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Quantifying conversion of linoleic to arachidonic and other n-6 polyunsaturated fatty acids in unanesthetized rats

2010; Elsevier BV; Volume: 51; Issue: 10 Linguagem: Inglês

10.1194/jlr.m005595

ISSN

1539-7262

Autores

Fei Gao, Dale O. Kiesewetter, Lisa Chang, Stanley I. Rapoport, Miki Igarashi,

Tópico(s)

Animal Nutrition and Physiology

Resumo

Isotope feeding studies report a wide range of conversion fractions of dietary shorter-chain polyunsaturated fatty acids (PUFAs) to long-chain PUFAs, which limits assessing nutritional requirements and organ effects of arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids. In this study, whole-body (largely liver) steady-state conversion coefficients and rates of circulating unesterified linoleic acid (LA, 18:2n-6) to esterified AA and other elongated n-6 PUFAs were quantified directly using operational equations, in unanesthetized adult rats on a high-DHA but AA-free diet, using 2 h of intravenous [U-13C]LA infusion. Unesterified LA was converted to esterified LA in plasma at a greater rate than to esterified γ-linolenic (γ-LNA, 18:3n-6), eicosatrienoic acid (ETA, 20:3n-6), or AA. The steady-state whole-body synthesis-secretion (conversion) coefficient to AA equaled 5.4 × 10−3 min−1, while the conversion rate (coefficient × concentration) equaled 16.1 μmol/day. This rate exceeds the reported brain AA consumption rate by 27-fold. As brain and heart cannot synthesize significant AA from circulating LA, liver synthesis is necessary to maintain their homeostatic AA concentrations in the absence of dietary AA. The heavy-isotope intravenous infusion method could be used to quantify steady-state liver synthesis-secretion of AA from LA under different conditions in rodents and in humans. Isotope feeding studies report a wide range of conversion fractions of dietary shorter-chain polyunsaturated fatty acids (PUFAs) to long-chain PUFAs, which limits assessing nutritional requirements and organ effects of arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids. In this study, whole-body (largely liver) steady-state conversion coefficients and rates of circulating unesterified linoleic acid (LA, 18:2n-6) to esterified AA and other elongated n-6 PUFAs were quantified directly using operational equations, in unanesthetized adult rats on a high-DHA but AA-free diet, using 2 h of intravenous [U-13C]LA infusion. Unesterified LA was converted to esterified LA in plasma at a greater rate than to esterified γ-linolenic (γ-LNA, 18:3n-6), eicosatrienoic acid (ETA, 20:3n-6), or AA. The steady-state whole-body synthesis-secretion (conversion) coefficient to AA equaled 5.4 × 10−3 min−1, while the conversion rate (coefficient × concentration) equaled 16.1 μmol/day. This rate exceeds the reported brain AA consumption rate by 27-fold. As brain and heart cannot synthesize significant AA from circulating LA, liver synthesis is necessary to maintain their homeostatic AA concentrations in the absence of dietary AA. The heavy-isotope intravenous infusion method could be used to quantify steady-state liver synthesis-secretion of AA from LA under different conditions in rodents and in humans. WithdrawalsJournal of Lipid ResearchVol. 55Issue 5PreviewThe following three articles were withdrawn by Dr. Stanley Rapoport after an investigation by the National Institute of Health found that Dr. Fei Gao engaged in research misconduct by fabricating and/or falsifying data in this article. Please note that none of the other authors were implicated in any way. Full-Text PDF Open Access Linoleic acid (LA, 18:2n-6), a dietary essential n-6 polyunsaturated fatty acid (PUFA), is a precursor of arachidonic acid (AA, 20:4n-6) (1Carlson S.E. Carver J.D. House S.G. High fat diets varying in ratios of polyunsaturated to saturated fatty acid and linoleic to linolenic acid: a comparison of rat neural and red cell membrane phospholipids.J. Nutr. 1986; 116: 718-725Crossref PubMed Scopus (131) Google Scholar). 