Chromatographic and Enzymatic Method to Quantify Individual Plasma Free and Triacylglycerol Fatty Acids Miquel Martorell, Xavier Capó, Antoni Sureda, Josep A. Tur & Antoni Pons
Chromatographia An International Journal for Rapid Communication in Chromatography, Electrophoresis and Associated Techniques ISSN 0009-5893 Volume 78 Combined 3-4 Chromatographia (2015) 78:259-266 DOI 10.1007/s10337-014-2820-8
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Author's personal copy Chromatographia (2015) 78:259–266 DOI 10.1007/s10337-014-2820-8
ORIGINAL
Chromatographic and Enzymatic Method to Quantify Individual Plasma Free and Triacylglycerol Fatty Acids Miquel Martorell · Xavier Capó · Antoni Sureda · Josep A. Tur · Antoni Pons
Received: 23 July 2014 / Revised: 26 November 2014 / Accepted: 27 November 2014 / Published online: 12 December 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Non-esterified and triacylglyceride fatty acid quantification in plasma is important in order to know fatty acid plasma availability and the nutritional status. A reliable method to determine free, triacylglycerol, phospholipid, and cholesterol ester fatty acids in human plasma samples by combining two types of fatty acid isolation and enzymatic and chromatographic methods has been developed and made safer by avoiding diazomethane derivatization. Lipoprotein lipase was used to obtain hydrolyzed fatty acids from triacylglycerides, and chromatography performed with an active carbon column was used to isolate free fatty acids. Posterior safe derivatization to methylated fatty acids by transesterification, with m-trifluoromethylphenyl trimethylammonium hydroxide reagent (Meth-Prep™ II) and gas chromatography enabled us to identify and quantify a wide spectral range of non-esterified and triacylglyceride fatty acids in plasma. Additionally, the method allows the fatty acids of the phospholipid and cholesterol ester fraction to be determined after the determination of total plasma fatty acids. Determining the individual fatty acid recovery with respect to the C17:0 internal standard makes it possible to calculate the individual fatty acid concentration in each fraction of plasma. The method optimizes the derivatization reagent and chromatographic conditions and enables M. Martorell · X. Capó · A. Sureda · J. A. Tur · A. Pons (*) Laboratori de Ciències de l’Activitat Física, Grup de Nutrició Comunitaria i Estrés Oxidatiu, Departament de Biologia Fonamental i Ciències de la Salut, Universitat de les Illes Balears, Crtra. Valldemossa, km 7.5, 07122 Palma de Mallorca, Illes Balears, Spain e-mail:
[email protected] M. Martorell · X. Capó · A. Sureda · J. A. Tur · A. Pons CIBER: CB12/03/30038 Fisiopatología de la Obesidad y la Nutrición, CIBERobn, Instituto de Salud Carlos III (ISCIII), Madrid, Spain
quantification of additional fatty acids such as omega-3 and omega-6 of the C18:3 fatty acid and C20:1n9, C22:0, and C22:6n3 fatty acids. The procedure can be safely used to analyze a large number of samples. Keywords Gas chromatography · Lipoprotein lipase · Plasma · Fatty acid · Non-esterified fatty acid · Triacylglyceride fatty acid Abbreviations ANOVA Analysis of variance BHA Butylated hydroxyanisole CEFA Cholesterol ester fatty acid FID Flame ionization detector LPL Lipoprotein lipase MUFA Monounsaturated fatty acid NEFA Non-esterified fatty acid PLFA Phospholipid fatty acid PUFA Polyunsaturated fatty acid SFA Saturated fatty acid TAG Triacylglyceride TGFA Triacylglyceride fatty acid TPFA Total plasma fatty acids TSF p-Toluenesulphonyl fluoride
Introduction Cells, tissues, and biological fluids such as plasma contain tens of thousands of structurally different lipids with multiple roles in cellular signaling, in membrane structure, and as a fuel source and reserve for many cell types [1]. Total plasma fatty acids (TPFA) are distributed in glycerolipids (mainly triacylglycerides, TAG), glycerophospholipids (phospholipids), and sterol lipids which are transported in lipoprotein particles
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[2, 3]. Fatty acids in plasma are determined in different studies without differentiating between non-esterified fatty acids (NEFAs), triacylglyceride fatty acids (TGFAs), phospholipid fatty acids (PLFAs), and cholesterol ester fatty acids (CEFAs) [4–7]. The NEFA fraction indicates the plasma availability of fatty acids mobilized from tissue stores [8], whereas the TGFA fraction indicates the availability of lipoprotein fatty acids from liver; in this case, fatty acid availability is lipoprotein-lipase-dependent [9]. In muscle, the fatty acids from adipocyte lipolysis are the main fuel for muscular contraction and physical activity maintenance [10]. Plasma phospholipid C18:3n3 fatty acid is a biomarker of circulating levels of C18:3n3 [11], and C18:3n3 cholesteryl fatty acid is inversely associated with cardiovascular disease [12]. Few specific, simple, rapid methods exist for free and esterified fatty acid determination. One of the most specific and rapid methods contemplates NEFA and TGFA determination by combining the specificity of lipoprotein lipase (LPL) with chromatographic methods for fatty acid isolation and quantification [13]. In this method, plasma sample not treated with LPL is used for NEFA determination, and plasma sample treated with LPL is used for simultaneous NEFA and TGFA determination; however, this method presents the inconvenience of diazomethane derivatization [14, 15]. We aimed to develop a method to determine a wide range of NEFAs and TGFA in plasma that is safer and more effective to quantify a great number of individual fatty acids. The secondary aim was to quantify the fatty acids in the phospholipids and cholesterol ester fraction.
