Abstract
In the last years, there has been an increasing interest in evaluating possible relations between fatty acid (FA) patterns and the risk for chronic diseases. Due to the long life span (120 days) of red blood cells (RBCs), their FA profile reflects a longer term dietary intake and was recently suggested to be used as an appropriate biomarker to investigate correlations between FA metabolism and diseases. Therefore, the aim of this work was to develop and validate a simple and fast methodology for the quantification of a broad range of FAs in RBCs using gas chromatography with flame ionization detector, as a more common and affordable equipment suitable for biomedical and nutritional studies including a large number of samples. For this purpose, different sample preparation protocols were tested and compared, including a classic two-step method (Folch method) with modifications and different one-step methods, in which lipid extraction and derivatization were performed simultaneously. For the one-step methods, different methylation periods and the inclusion of a saponification reaction were evaluated. Differences in absolute FA concentrations were observed among the tested methods, in particular for some metabolically relevant FAs such as trans elaidic acid and eicosapentaenoic acid. The one-step method with saponification and 60 min of methylation time was selected since it allowed the identification of a higher number of FAs, and was further submitted to in-house validation. The proposed methodology provides a simple, fast and accurate tool to quantitatively analyse FAs in human RBCs, useful for clinical and nutritional studies.
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References
Rustan AC, Drevon CA (2001) Fatty acids: structures and properties. eLS. Wiley
Berry EM, Eisenberg S, Haratz D, Friedlander Y, Norman Y, Kaufmann NA, Stein Y (1991) Effects of diets rich in monounsaturated fatty acids on plasma lipoproteins-the Jerusalem Nutrition Study: high MUFAs vs high PUFAs. Am J Clin Nutr 53(4):899–907
Djousse L, Matthan NR, Lichtenstein AH, Gaziano JM (2012) Red blood cell membrane concentration of cis-palmitoleic and cis-vaccenic acids and risk of coronary heart disease. Am J Cardiol 110(4):539–544
Novgorodtseva T, Kantur T, Karaman Y, Antonyuk M, Zhukova N (2011) Modification of fatty acids composition in erythrocytes lipids in arterial hypertension associated with dyslipidemia. Lipids Health Dis 10(1):18
Shearer GC, Pottala JV, Spertus JA, Harris WS (2009) Red Blood cell fatty acid patterns and acute coronary syndrome. PLoS ONE 4(5):e5444
Sertoglu E, Kurt I, Tapan S, Uyanik M, Serdar MA, Kayadibi H, El-Fawaeir S (2014) Comparison of plasma and erythrocyte membrane fatty acid compositions in patients with end-stage renal disease and type 2 diabetes mellitus. Chem Phys Lipids 178:11–17
Yu DX, Sun Q, Ye XW, Pan A, Zong G, Zhou YH, Li HX, Hu FB, Lin X (2012) Erythrocyte trans-fatty acids, type 2 diabetes and cardiovascular risk factors in middle-aged and older Chinese individuals. Diabetologia 55(11):2954–2962
Yue KT, Davis JW, Phillips PE, Graham BA (1981) Effect of glucose on plasma concentrations of individual non-esterified fatty acids of non-diabetic and insulin-independent diabetic men. Postgrad Med J 57(672):622–626
Harris RB, Foote JA, Hakim IA, Bronson DL, Alberts DS (2005) Fatty acid composition of red blood cell membranes and risk of squamous cell carcinoma of the skin. Cancer Epidemiol Biomarkers Prev 14(4):906–912
Conquer J, Tierney M, Zecevic J, Bettger W, Fisher R (2000) Fatty acid analysis of blood plasma of patients with alzheimer’s disease, other types of dementia, and cognitive impairment. Lipids 35(12):1305–1312
Freeman MP (2006) Omega-3 fatty acids and perinatal depression: a review of the literature and recommendations for future research. Prostaglandins LeukotEssent Fatty Acids 75(4–5):291–297
