Whole blood glycerophospholipids in dried blood spots - a reliable marker for the fatty acid status.

BACKGROUND: Whole blood total fatty acid analysis in capillary blood has recently been proposed for fatty acid status determination, but the accuracy of this method is affected by the fast turnover of triaclyglyceride (TAG) fatty acids, the individual hematocrit and postprandial state. METHOD: An established method for the glycerophospholipid (GPL) analysis in plasma was adapted for the analysis of whole blood GPL and tested in a fat challenge test. Blood samples were collected from nine participants after receiving a standardised breakfast containing 42g of sunflower oil. Whole blood GPL fatty acids were compared against whole blood total lipid, plasma TAG and phospholipid fatty acids. RESULTS: All fatty acid concentrations in dried blood samples showed a coefficient of variation <5.7%. The fat challenge test induced a significant increase of TAG fatty acid concentration (mean Δ=42.3%±35.7) and whole blood total fatty acid concentration (mean Δ=5.2%±3.7) whereas whole blood GPL fatty acids were hardly changed (mean Δ=1.3%±1.6). CONCLUSION: Whole blood GPL fatty acids are a robust biological marker for the fatty acid status of fasted and non-fasted subjects. The influence of very recent dietary intake on whole blood GPL is smaller than on whole blood total lipids.

Changes of molecular glycerophospholipid species in plasma and red blood cells during docosahexaenoic acid supplementation.

Docosahexaenoic acid (DHA) status is related to health and disease risk. DHA status is mainly determined by dietary DHA intake, since endogenous synthesis of DHA is limited. We aimed to investigate the changes of different molecular glycerophospholipid species containing DHA in plasma and red blood cells (RBC) in response to increased DHA intake. Thirteen healthy adults had their diet supplemented with 510 mg DHA/day for 29 days. Fasted blood samples were taken at 11 time points and glycerophospholipid species were analyzed by liquid chromatography mass spectrometry. In plasma, percentages of glycerophospholipid species containing DHA increased significantly by 64-104 % relative to baseline values during supplementation, but the relative distribution between species was not markedly altered. In RBC, phosphatidylcholine (PtdCho) species containing DHA increased to a similar extent as in plasma, while phosphatidylethanolamine and phosphatidylserine species with DHA increased by only 12-25 %, respectively, which was significantly different compared to PtdCho species (p < 0.01). Despite the high increase, the contribution of DHA PtdCho species to total DHA remained minor (14 % after supplementation). In conclusion, DHA supplementation does not alter the relative distribution of DHA among glycerophospholipid species in plasma. A majority of PtdCho species are rapidly exchanged between plasma lipoproteins and RBC membrane lipids, while there is a minor exchange of phosphatidylethanolamine and phosphatidylserine species.

Comparison of the incorporation of orally administered DHA into plasma, erythrocyte and cheek cell glycerophospholipids.

Adequate intake of n-3 fatty acids plays an important role in human health. The analysis of various blood lipids is used as a measure of fatty acid status in humans. Cheek cell phospholipids (PL) have also been proposed as biological markers, but are rarely used in clinical studies due to limitations in sample quality and quantity. An improved method for the analysis of cheek cell glycerophospholipid fatty acids is applied in a 29 d supplementation trial with 510 mg DHA daily. The DHA increases in cheek cell, plasma and erythrocyte glycerophospholipids are compared. High correlations are shown for glycerophospholipid DHA between cheek cells and plasma (r 0·88) and erythrocytes (r 0·76) before study commencement. After the daily supplementation of DHA, the half-maximal glycerophospholipid DHA level is reached after about 4 d in plasma, 6 d in erythrocytes and 10 d in cheek cells. The mean DHA increase (mol%) relative to baseline was most prominent in plasma (186 %), followed by cheek cells (180 %) and erythrocytes (130 %). Considering a lag phase of about 5 d, cheek cells reflect short-term changes in dietary fat uptake. Based on the data of the present study, they can be used alternatively to plasma and erythrocyte PL as non-invasive n-3 fatty acid status markers.

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

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.

Docosahexaenoic acid supply in pregnancy affects placental expression of fatty acid transport proteins.

BACKGROUND: Better understanding of the mechanisms involved in docosahexaenoic acid (DHA) transfer to the neonate may contribute to improve dietary support for infants born prematurely to mothers with placental lipid transport disorders. OBJECTIVE: We studied whether DHA supplements modify the messenger RNA (mRNA) expression of placental lipid transport proteins to allow a selective transfer of DHA to the fetus. DESIGN: Healthy pregnant women (n = 136) received, in a double-blind randomized trial, 500 mg DHA + 150 mg eicosapentaenoic acid, 400 microg 5-methyl-tetrahydrofolic acid, 500 mg DHA + 400 microg 5-methyl-tetrahydrofolic acid, or placebo during the second half of gestation. We analyzed the fatty acid composition of maternal and cord blood phospholipids and of placenta; we quantified placental mRNA expression of fatty acid-transport protein 1 (FATP-1), FATP-4, FATP-6, fatty acid translocase, fatty acid-binding protein (FABP) plasma membrane, heart-FABP, adipocyte-FABP, and brain-FABP. RESULTS: The mRNA expression of the lipid carriers assayed did not differ significantly between the 4 groups. However, the mRNA expression of FATP-1 and FATP-4 in placenta was correlated with DHA in both maternal plasma and placental phospholipids, although only FATP-4 expression was significantly correlated with DHA in cord blood phospholipids. Additionally, the mRNA expression of several membrane lipid carriers was correlated with EPA and DHA in placental triacylglycerols and with EPA in placental free fatty acids. CONCLUSIONS: Correlation of the mRNA expression of the membrane placental proteins FATP-1 and especially of FATP-4 with maternal and cord DHA leads us to conclude that these lipid carriers are involved in placental transfer of long-chain polyunsaturated fatty acids.

The effect of docosahexaenoic acid and folic acid supplementation on placental apoptosis and proliferation.

The hypothesis was tested that the additional dietary uptake of n-3 fatty acids, in particular of DHA and 5-methyltetrahydrofolate (5-MTHF), during the second half of pregnancy would influence proliferation and apoptosis in the full-term human placenta. The diets of pregnant women from Spain (n 55) were supplemented with modified fish oil and/or 5-MTHF or placebo, and assigned in a random, double-blind manner to one of the four groups. Immunohistochemistry and immunoblotting were used to detect placental proliferation and apoptosis with monoclonal antibodies for key proteins that reflected the extent of both processes: proliferation cell nuclear antigen (PCNA), p53, cytokeratin 18 neoepitope. The PCNA level in the fish oil/5-MTHF-treated group was higher by 66 % (P < 0.05) than that of the placebo group, whereas the levels of p53 and cytokeratin 18 neoepitope were unaffected by treatment. PCNA expression was altered only in the trophoblast compartment (placebo 11.1 (se 0.5) % v. combination 21.5 (se 1.2) %; P < 0.05), whereas the proportion of nuclei stained in endothelial and other stromal cells was similar in the placebo and combined treatment groups. No correlation was found between fish oil or 5-MTHF supplementation and the levels of the proteins. The present data suggest that supplementation with fish oil and/or 5-MTHF had no effect on the parameters reflecting placental proliferation and apoptosis. A defined combination of DHA and 5-MTHF may, however, affect placental proliferation.