Synthesis and isolation of trans-7,cis-9 octadecadienoic acid and other CLA isomers by base conjugation of partially hydrogenated gamma-linolenic acid

Abstract

CLA is of considerable interest because of reported potentially beneficial effects in animal studies. CLA, while not yet unambiguously defined, is a mixture of octadecadienoic acids with conjugated double bonds. The major isomer in natural products is generally considered to be cis-9,trans-11-octadecadienoic acid (c9,t11), which represents > 75% of the total CLA in most cases. Other isomers are drawing increased attention. The t7,c9 isomer, which is often the second-most prevalent CLA in natural products, has been reported to represent as much as 40% of total CLA in milk from cows fed a high-fat diet. The need for a reference material became apparent in a recent study directed specifically at measuring t7,c9-CLA in milk, plasma, and rumen. A suitable standard mixture was produced by stirring 0.5 g of gamma-linolenic acid (all cis-6,9,12-C18.3) with 100 mL of 10% hydrazine hydrate in methanol for 2.5 h at 45 degrees C. The solution was diluted with H2O and acidified with HCI. The resulting partially hydrogenated FA were extracted with ether/petroleum ether, dried with Na2SO4, and conjugated by adding of 6.6% KOH in ethlylene glycol and heating for 1.5 h at 150-160 degrees C. Approximately 20 mg each of cis-6,trans-8; trans-7,cis-9; cis-9,trans-11; and trans- 10,cis-12 were obtained along with other FA. Methyl esters (FAME) of these four cis/trans isomers were resolved by Ag+ HPLC (UV 233) and partially resolved by GC/(MS or FID) (CP-Sil 88). Treatment of these FAME with 12 yielded all possible cis/trans (geometric) isomers for the four positions 6,8; 7,9; 9,11; and 10,12.