Hodson L, Skeaff CM, Fielding BA (2008) Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake. Prog Lipid Res 47(5):348–380
Acar N, Berdeaux O, Grégoire S, Cabaret S, Martine L, Gain P, Thuret G, Creuzot-Garcher CP, Bron AM, Bretillon L (2012) Lipid Composition of the human eye: are red blood cells a good mirror of retinal and optic nerve fatty acids? PLoS ONE 7(4):e35102
Catalan U, Rodriguez MA, Ras MR, Macia A, Mallol R, Vinaixa M, Fernandez-Castillejo S, Valls RM, Pedret A, Griffin JL, Salek R, Correig X, Motilva MJ, Sola R (2013) Biomarkers of food intake and metabolite differences between plasma and red blood cell matrices; a human metabolomic profile approach. Mol BioSyst 9(6):1411–1422
Shemin D, Rittenberg D (1946) The life span of the human red blood cell. J Biol Chem 166(2):627–636
Clayton E, Gulliver C, Piltz J, Taylor R, Blake R, Meyer R (2012) Improved extraction of saturated fatty acids but not omega-3 fatty acids from sheep red blood cells using a one-step extraction procedure. Lipids 47(7):719–727
Cao J, Schwichtenberg KA, Hanson NQ, Tsai MY (2006) Incorporation and clearance of omega-3 fatty acids in erythrocyte membranes and plasma phospholipids. Clin Chem 52(12):2265–2272
Courville AB, Keplinger MR, Judge MP, Lammi-Keefe CJ (2009) Plasma or red blood cell phospholipids can be used to assess docosahexaenoic acid status in women during pregnancy. Nutr Res 29(3):151–155
Firl N, Kienberger H, Hauser T, Rychlik M (2013) Determination of the fatty acid profile of neutral lipids, free fatty acids and phospholipids in human plasma. Clin Chem Lab Med 51(4):799–810
Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1):497–509
Rizzo A, Montorfano G, Negroni M, Adorni L, Berselli P, Corsetto P, Wahle K, Berra B (2010) A rapid method for determining arachidonic:eicosapentaenoic acid ratios in whole blood lipids: correlation with erythrocyte membrane ratios and validation in a large Italian population of various ages and pathologies. Lipids Health Dis 9(1):7
Bailey-Hall E, Nelson EB, Ryan AS (2008) Validation of a rapid measure of blood PUFA levels in humans. Lipids 43(2):181–186
Bell JG, Mackinlay EE, Dick JR, Younger I, Lands B, Gilhooly T (2011) Using a fingertip whole blood sample for rapid fatty acid measurement: method validation and correlation with erythrocyte polar lipid compositions in UK subjects. Br J Nutr 106(9):1408–1415
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem 37(8):911–917
Araujo P, Nguyen TT, Froyland L, Wang J, Kang JX (2008) Evaluation of a rapid method for the quantitative analysis of fatty acids in various matrices. J Chromatogr A 1212(1–2):106–113
Kang J, Wang J (2005) A simplified method for analysis of polyunsaturated fatty acids. BMC Biochem 6(1):5
Bondia-Pons I, Castellote AI, López-Sabater MC (2004) Comparison of conventional and fast gas chromatography in human plasma fatty acid determination. J Chromatogr B 809(2):339–344
Dole VP, Meinertz H (1960) Microdetermination of long-chain fatty acids in plasma and tissues. J Biol Chem 235(9):2595–2599
Kangani CO, Kelley DE, Delany JP (2008) New method for GC/FID and GC-C-IRMS analysis of plasma free fatty acid concentration and isotopic enrichment. J Chromatogr B Analyt Technol Biomed Life Sci 873(1):95–101
Kopf T, Schmitz G (2013) Analysis of non-esterified fatty acids in human samples by solid-phase-extraction and gas chromatography/mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 938:22–26
Lepage G, Roy CC (1986) Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 27(1):114–120
Bicalho B, David F, Rumplel K, Kindt E, Sandra P (2008) Creating a fatty acid methyl ester database for lipid profiling in a single drop of human blood using high resolution capillary gas chromatography and mass spectrometry. J Chromatogr A 1211(1–2):120–128
