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 ¨C 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

 

2. Palmitate or stearate universally labeled with ¹³C.  With these labels, the masses scanned are the m0 cluster, and the m16 or m18 cluster.

 

Masses, time windows and retention times:  

Compound   time window       m/z cluster          retention time

C14 group    3 to 7 min    m/z 242, 256 clusters C14  6.3 min

C16 group   7 to 10 min   m/z 270, 286 clusters for palmitate, 8.72 min

m/z 239, 255 clusters for C16:1,  9.51 min

C18 group   10 to 13 min m/z 298, 314 clusters for stearate, 11.33 min

m/z 263, 289 clusters for C18:1,  11.73 min

C20 group   13 to 15 min m/z 326, 342 clusters C20  13.85 min

C22 group    15 to 17 min m/z 354, 370 clusters C22  16.23 min

 

 

More Fatty Acid Assay Protocols