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Nevertheless, there is no general agreement whether AA or DHA is required in the diet and, if so, to what extent, as whole-body conversion estimates from their precursors range from 1 to 10% (7Brenna J.T. Salem Jr., N. Sinclair A.J. Cunnane S.C. alpha-Linolenic acid supplementation and conversion to n-3 long-chain polyunsaturated fatty acids in humans.Prostaglandins Leukot. Essent. Fatty Acids. 2009; 80: 85-91Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 8Das U.N. Long-chain polyunsaturated fatty acids in the growth and development of the brain and memory.Nutrition. 2003; 19: 62-65Crossref PubMed Scopus (142) Google Scholar, 9Innis S.M. The role of dietary n-6 and n-3 fatty acids in the developing brain.Dev. Neurosci. 2000; 22: 474-480Crossref PubMed Scopus (196) Google Scholar, 10Kris-Etherton P.M. Hill A.M. N-3 fatty acids: food or supplements?.J. Am. Diet. Assoc. 2008; 108: 1125-1130Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 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Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified {alpha}-linolenic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 749-758Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Gao F. Kiesewetter D. Chang L. Ma K. Rapoport S.I. Igarashi M. Whole-body synthesis secretion of docosahexaenoic acid from circulating eicosapentaenoic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 2463-2470Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 13Lehninger A.L. Nelson D.L. Cox M.M. Principles of Biochemistry. Worth, New York1993Google Scholar, 14Scott B.L. Bazan N.G. Membrane docosahexaenoate is supplied to the developing brain and retina by the liver.Proc. Natl. Acad. Sci. USA. 1989; 86: 2903-2907Crossref PubMed Scopus (382) Google Scholar, 15Vance J.E. Vance D.E. Lipoprotein assembly and secretion by hepatocytes.Annu. Rev. Nutr. 1990; 10: 337-356Crossref PubMed Scopus (55) Google Scholar). 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However, they do so at much lower rates, because their enzyme activities are at lower levels (21Bezard J. Blond J.P. Bernard A. Clouet P. The metabolism and availability of essential fatty acids in animal and human tissues.Reprod. Nutr. Dev. 1994; 34: 539-568Crossref PubMed Scopus (192) Google Scholar, 22Brenner R.R. The desaturation step in the animal biosynthesis of polyunsaturated fatty acids.Lipids. 1971; 6: 567-575Crossref PubMed Scopus (200) Google Scholar, 23Igarashi M. Ma K. Chang L. Bell J.M. Rapoport S.I. Dietary n-3 PUFA deprivation for 15 weeks upregulates elongase and desaturase expression in rat liver but not brain.J. Lipid Res. 2007; 48: 2463-2470Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 24Rapoport S.I. Rao J.S. Igarashi M. Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver.Prostaglandins Leukot. Essent. Fatty Acids. 2007; 77: 251-261Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Rat heart and brain have low capacities of synthesizing DHA and possibly AA from circulating unesterified α-LNA or LA, respectively, due to their low expression of synthesizing enzymes (23Igarashi M. Ma K. Chang L. Bell J.M. Rapoport S.I. Dietary n-3 PUFA deprivation for 15 weeks upregulates elongase and desaturase expression in rat liver but not brain.J. Lipid Res. 2007; 48: 2463-2470Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 25Igarashi M. Ma K. Chang L. Bell J.M. Rapoport S.I. Rat heart cannot synthesize docosahexaenoic acid from circulating alpha-linolenic acid because it lacks elongase-2.J. Lipid Res. 2008; 49: 1735-1745Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 26DeMar Jr., J.C. Lee H.J. Ma K. Chang L. Bell J.M. Rapoport S.I. Bazinet R.P. Brain elongation of linoleic acid is a negligible source of the arachidonate in brain phospholipids of adult rats.Biochim. Biophys. Acta. 2006; 1761: 1050-1059Crossref PubMed Scopus (129) Google Scholar, 27DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. alpha-Linolenic acid does not contribute appreciably to docosahexaenoic acid within brain phospholipids of adult rats fed a diet enriched in docosahexaenoic acid.J. Neurochem. 