RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2004; 18: 2593–2600 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1662 A streamlined approach to the analysis of volatile fatty acids and its application to the measurement of whole-body flux Douglas J. Morrison 1,2 *, Karen Cooper 2 , Susan Waldron 2 , Christine Slater 1 , Lawrence T. Weaver 1 and Tom Preston 2 1 Division of Developmental Medicine, University of Glasgow, Royal Hospital for Sick Children, Yorkhill, Glasgow G3 8SJ, UK 2 Scottish Universities Environmental Research Centre, Rankine Avenue, East Kilbride G75 0QF, UK Received 5 July 2004; Revised 1 September 2004; Accepted 1 September 2004 Volatile fatty acids (VFAs) are produced in the human colon by the bacterial breakdown of carbo- hydrates that escape digestion and absorption in the small intestine. They have important local and systemic effects on gastrointestinal and nutritional functions. Measuring their production is diffi- cult because of inaccessibility of sampling sites and low circulating concentrations. Stable isotope tracer techniques are a way to measure VFA production but require measurement of isotope dilu- tion in blood and other biological fluids. We have developed a streamlined and robust method to measure the concentration and enrichment of [ 2 H]-labelled VFAs by gas chromatography/mass spectrometry (GC/MS) and [ 13 C]-labelled VFAs by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Both types of analysis were carried out on the same samples allowing multiple tracer studies to be conducted. Good accuracy and repeatability were found for GC/MS analysis of [ 2 H]-labelled VFAs. Careful handling of the background contribution, especially acetate, allowed quantitation of concentration and enrichment within the analysis. GC/C/IRMS analysis of [ 13 C] VFAs was also achieved with good accuracy and repeatability. This methodology was used to determine whole-body acetate production in two subjects using multiple tracers ([ 2 H 3 ]- and [1- 13 C]acetate) and blood and urine sampling. Whole-body acetate flux was similar when measured either with [ 2 H 3 ]- or [1- 13 C]acetate, and when flux was determined from plasma or urine tracer enrichment. This new method will permit rapid and accurate measurement of VFA flux using [ 2 H]- and/or [ 13 C]-labelled VFAs as tracers. Measurements of the contribution of colonic VFA production to whole-body VFA flux are now possible. Copyright # 2004 John Wiley & Sons, Ltd. Volatile fatty acids (VFAs) are important biological products in the human colon and in the rumen of farm animals. They refer to linear fatty acids of up to 4 – 6 carbons in chain length. Short-chain fatty acids (SCFAs) refer to broadly similar fatty acids in human nutrition, up to 6 carbons in length. Both defi- nitions refer primarily to the acids, acetic (ethanoic), propio- nic (propanoic) and butyric (butanoic), because they are the most abundant substrates of physiological significance, although lactic acid (2-hydroxypropionic acid) can be more abundant in the colon of infants. 1 In addition to dietary sources, exogenous VFAs are produced by the bacterial degradation of carbohydrates and proteins that escape diges- tion and absorption in the small intestine. 2 This fermentation process is carried out by the predominantly anaerobic bacter- ial flora in the colon. The main substrates for fermentation are dietary fibre, resistant starches, non-digestible oligosacchar- ides, plant cell and cell wall remnants—collectively referred to as non-digestible carbohydrates (NDC). Recent evidence suggests that VFAs produced in the colon have several local and systemic effects. Butyric acid and, to a lesser extent, propionic and acetic acids, are oxidised by the colonocytes and account for the majority of their energy needs. 3 Butyric acid is also a key mediator in the inflamma- tory process in the large intestine. 4 VFAs produced from fermentation stimulate water absorption and mineral uptake and therefore may prevent mineral deficiency 5 and hasten recovery from diarrhoea. 6 Acetic and propionic acids are mostly absorbed across the colonic epithelium and trans- ported to the liver. A substantial fraction of exogenous acetate production escapes hepatic metabolism but little propionate appears in the circulation. 7 Acetate can be used as an energy source and induces an insulin response, 8 while propionate may reduce hepatic cholesterol synthesis. 9 The optimum VFA production to maintain health has not been determined, Copyright # 2004 John Wiley & Sons, Ltd. *Correspondence to: D. J. Morrison, Division of Developmental Medicine, Child Health Section, University of Glasgow, Royal Hospital for Sick Children, Yorkhill, Glasgow G3 8SJ, UK. E-mail: douglas.morrison@clinmed.gla.ac.uk Contract/grant sponsor: Mars Nutrition Research Council. Contract/grant sponsor: Commission of the European Commu- nities; contract/grant number: QLK 1-2001-00431.