Efficient and Specific Analysis of Red Blood Cell Glycerophospholipid Fatty Acid Composition

Abstract

BackgroundRed blood cell (RBC) n-3 fatty acid status is related to various health outcomes. Accepted biological markers for the fatty acid status determination are RBC phospholipids, phosphatidylcholine, and phosphatidyletholamine. The analysis of these lipid fractions is demanding and time consuming and total phospholipid n-3 fatty acid levels might be affected by changes of sphingomyelin contents in the RBC membrane during n-3 supplementation.AimWe developed a method for the specific analysis of RBC glycerophospholipids. The application of the new method in a DHA supplementation trial and the comparison to established markers will determine the relevance of RBC GPL as a valid fatty acid status marker in humans.MethodsMethyl esters of glycerophospholipid fatty acids are selectively generated by a two step procedure involving methanolic protein precipitation and base-catalysed methyl ester synthesis. RBC GPL solubilisation is facilitated by ultrasound treatment. Fatty acid status in RBC glycerophospholipids and other established markers were evaluated in thirteen subjects participating in a 30 days supplementation trial (510 mg DHA/d).OutcomeThe intra-assay CV for GPL fatty acids ranged from 1.0 to 10.5% and the inter-assay CV from 1.3 to 10.9%. Docosahexaenoic acid supplementation significantly increased the docosahexaenoic acid contents in all analysed lipid fractions. High correlations were observed for most of the mono- and polyunsaturated fatty acids, and for the omega-3 index (r = 0.924) between RBC phospholipids and glycerophospholipids. The analysis of RBC glycerophospholipid fatty acids yields faster, easier and less costly results equivalent to the conventional analysis of RBC total phospholipids.

Phospholipid composition of packed red blood cells and that of extracellular vesicles show a high resemblance and stability during storage

“…Large amounts of carbon chains this long with only one double bond may lower membrane fluidity and would not be expected to be favorable in cell membranes. In addition, 20-or 22-carbon saturated or monounsaturated fatty acids, which most likely comprise one of the two chains in PS 38:1, have not been found in significant amounts in RBCs [3,23,35,38,39]. Moreover, we did not detect a significant amount of this PS species in RBCs at any time point, but instead a large relative amount of PS 38:4, in line with another study [26].…”

Hexadecenoic Fatty Acid Isomers in Human Blood Lipids and Their Relevance for the Interpretation of Lipidomic Profiles

“…Thin layer chromatography ( n -hexane/diethyl ether/acetic acid 70/30/1) evidenced the presence of PL and cholesterol as main components. Then, using proper eluent systems and references of all lipid classes [ 31 ], the identification of membrane phospholipids, including plasmalogens and sphingomyelin, (SM) was performed. Other components, such as phosphatidic acid (PA), lysophosphatidyl choline (LPC) or lysophosphatidyl ethanolamine (LPE), if present, were not detectable under our conditions.…”

Fatty acid supply with complementary foods and LC-PUFA status in healthy infants: results of a randomised controlled trial

“…Fatty acid methyl esters (FAMEs) were extracted into hexane (containing 0.2 g/l BHT) for gas chromatographic (GC) analysis. For the analysis of RBC GP fatty acids, the method described for plasma GP was adapted as described by Klem et al [27] introducing a 5-min ultrasound (40 kHz, 120 W) treatment to ensure dissolution of GP. FAME of plasma and RBC was quantified by GC (Agilent 7890, Waldbronn, Germany) with a programmable temperature vaporizer (Gerstel, Mülheim, Germany) and a flame ionisation detector using BPX-70 column (60 m, 0.25 mm ID, SGE, Weiterstadt, Germany).…”