Determination of c9,t11‐CLA in major human plasma lipid classes using a combination of methylating methodologies

• Published in: Lipids 2003-07, Vol.38 (7), p.793-800
• Main Authors: Shahin, Alam M., McGuire, Michelle K., McGuire, Mark A., Ritzenthaler, Kristin L., Shultz, Terry D.
• Format: Article
• Language: English
• Subjects: Adult; Cholesterol; Chromatography, Gas; Chromatography, Thin Layer; Esters; Female; Humans; Indicators and Reagents; Linoleic Acids, Conjugated - blood; Lipids; Lipids - blood; Lipids - chemistry; Lipids - classification; Methods; Methylation; Plasma; Reagents; Sodium
• ISSN: 0024-4201; 1558-9307
• DOI: 10.1007/s11745-003-1128-3

Isomeric CLA exhibit several significant biological activities in animals and humans and are easily isomerized to their corresponding t,t‐CLA isomers during methylation with various acid‐catalyzed reagents. To minimize such isomerization and provide a valid quantification of human plasma CLA content, several methylation methods were tested. Plasma neutral lipid, nonesterified FA (NEFA), and polar lipid classes were separated into the following fractions: (i) cholesteryl ester (CE, 1.2 mg/12 mL, 37.5% lipids), (ii) TAG (0.8 mg/12 mL, 25% lipids), (iii) NFFA (0.2 mg/12 mL, 6.2% lipids), (iv) MAG/DAG/cholesterol (0.3 mg/12 mL, 9.4% lipids), and (v) phospholipid (PL, 0.5 mg/20 mL, 15.6% lipids). Data showed that c9,t11‐CLA found in TAG, MAG/DAG/cholesterol, and PL fractions were converted to methyl esters with sodium methoxide within 2 h at 55°C. However, the c9,t11‐CLA in the CE fraction could not be completely converted to methyl esters by sodium methoxide/acetylchloride in methanol or methanolic KOH; instead, CE was treated with sodium methoxide and methyl acetate in diethyl ether for 1 h. NEFA were converted to methyl esters with trimethylsilyldiazomethane (TMSDAM). All reaction mixtures were monitored by TLC prior to GLC analysis. The highest enrichment of c9,t11‐18∶2 (% FA) was in TAG (0.31%), followed by CE (0.14%) and PL (0.13%). The above methylation methods were then applied to a small subset (n=10) of nonfasting plasma lipid fractions to confirm the applicability of these data. Results from this subset of samples also indicated that the greatest enrichment of c9,t11‐CLA was present in the TAG fraction (0.39%), followed by CE (0.27%) and PL (0.22%). These data indicate that different plasma fractions have different c9,t11‐CLA contents.