BioProtocols--Met7

Lipids

Fatty acids

Fatty acid derivative: methyl ester.

Reagent:

0.5N HCl in methanol (Supelco 3-3095)

Procedure:

Fatty acids are extracted by petroleum ether after acidification of a saponified tissue. To the dried fatty acid fraction add 200mL to 500 mL methanolic HCl and heat at 70^oC for at least 1 hour. Dry with nitrogen. Add hexane as solvent for GC/MS analysis.

GC/MS:

GC column: bpx70 (SGE), length-30m, 250mM, programmed at a constant He flow of 1ml/min.

Inlet temp. 250^oC, split ratio 1:100 adjusted depending on sample concentration.

Oven temperature programmed as follows: 120^oC for 1 min, then a 5^oC/min ramp to 220^oC and hold for 1min.

We use three different labels to study fatty acid metabolism:

1. Deuterium label from deuterium oxide, to measure fractional new synthesis. (ref 1.)

2. Palmitate or stearate universally labeled with¹³C, to measure chain shortening, chain elongation and desaturation (ref 2.)

3. Perdeuterated palmitate and stearate to measure chain shortening, chain elongation and desaturation (ref 3.)

The masses scanned and retention times for the unlabeled and labeled fatty acids depend on the method used. The following three tables summarize these three methods of analysis.

1. Deuterium label from deuterium oxide 每 scan for m0, m1,m2,?shy;.m10.

Masses, time windows and retention times:

Compound time window m/z cluster retention time

C14 group 3 to 7 min m/z 242 cluster C14 6.3 min

C16 group 7 to 10 min m/z 270 cluster for palmitate, 8.72 min

m/z 239 cluster for C16:1, 9.51 min

C18 group 10 to 13 min m/z 298 cluster for stearate, 11.33 min

m/z 263 cluster for C18:1, 11.73 min

C18:2, 12.51 min.

C20 group 13 to 15 min m/z 326 cluster C20 13.85 min

C22 group 15 to 17 min m/z 354 cluster C22 16.23 min

C24 group 17 to 24 min m/z 382 cluster C24 18.45 min