A simplified and efficient method for the analysis of fatty acid methyl esters suitable for large clinical studies

Conventional sample preparation for fatty acid analysis is a complicated, multiple-step process, and gas chromatography (GC) analysis alone can require >1 h per sample to resolve fatty acid methyl esters (FAMEs). Fast GC analysis was adapted to human plasma FAME analysis using a modified polyethylene glycol column with smaller internal diameters, thinner stationary phase films, increased carrier gas linear velocity, and faster temperature ramping. Our results indicated that fast GC analyses were comparable to conventional GC in peak resolution. A conventional transesterification method based on Lepage and Roy was simplified to a one-step method with the elimination of the neutralization and centrifugation steps. A robotics-amenable method was also developed, with lower methylation temperatures and in an open-tube format using multiple reagent additions. The simplified methods produced results that were quantitatively similar and with similar coefficients of variation as compared with the original Lepage and Roy method.The present streamlined methodology is suitable for the direct fatty acid analysis of human plasma, is appropriate for research studies, and will facilitate large clinical trials and make possible population studies. Conventional sample preparation for fatty acid analysis is a complicated, multiple-step process, and gas chromatography (GC) analysis alone can require >1 h per sample to resolve fatty acid methyl esters (FAMEs). Fast GC analysis was adapted to human plasma FAME analysis using a modified polyethylene glycol column with smaller internal diameters, thinner stationary phase films, increased carrier gas linear velocity, and faster temperature ramping. Our results indicated that fast GC analyses were comparable to conventional GC in peak resolution. A conventional transesterification method based on Lepage and Roy was simplified to a one-step method with the elimination of the neutralization and centrifugation steps. A robotics-amenable method was also developed, with lower methylation temperatures and in an open-tube format using multiple reagent additions. The simplified methods produced results that were quantitatively similar and with similar coefficients of variation as compared with the original Lepage and Roy method. The present streamlined methodology is suitable for the direct fatty acid analysis of human plasma, is appropriate for research studies, and will facilitate large clinical trials and make possible population studies. The consumption of n-3 polyunsaturated fatty acids (PUFAs), particularly eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), is implicated in various aspects of human health (1Salem Jr, N. Omega-3 fatty acids: molecular and biochemical aspects.in: Spiller G. Scala J. New Protective Roles of Selective Nutrients in Human Nutrition. Alan R. Liss, New York1989: 263-317Google Scholar, 2Simopoulos A.P. Essential fatty acids in health and chronic disease.Am. J. Clin. Nutr. 1999; 70: 560-569Google Scholar). Presently, analysis of the fatty acid composition of human blood and plasma samples involves tedious and time-consuming extraction and transesterification procedures (3Dacremont G. Vincent G. Assay of plasmalogens and polyunsaturated fatty acids (PUFA) in erythrocytes and fibroblasts.J. Inherit. Metab. Dis. 1995; 18: 84-89Google Scholar, 4Garces R. Mancha M. One-step lipid extraction and fatty acid methyl esters preparation from fresh plant tissues.Anal. Biochem. 1993; 211: 139-143Google Scholar, 5Lepage G. Roy C.C. Improved recovery of fatty acid through direct transesterification without prior extraction or purification.J. Lipid Res. 1984; 25: 1391-1396Google Scholar, 6Lepage G. Roy C.C. Direct transesterification of all classes of lipids in a one-step reaction.J. Lipid Res. 1986; 27: 114-120Google Scholar, 7Morrison W.R. Smith L.M. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol.J. Lipid Res. 1964; 5: 600-608Google Scholar, 8Rodriguez-Ruiz J. Belarbi E.H. Sanchez J.L.G. Alonso D.L. Rapid simultaneous lipid extraction and transesterification for fatty acid analyses.Biotechnol. Tech. 1998; 12: 689-691Google Scholar). In addition, gas chromatography (GC), which is the analytical technique of choice for fatty acid methyl ester (FAME) analysis, requires long run times in order to resolve all of the commonly occurring mammalian fatty acids (9Greiner R.S. Catalan J.N. Moriguchi T. Salem Jr, N. Docosapentaenoic acid does not completely replace DHA in n-3 FA-deficient rats during early development.Lipids. 