Community Research and Development Information Service - CORDIS

Availability of technique to measure 13C-short chain fatty acids in blood

Colonic fermentation of dietary fibres, non digestible oligosaccharides or resistant starches by the bacterial microflora results in a production of different organic acids and mainly short chain fatty acids (acetate, propionate and butyrate). These short chain fatty acids are mostly absorbed and partly metabolized in the colonic epithelium and the liver. As a consequence they appear at very low concentration in the stools but can be quantified in the peripheral blood before their captation by different organs. Several methods (long and tedious) are available to quantify SCFAs in the plasma but a precise quantification of acetate (the main SCFA) remains very difficult.

Stable isotope techniques are increasingly used to understand metabolism of SCFAs in human. GC/MS is a powerful analytical tool for the stable isotope studies but requires high isotopic enrichments of 13C-labelled SCFA (> 1MPE) in the plasma sample. To measure low isotopic enrichments of 13C-SCFA (or natural abundance < 1 MPE), we have developed a method in GC-C-IRMS using a Solid Phase Micro-Extraction (SPME). After removal of plasma proteins, the SPME fiber was plunged in the liquid sample during 40 min at 40°C.

Then, acetate was directly desorbed into GC-C-IRMS. The accuracy of isotopic enrichment measurement was determined using plasma spiked with 13C-acetate and 13C-butyrate solution from 0 to 1 Mol Percent Excess (MPE). Good linearity and repeatability (RSD < 5%) were obtained for acetate and butyrate. Plasma acetate, propionate and butyrate concentrations were also determined relative to 3-Methylvalerate (Internal Standard). Good linearity and repeatability were observed from 0 to 400 µM for acetate, from 0 to 20 µM for propionate and from 0 to 10 µM for butyrate.

This method was applied to determine plasma acetate production obtained from the lactulose fermentation in one healthy volunteer over 3 hours.. Acetate concentration was increased twofold, 2 hours after oral lactulose intake. These results are in agreement with those measured by GC/MS in healthy and overweight adults following a lactulose intake by using higher amount of labelled tracers. Natural abundance of 13C-acetate was of 29.93 +/- 0.78 0 (mean n=8) at basal state and 25.87 +/- 0.80 0 (mean n=8) during the production of lactulose. These results illustrate the accuracy of the SPME/GC-C-IRMS method.

In conclusion, the combination of SPME in liquid sampling mode with the GC-C-IRMS allows to measure very low enrichments (in the range of natural abundance carbon isotope) of VFAs in human plasma within physiological concentrations. This new method is now used to study the colonic fermentation of 13C-starch in an integrated protocol into human volunteers in collaboration with other Eurostarch partners (HNRC-Nantes, U. Glasgow, RUG, KULRD).

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44093 Nantes
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