Materials and Methods Chemicals and Reagents Solvents of analytical grade were from Panreac Quimica (Spain). Lipase inhibitor (p-toluenesulphonyl fluoride, TSF), butylated hydroxyanisole (BHA), and Triglyceride Reagent containing LPL were from Sigma (St. Louis, MO, USA). The Triglyceride Reagent, when reconstituted according to the manufacturer’s instructions, contains 250,000 units L−1 Lipase (microbial) and 0.05 % azide as preservative. The transesterification reagent to methylate fatty acids was a 0.2 N methanolic solution of m-trifluoromethylphenyl trimethylammonium hydroxide (Meth-Prep™ II, Grace Davison, Columbia, MD, USA). Carbopack™ 60/80, fatty acid methyl standards mixtures, single fatty acids, and gas chromatography column were supplied by Supelco (Bellefonte, PA, USA). The glass columns and Pyrex tubes were supplied by ANORSA (Spain), and chromatographic vials by Agillent Technologies (Santa Clara, CA, USA).
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Plasma Collection Plasma was obtained from 24 healthy men in resting conditions for NEFAs and TGFAs determination. Prior to blood collection, anthropometrical measurements were carried out and dietary habits were assessed using a 7-day dietary record questionnaire. A qualified dietician verified and quantified the food records. All food items consumed were transformed into nutrients using a computerized program based on European and Spanish food composition tables [16, 17]. The subjects were participating in a clinical trial (ClinicalTrials. gov NCT02177383) and were informed of the purpose and demands of the study before giving their written consent to participate. The study protocol was in accordance with the Helsinki Declaration for research on human subjects and was approved by the Ethical Committee of Clinical Investigation of the Comunidad Autónoma de les Illes Balears N° IB 994/08 PI (Palma de Mallorca, Balearic Islands, Spain). Venous blood samples were obtained after 12 h, overnight, fasted conditions, from the antecubital vein with suitable vacutainers (BD Vacutainer, Plymouth, UK) containing EDTA as anticoagulant following an adaptation of the method described elsewhere [18, 19]. An inhibitor of LPL, 2 µL 50 mM of TSF, was previously added to the vacutainer designated for NEFA determination. Plasma was obtained after centrifugation (900×g, 30 min, 4 °C). NEFA and TGFA Isolations For NEFAs quantification, 100 µL of plasma was diluted in a Pyrex tube with 5 mL methanol:water (1:1 v/v) containing 0.01 % BHA and 2 µL n-heptadecanoic acid (C17:0, 15 mM in methanol) as internal standard. For TGFA quantification 10 µL of plasma (without LPL inhibitor) was used for reaction, 5 min at 37 °C, with 200 µL Triglyceride Reagent containing LPL and subsequently diluted in 4 mL methanol:water (5:3 v/v) containing 0.01 % BHA and 2 µL of C17:0 (15 mM). These samples were a mixture of free fatty acids from the NEFA and TGFA (NEFAs–TGFAs mixture). All the glass material was cleaned with acetone prior to usage. The plasma dilution of LPL and non-LPL treated samples were eluted in a chromatographic column of 0.25 g Carbopack™ 60/80 as a stationary phase and 10 mL chloroform:methanol (1:1 v/v) as eluent. The column (15 mL capacity, 10 mM diameter of filter plate) was initially cleaned by 5 mL chloroform, 3 mL methanol and 3 mL distilled water. The sample was then percolated through the column and the remaining material in the Pyrex tube containing the sample was recovered by rinsing the tube with 5 mL 3 mM HCl solution, which was also eluted through the column. Then the column was washed with 1.5 mL methanol. Finally, free fatty acids retained inside the Carbopack™ 60/80 column were eluted with 10 mL
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chloroform–methanol solution (1:1 v:v) [20]. The resultant eluted organic phases were evaporated under a nitrogen stream at 55 °C. To recycle the same column, the adsorbent had to be washed sequentially with 5 mL chloroform, 3 mL methanol, and 3 mL distilled water.
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