2003; 38: 431-435Google Scholar, 10Lands W.E. Morris A. Libelt B. Quantitative effects of dietary polyunsaturated fats on the composition of fatty acids in rat tissues.Lipids. 1990; 25: 505-516Google Scholar, 11Marangoni F. Colombo C. Galli C. A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies.Anal. Biochem. 2004; 326: 267-272Google Scholar, 12Moriguchi T. Lim S.Y. Greiner R. Lefkowitz W. Loewke J. Hoshiba J. Salem Jr, N. Effects of an n-3-deficient diet on brain, retina, and liver fatty acyl composition in artificially reared rats.J. Lipid Res. 2004; 45: 1437-1445Google Scholar, 13Ohta A. Mayo M.C. Kramer N. Lands W.E. Rapid analysis of fatty acids in plasma lipids.Lipids. 1990; 25: 742-747Google Scholar, 14Salem Jr., N. Reyzer M. Karanian J. Losses of arachidonic acid in rat liver after alcohol inhalation.Lipids. 1996; 31: 153-156Google Scholar). Streamlined, cost-effective methodology is needed to facilitate large clinical trials that examine the effects of n-3 PUFAs on chronic health conditions as well as population-based nutritional surveys. Currently, there is evidence that n-3 PUFA intake reduces the risk of cardiovascular disease. Supplementation of approximately 1 g of EPA/DHA to patients surviving myocardial infarction has been demonstrated to reduce the risk of cardiovascular and total death, with a profound 45% reduction in sudden cardiac death (15GISSI-Prevenzione InvestigatorsDietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione Trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico.Lancet. 1999; 354: 447-455Google Scholar). Observational studies have also demonstrated that blood levels of n-3 highly unsaturated fatty acids (HUFAs) are inversely related to the risk of coronary heart disease (16Simon J.A. Hodgkins M.L. Browner W.S. Neuhaus J.M. Bernert Jr., J.T. Hulley S.B. Serum fatty acids and the risk of coronary heart disease.Am. J. Epidemiol. 1995; 142: 469-476Google Scholar), sudden cardiac death (17Albert C.M. Campos H. Stampfer M.J. Ridker P.M. Manson J.E. Willett W.C. Ma J. Blood levels of long-chain n-3 fatty acids and the risk of sudden death.N. Engl. J. Med. 2002; 346: 1113-1118Google Scholar), and fatal ischemic heart disease (18Lemaitre R.N. King I.B. Mozaffarian D. Kuller L.H. Tracy R.P. Siscovick D.S. n-3 Polyunsaturated fatty acids, fatal ischemic heart disease, and nonfatal myocardial infarction in older adults: the Cardiovascular Health Study.Am. J. Clin. Nutr. 2003; 77: 319-325Scopus (351) Google Scholar). In as much as dietary intakes of n-3 PUFA are well indicated in the blood compartment (19Kobayashi M. Sasaki S. Kawabata T. Hasegawa K. Akabane M. Tsugane S. Single measurement of serum phospholipid fatty acid as a biomarker of specific fatty acid intake in middle-aged Japanese men.Eur. J. Clin. Nutr. 2001; 55: 643-650Google Scholar, 20Kuriki K. Nagaya T. Tokudome Y. Imaeda N. Fujiwara N. Sato J. Goto C. Ikeda M. Maki S. Tajima K. et al.Plasma concentrations of (n-3) highly unsaturated fatty acids are good biomarkers of relative dietary fatty acid intakes: a cross-sectional study.J. Nutr. 2003; 133: 3643-3650Google Scholar), it has been proposed that n-3 HUFA status in blood may be an important, modifiable, clinical biomarker for the prevention of various chronic diseases (21Harris W.S. Schacky C. Von The Omega-3 Index: a new risk factor for death from coronary heart disease?.Prev. Med. 2004; 39: 212-220Google Scholar, 22Lands W.E. Long-term fat intake and biomarkers.Am. J. Clin. Nutr. 1995; 61: 721-725Google Scholar). Increased blood status of DHA has also been associated with visual and cognitive functions, including improved visual acuity in infants (23Carlson S.E. Ford A.J. Werkman S.H. Peeples J.M. Koo W.W. Visual acuity and fatty acid status of term infants fed human milk and formulas with and without docosahexaenoate and arachidonate from egg yolk lecithin.Pediatr. Res. 1996; 39: 882-888Google Scholar) and decreased risk of suicide (24Huan M. Hamazaki K. Sun Y. Itomura M. Liu H. Kang W. Watanabe S. Terasawa K. Hamazaki T. Suicide attempt and n-3 fatty acid levels in red blood cells: a case control study in China.Biol. Psychiatry. 2004; 56: 490-496Google Scholar) and postpartum depression (25Otto S.J. de Groot R.H. Hornstra G. Increased risk of postpartum depressive symptoms is associated with slower normalization after pregnancy of the functional docosahexaenoic acid status.