Frozen 50 g faecal samples were thawed at 4°C for 4 h and prepared as described by Otto
et al.(64 ). Briefly, 2 g of thawed faeces were mixed with 8 ml deionised water
and 0·5 ml concentrated HCl (Fisher-Scientific), vortexed for 10 s, and then centrifuged
at 25314g for 20 min. The supernatant fraction was filtered through a 0·22 µm filter
(Millipore Co.) and stored in 3·7 ml (1 fluid dram; DR) glass vials (no. 0333922B; Fisher
Scientific). Samples were pooled to combine by day within horse and stored at –80°C until
VFA analysis. Thawed pooled plasma and faecal supernatant fraction samples were spiked
with 100 µl internal standard/volume marker
(2·5 mm -[1,2-13C2]sodium acetate,
1 mm -[1,2,3-13C3]propionic acid,
1 mm -[1,2,3,4-13C4]sodium butyrate) then derivatised
using a water, acetonitrile and 2-chloroethanol solution adapted from
Kristensen(65 ). Faecal preparations were analysed for acetate, propionate and
butyrate, and plasma was analysed for acetate by GC and MS(65 ).
et al.(
and 0·5 ml concentrated HCl (Fisher-Scientific), vortexed for 10 s, and then centrifuged
at 25314
(Millipore Co.) and stored in 3·7 ml (1 fluid dram; DR) glass vials (no. 0333922B; Fisher
Scientific). Samples were pooled to combine by day within horse and stored at –80°C until
VFA analysis. Thawed pooled plasma and faecal supernatant fraction samples were spiked
with 100 µl internal standard/volume marker
(2·5 m
1 m
1 m
using a water, acetonitrile and 2-chloroethanol solution adapted from
Kristensen(
butyrate, and plasma was analysed for acetate by GC and MS(