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Efficient and specific analysis of red blood cell glycerophospholipid fatty acid composition.

Klem S, Klingler M, Demmelmair H, Koletzko B - PLoS ONE (2012)

Bottom Line: Red blood cell (RBC) n-3 fatty acid status is related to various health outcomes.Docosahexaenoic acid supplementation significantly increased the docosahexaenoic acid contents in all analysed lipid fractions.The analysis of RBC glycerophospholipid fatty acids yields faster, easier and less costly results equivalent to the conventional analysis of RBC total phospholipids.

View Article: PubMed Central - PubMed

Affiliation: Division of Metabolic and Nutritional Medicine, Dr von Hauner Children's Hospital, University of Munich Medical Center, Munich, Germany.

ABSTRACT

Background: Red 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.

Aim: We 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.

Methods: Methyl 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).

Outcome: The 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.

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Recovery of total RBC fatty acids using different extraction procedures (P<0.05).
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pone-0033874-g001: Recovery of total RBC fatty acids using different extraction procedures (P<0.05).

Mentions: The extraction efficiency for of GPL was tested by applying 4 different extraction procedures (Figure 1). In total 16 aliquots (4×4) of an RBC sample were tested. Continuous shaking of RBC dissolved in methanol for 5 min yielded in 185±120 µg total GPL fatty acids per 100 µl RBC, which was similar to prolonged shaking for 10 min (241±63 µg) or additional ultrasound treatment for another 5 min (301±46 µg). Adding methanol to RBC without shaking caused clotting of RBC and lower recovery (37±24 µg). Partial clotting was also observed after shaking the samples for 5 or 10 min, but not when samples were treated with ultrasound. The fatty acid pattern of the 16 RBC aliquots did not differ to any appreciable extent (data not shown).


Efficient and specific analysis of red blood cell glycerophospholipid fatty acid composition.

Klem S, Klingler M, Demmelmair H, Koletzko B - PLoS ONE (2012)

Recovery of total RBC fatty acids using different extraction procedures (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316509&req=5

pone-0033874-g001: Recovery of total RBC fatty acids using different extraction procedures (P<0.05).
Mentions: The extraction efficiency for of GPL was tested by applying 4 different extraction procedures (Figure 1). In total 16 aliquots (4×4) of an RBC sample were tested. Continuous shaking of RBC dissolved in methanol for 5 min yielded in 185±120 µg total GPL fatty acids per 100 µl RBC, which was similar to prolonged shaking for 10 min (241±63 µg) or additional ultrasound treatment for another 5 min (301±46 µg). Adding methanol to RBC without shaking caused clotting of RBC and lower recovery (37±24 µg). Partial clotting was also observed after shaking the samples for 5 or 10 min, but not when samples were treated with ultrasound. The fatty acid pattern of the 16 RBC aliquots did not differ to any appreciable extent (data not shown).

Bottom Line: Red blood cell (RBC) n-3 fatty acid status is related to various health outcomes.Docosahexaenoic acid supplementation significantly increased the docosahexaenoic acid contents in all analysed lipid fractions.The analysis of RBC glycerophospholipid fatty acids yields faster, easier and less costly results equivalent to the conventional analysis of RBC total phospholipids.

View Article: PubMed Central - PubMed

Affiliation: Division of Metabolic and Nutritional Medicine, Dr von Hauner Children's Hospital, University of Munich Medical Center, Munich, Germany.

ABSTRACT

Background: Red 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.

Aim: We 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.

Methods: Methyl 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).

Outcome: The 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.

Show MeSH