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 69: 237-243Google Scholar). A rapid, cost-effective, technique for determining n-3 fatty acids in blood would allow evidence to be collected regarding n-3 PUFA status and several aspects of health, including infant development (26Innis S.M. Gilley J. Werker J. Are human milk long-chain polyunsaturated fatty acids related to visual and neural development in breast-fed term infants?.J. Pediatr. 2001; 139: 532-538Google Scholar, 27Larque E. Demmelmair H. Koletzko B. Perinatal supply and metabolism of long-chain polyunsaturated fatty acids: importance for the early development of the nervous system.Ann. N.Y. Acad. Sci. 2002; 967: 299-310Google Scholar, 28Innis S.M. Essential fatty acids in growth and development.Prog. Lipid Res. 1991; 30: 39-103Google Scholar, 29Nettleton J.A. Are n-3 fatty acids essential nutrients for fetal and infant development?.J. Am. Diet. Assoc. 1993; 93: 58-64Google Scholar, 30Simopoulos A.P. Omega-3 fatty acids in health and disease and in growth and development.Am. J. Clin. Nutr. 1991; 54: 438-463Google Scholar, 31Uauy R. Hoffman D.R. Mena P. Llanos A. Birch E.E. Term infant studies of DHA and ARA supplementation on neurodevelopment: results of randomized controlled trials.J. Pediatr. 2003; 143: 17-25Google Scholar), neurodegenerative disease prevention (32Conquer J.A. Tierney M.C. Zecevic J. Bettger W.J. Fisher R.H. Fatty acid analysis of blood plasma of patients with Alzheimer's disease, other types of dementia, and cognitive impairment.Lipids. 2000; 35: 1305-1312Google Scholar), depression and behavioral disorders (33Brunner J. Parhofer K.G. Schwandt P. Bronisch T. Cholesterol, essential fatty acids, and suicide.Pharmacopsychiatry. 2002; 35: 1-5Google Scholar), enhancement of immune defenses (34Kew S. Banerjee T. Minihane A.M. Finnegan Y.E. Muggli R. Albers R. Williams C.M. Calder P.C. Lack of effect of foods enriched with plant- or marine-derived n-3 fatty acids on human immune function.Am. J. Clin. Nutr. 2003; 77: 1287-1295Google Scholar), and chronic inflammatory diseases (35Mori T.A. Beilin L.J. Omega-3 fatty acids and inflammation.Curr. Atheroscler. Rep. 2004; 6: 461-467Google Scholar). The present study presents two modifications of standard FAME analytical techniques. First, a fast GC method (36Mondello L. Casilli A. Tranchida P.Q. Cicero L. Dugo P. Dugo G. Comparison of fast and conventional GC analysis for citrus essential oils.J. Agric. Food Chem. 2003; 51: 5602-5606Google Scholar) was adapted to FAME analysis of human plasma samples to reduce the analysis time from >70 min per sample to <12 min and thereby allow for high throughput. Second, the tedious plasma lipid transesterification and extraction procedure was streamlined to a one-step procedure. These procedures include a simple and convenient “bench-top” procedure to increase the efficiency of FAME analysis in a traditional laboratory, as well as a modification that is amenable to automated robotic transmethylation reactions. The application of the present methodological advances promises the ability to perform FAME analysis on a greatly increased number of clinical and research samples. Acetyl chloride, 2[6]di-tert-butyl-p-cresol [butylated hydroxytoluene (BHT)], potassium carbonate, analytical-grade methanol, hexane, and toluene were purchased from Sigma-Aldrich Chemical Co. Porcine brain ceramide and sphingomyelin were purchased from Avanti Polar Lipids, Inc. BHT was added to methanol (50 μg BHT/ml methanol) to prevent fatty acid oxidation. The internal (23:0 methyl ester) and external (GLC-462) fatty acid standards were purchased from Nu-Chek The internal standard was in the a of Blood the was collected from an and was for min The plasma was in of and using the method of Lepage and Roy G. Roy C.C. Direct transesterification of all classes of lipids in a one-step reaction.J. Lipid Res. 1986; 27: 114-120Google Scholar) were the conventional GC in Jr., N. Reyzer M. Karanian J. Losses of arachidonic acid in rat liver after alcohol inhalation.Lipids. 