Comprehensive SCFA Profiling in Fecal Samples

A panel of 5 straight-chain SCFAs (acetate, propionate, butyrate, valerate, caproate) and 4 branched-chain SCFAs (isobutyrate, 2-methylbutyrate, isovalerate, 4-methylvalerate) were assessed by LC-MS, as we described previously [39 (link)]. In brief, fecal samples were homogenized in 1:1 acetonitrile/water using a Bead Mill 24 Homogenizer (Fisherbrand, Waltham, MA, USA) in tubes containing 1.0 mm zirconia/silica beads (ThermoFisher Scientific) and centrifuged (15,000× g, 10 min, 10 °C). The supernatant was mixed with internal standard (¹³C₄-butyrate; Cambridge Isotopes Laboratories) and derivatized with 3-nitrophenylhydrazine to convert SCFAs to their corresponding 3-nitrophenylhydrazones prior to LC-MS analysis.

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Pokala A., Quarles W.R., Ortega-Anaya J., Jimenez-Flores R., Cao S., Zeng M., Hodges J.K, & Bruno R.S. (2023). Milk-Fat-Globule-Membrane-Enriched Dairy Milk Compared with a Soy-Lecithin-Enriched Beverage Did Not Adversely Affect Endotoxemia or Biomarkers of Gut Barrier Function and Cardiometabolic Risk in Adults with Metabolic Syndrome: A Randomized Controlled Crossover Trial. Nutrients, 15(14), 3259.

Publication 2023

Bead Mill 24 Homogenizer zirconia/silica beads

2 methylbutyrate Acetate Acetonitrile Butyrate Caproate Fecal Isotopes Propionate Silica Valerate Zirconia

Corresponding Organization : The Ohio State University

Other organizations : Pennsylvania State University

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Variable analysis

independent variables

Panel of 5 straight-chain SCFAs (acetate, propionate, butyrate, valerate, caproate)
Panel of 4 branched-chain SCFAs (isobutyrate, 2-methylbutyrate, isovalerate, 4-methylvalerate)

dependent variables

SCFA concentrations in fecal samples as measured by LC-MS

control variables

Homogenization of fecal samples in 1:1 acetonitrile/water using a Bead Mill 24 Homogenizer
Centrifugation of samples (15,000× g, 10 min, 10 °C)
Addition of internal standard (13C4-butyrate; Cambridge Isotopes Laboratories)
Derivatization of SCFAs with 3-nitrophenylhydrazine to form 3-nitrophenylhydrazones prior to LC-MS analysis

controls

Positive control: internal standard (13C4-butyrate)
Negative control: not specified

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