may affect plasma and serum SCFA concentrations. For that reason, we investigated the influence of different blood collection tubes on plasma and serum SCFA concentrations.