1996; 31: 153-156Google Scholar) and a fast GC method for the present study from a method for the analysis of citrus essential (36Mondello L. Casilli A. Tranchida P.Q. Cicero L. Dugo P. Dugo G. Comparison of fast and conventional GC analysis for citrus essential oils.J. Agric. Food Chem. 2003; 51: 5602-5606Google Scholar). Conventional analyses were on an and fast GC on an with a and were controlled and was a GC The fast GC was with a supply to fast temperature ramping. The and application procedure was with a The transesterification method Lepage and Roy G. Roy C.C. Direct transesterification of all classes of lipids in a one-step reaction.J. Lipid Res. 1986; 27: 114-120Google Scholar) was as the for of new of the internal standard μg of methyl ester) and of plasma were added to a were and the were on Acetyl was added the The were and to a The samples were for min and were to prevent and samples were to the for the samples were on to and an of of a of The were and for 1 centrifugation for min to and the two The phase was and the extraction procedure was on the lower phase of hexane, and The were and to a of was to a GC and the was for FAME analysis were in in a A of the in the method of Lepage and Roy G. Roy C.C. Direct transesterification of all classes of lipids in a one-step reaction.J. Lipid Res. 1986; 27: 114-120Google Scholar) was prior to The of for sample of methanol, of chloride, and of the internal standard μg of methyl of plasma and of the were in The were and for The were to to was and the were for The phase was collected with a extraction procedure was as in order to lipid The was to and the was in of hexane, to GC and The for the from that in the simplified procedure The of for sample of methanol, of chloride, of the internal standard μg of methyl and of was added to prevent of the in the The transesterification was similar to the one-step method with the The plasma and with toluene were on a in for The of without internal was added a total of times during the h approximately The were and to to and 1 of was added to The were on a robotic and the using the The on the were the and of was was the of the procedure was a 1 min procedure was times for a total of The and of the phase was to the procedure for the simplified method. Porcine brain ceramide and sphingomyelin were also the and open-tube The effect of times from min to min for transesterification on concentrations of fatty acids as fast GC FAME analysis was also In of the μg of brain ceramide or sphingomyelin was with of the simple or the with the with toluene were in for min or for the without toluene were and for min or for The samples were to to and the were collected and for fast GC analyses as Conventional analyses were with a column and a a and with a 1 min to to and to with a min gas was with a linear of a of was Fatty acid analysis was of 1 of sample a of The temperature was with and gas of and The was The run time for a sample was with a sample time of fast GC the column was a of and from was as with a min to to with a min and to with a min conditions were as carrier gas was a of and a of and gas of and of of of time for a sample was with a sample time of The of fatty acids were using the is the of the fatty acids is to be is the for the internal is the of the internal standard in the in of and is the in of plasma sample in an The relative factor for peak was from a standard of fatty acids Nu-Chek The was from the from the for The application of fast GC demonstrated for citrus analysis (36Mondello L. Casilli A. Tranchida P.Q. Cicero L. Dugo P. Dugo G. Comparison of fast and conventional GC analysis for citrus essential oils.J. Agric. Food Chem. 2003; 51: 5602-5606Google Scholar) to FAME analysis of human plasma samples was in the with conventional GC in Jr., N. Reyzer M. Karanian J. Losses of arachidonic acid in rat liver after alcohol inhalation.Lipids. 1996; 31: 153-156Google Scholar), as demonstrated in The from the conventional and fast GC similar with a much faster analysis time for the fast Fast GC can the results in approximately of the time it with conventional run times of would allow the analysis of approximately samples per run times can be with column for using faster temperature ramping. an method of from to results in a run time of in of with on the present column may be for analyses of that not of fatty acid The smaller peak with the fast GC technique were of the high in conventional The of fast GC is gas an of various A. 1999; Scholar). is column diameters, and column and requires increased and faster temperature ramping. is and high are to reduce the risk of column The column was from of column is that it is the conventional column the for The fast GC were fast GC results and the GC The temperature which from an temperature of and min was and to results with a conventional GC in of peak order and of fatty and was the fast run The concentrations of fatty acids in human plasma as fast GC and conventional GC are in may be from and may be for clinical The and the of were similar in the and were for the plasma fatty acids and The were for fatty acids, were not would be for that of the total fatty The in were These