Burrows T, Berthon B, Garg ML, Collins CE (2012) A comparative validation of a child food frequency questionnaire using red blood cell membrane fatty acids. Eur J Clin Nutr 66(7):825–829
Magnusardottir AR, Steingrimsdottir L, Thorgeirsdottir H, Gunnlaugsson G, Skuladottir GV (2009) Docosahexaenoic acid in red blood cells of women of reproductive age is positively associated with oral contraceptive use and physical activity. Prostaglandins Leukot Essent Fatty Acids 80(1):27–32
Meneses F, Ney JG, Torres AG, Trugo NM (2009) Erythrocyte membrane and plasma non-esterified n-3 and n-6 polyunsaturated fatty acids of pregnant and non-pregnant Brazilian adolescents. Prostaglandins Leukot Essent Fatty Acids 80(2–3):137–142
Sheppard AJ, Food US, Administration D (1992) Lipid manual: methodology suitable for fatty acid-cholesterol analysis. Wm. C. Brown Publishers, Iowa
Taverniers I, De Loose M, Van Bockstaele E (2004) Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance. TrAC, Trends. Anal Chem 23(8):535–552
International Conference on Harmonisation (October 1994/November 1996) ICH harmonised tripartite guideline validation of analytical procedure: text and methodology Q2(R1), vol Q2(R1)
Amaral J, Casal S, Citová I, Santos A, Seabra R, Oliveira BP (2006) Characterization of several hazelnut (Corylus avellana L.) cultivars based in chemical, fatty acid and sterol composition. Eur Food Res Technol 222(3–4):274–280
ISO (2011) 12966-2:2011 Animal and vegetable fats and oils—gas chromatography of fatty acid methyl esters—part 2: preparation of methyl esters of fatty acids
Phillips KM, Ruggio DM, Howe JC, Leheska JM, Smith SB, Engle T, Rasor AS, Conley NA (2010) Preparation and characterization of control materials for the analysis of conjugated linoleic acid and trans-vaccenic acid in beef. Food Res Int 43(9):2253–2261
Magnusardottir AR, Skuladottir GV (2006) Effects of storage time and added antioxidant on fatty acid composition of red blood cells at -20 degrees C. Lipids 41(4):401–404
Schreiner M (2006) Principles for the Analysis of Omega-3 Fatty Acids. In: Teale MC (ed) Omega 3 fatty acid research. Nova Science Publishers, New York, pp 1–25
Morrison WR, Smith LM (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride–methanol. J Lipid Res 5(4):600–608
IUPAC (1987) Standard methods for analysis of oils, fats and derivatives. IUPAC Method 2.301, Seventh edn. Blackwell Scientific Publications, Oxford
AOAC (1997) Fatty acids in oils and fats; preparation of methyl ester boron trifluoride method. AOAC Official Method 969.33. AOAC International
Bocking C, Nockher WA, Schreiner M, Renz H, Pfefferle PI (2010) Development and validation of a combined method for the biomonitoring of omega-3/-6 fatty acids and conjugated linoleic acids in different matrices from human and nutritional sources. Clin Chem Lab Med 48(12):1757–1763
Bhardwaj S, Passi SJ, Misra A (2011) Overview of trans fatty acids: biochemistry and health effects. Diabetes Metab Syndr 5(3):161–164
Kvannes J, Eikhom TS, Flatmark T (1995) On the mechanism of stimulation of peroxisomal β-oxidation in rat heart by partially hydrogenated fish oil. BBA-Lipid Lipid Met 1255(1):39–49
Acknowledgments
This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project PTDC/SAU-ENB/116929/2010 and EXPL/EMS-SIS/2215/2013. ROR acknowledges PhD scholarship SFRH/BD/97658/2013 attributed by FCT (Fundação para a Ciência e Tecnologia).
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R. O. Rodrigues and H. Costa have contributed equally to this work.
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Rodrigues, R.O., Costa, H., Lima, R. et al. Simple Methodology for the Quantitative Analysis of Fatty Acids in Human Red Blood Cells. Chromatographia 78, 1271–1281 (2015). https://doi.org/10.1007/s10337-015-2947-2
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DOI: https://doi.org/10.1007/s10337-015-2947-2