Milk and betaine lipids were extracted using 2:1
chloroform:methanol (Clark et al., 1982) and methylated
using base-catalyzed transesterification (Christie,
1982). Ground freeze-dried TMR and betaine samples
were methylated in a 2-step procedure using methanolic
HCl and sodium methoxide according to Kramer et
al. (1997).
The FA composition was determined using a gas
chromatograph (Agilent 7890a series with autoinjector;
Agilent Technologies Inc., Santa Clara, CA) fitted
with a flame ionization detector and a 100-m × 0.25-mm (with 0.2-μm film) capillary column coated with (88%-cyanopropyl)-methylarylpolysiloxane (Agilent Technologies Inc.). The carrier gas was hydrogen at a
column flow rate of 2 mL/min and hydrogen flow to the
detector was 40 mL/min with a detector temperature
of 280°C. After sample injection, the oven temperature
was 120°C for 1 min and then increased to 175°C at
a rate of 10°C/min and held for 10 min, and then increased
to 210°C and held for 5 min, and then increased
to 230°C at a rate of 5°C/min and held for 5 min. To
identify FA, retention times were compared with a