that the present fast GC method is suitable for fatty acid analyses of human plasma and can reduce analytical run The reduction in GC analysis time is without methodological sample preparation time and is the for fatty acid of human plasma fatty acid concentrations using conventional and fast GC n-3 fatty of gas polyunsaturated fatty in a new of gas polyunsaturated fatty to the development of a fast GC method for mammalian fatty acid analysis, studies to the Lepage and Roy method G. Roy C.C. Direct transesterification of all classes of lipids in a one-step reaction.J. Lipid Res. 1986; 27: 114-120Google Scholar) of sample preparation were The results from the simplified and the robotic of the simplified method for transesterification are compared with the standard Lepage and Roy methodology with methods were using the fast GC The total fatty acid concentrations were similar of the method of the simplified and robotic transesterification methods a methodological in that all were a simple that be added in is a from the Lepage and Roy technique of the of the sample and the internal and and of after the to a are and the phase is to the Lepage and Roy method were the of the one-step without the for neutralization or centrifugation to A is amenable for application to and is in as much as several with the of are of fatty acid composition using various transesterification methods fast n-3 fatty in a new The fatty acid from the simplified produced results to of the standard Lepage and Roy The robotic method also results similar to of the standard Lepage and Roy The min of with the multiple of and the of the toluene in the open-tube for the method was for application to an automated method for preparation of using the The open-tube and the elimination of and centrifugation will greatly a robotic method. with the open-tube methodology were for min for fatty acids, as and a of approximately as compared with results with the Lepage and Roy method or the simplified These fatty acids to be associated with the sphingomyelin and ceramide of human plasma G. of and sphingomyelin in rat Scholar). and ceramide that are to and and as temperatures and times W.W. Lipid The Scholar), may be for with brain ceramide and sphingomyelin temperatures of and and times of h in the fatty acid concentrations of and and in the total fatty acid not conditions were for which of all fatty acids be that of the of reagent in the the was not to for fatty acid the to min and the temperature to with of reagent times of approximately These conditions to of all lipid that the were the as with the standard Lepage technique A extraction with sample a would be time-consuming for a the of was methodology and a as the of the from the standard Lepage and robotic in In the standard Lepage and the simplified method the robotic method The in fatty acid concentrations samples or were and were present a simplified and method for the analysis of fatty acids in human method was fast GC methodology for human plasma FAME analysis with a method the peak peak and similar the results with conventional GC as has been in the present for Jr., N. Reyzer M. Karanian J. Losses of arachidonic acid in rat liver after alcohol inhalation.Lipids. 1996; 31: 153-156Google Scholar). The present study also for the a simple transesterification procedure that is to perform and suitable for high FAME preparation from plasma samples in The of a and the elimination of several much of the and of methods G. Roy C.C. Improved recovery of fatty acid through direct transesterification without prior extraction or purification.J. Lipid Res. 1984; 25: 1391-1396Google Scholar, 8Rodriguez-Ruiz J. Belarbi E.H. Sanchez J.L.G. Alonso D.L. Rapid simultaneous lipid extraction and transesterification for fatty acid analyses.Biotechnol. Tech. 1998; 12: 689-691Google Scholar, 11Marangoni F. Colombo C. Galli C. A method for the direct evaluation of the fatty acid status in a drop of blood from a fingertip in humans: applicability to nutritional and epidemiological studies.Anal. Biochem. 2004; 326: 267-272Google Scholar). The robotic method produced results to with traditional the method may make possible clinical studies and population-based nutritional In have a procedure for transesterification of fatty acids from plasma samples that can be in and is amenable to for with the fast GC analytical the reduction in analytical and time promises to greatly fatty acid analyses for clinical research and in the research was in the of the on and of was in a

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