2005; 94: 1063-1076Crossref PubMed Scopus (163) Google Scholar). It is not clear whether the liver has a higher efficiency of conversion of α-LNA to DHA than of LA to AA, but data suggest this to be the case (28Brenner R.R. Peluffo R.O. Effect of saturated and unsaturated fatty acids on the desaturation in vitro of palmitic, stearic, oleic, linoleic, and linolenic acids.J. Biol. Chem. 1966; 241: 5213-5219Abstract Full Text PDF PubMed Google Scholar, 29El-Badry A.M. Graf R. Clavien P.A. Omega 3 - Omega 6: what is right for the liver?.J. Hepatol. 2007; 47: 718-725Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 30Gurr M.I. Harwood J.L. Frayn K.N. Lipid Biochemistry. Blackwell Science, Oxford, UK2002Crossref Google Scholar, 31Salem Jr., N. Wegher B. Mena P. Uauy R. Arachidonic and docosahexaenoic acids are biosynthesized from their 18- carbon precursors in human infants.Proc. Natl. Acad. Sci. USA. 1996; 93: 49-54Crossref PubMed Scopus (421) Google Scholar, 32Sanders T.A. Rana S.K. Comparison of the metabolism of linoleic and linolenic acids in the fetal rat.Ann. Nutr. Metab. 1987; 31: 349-353Crossref PubMed Scopus (39) Google Scholar). Exact rates of synthesis in the intact organism are not known, yet knowing them would help to estimate daily dietary requirements of α-LNA and LA compared with their elongated products and would provide a firmer basis for making nutritional recommendations (8Das U.N. Long-chain polyunsaturated fatty acids in the growth and development of the brain and memory.Nutrition. 2003; 19: 62-65Crossref PubMed Scopus (142) Google Scholar, 9Innis S.M. The role of dietary n-6 and n-3 fatty acids in the developing brain.Dev. Neurosci. 2000; 22: 474-480Crossref PubMed Scopus (196) Google Scholar). To address this issue, we developed a heavy-isotope intravenous infusion method to determine rates of whole-body, steady-state DHA synthesis from circulating unesterified α-LNA and eicosapentaenoic acid (EPA, 20:5n-3) in the unanesthetized rat (11Gao F. Kiesewetter D. Chang L. Ma K. Bell J.M. Rapoport S.I. Igarashi M. Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified {alpha}-linolenic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 749-758Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Gao F. Kiesewetter D. Chang L. Ma K. Rapoport S.I. Igarashi M. Whole-body synthesis secretion of docosahexaenoic acid from circulating eicosapentaenoic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 2463-2470Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar); this method might be extended for human studies. Briefly, the method involves infusing the isotopically labeled precursor intravenously at a constant rate, sampling arterial plasma concentrations of unesterified and esterified labeled precursor and elongation products as a function of time, and then applying operational equations to calculate steady-state synthesis-secretion coefficients and rates of the esterified products when taking plasma volume into account. With this method, we reported that whole-body steady-state rate of conversion of unesterified α-LNA to esterified DHA, in rats fed a high DHA-containing diet, equaled 9.84 μmol/day. To compare this rate to the rate of AA synthesis from LA in rats fed the same diet, in the present study we infused [U-13C]LA intravenously for 2 h and determined rates of appearance of esterified AA in arterial plasma, as well as of other n-6 PUFA elongation products, γ-LNA and ETA. We applied our operational equations to estimate steady-state synthesis coefficients and rates, and plasma turnovers and half-lives (11Gao F. Kiesewetter D. Chang L. Ma K. Bell J.M. Rapoport S.I. Igarashi M. Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified {alpha}-linolenic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 749-758Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Gao F. Kiesewetter D. Chang L. Ma K. Rapoport S.I. Igarashi M. Whole-body synthesis secretion of docosahexaenoic acid from circulating eicosapentaenoic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 2463-2470Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). [U-13C]LA was purchased from Cambridge Isotope Laboratories (Andover, MA), and was purified by high-performance liquid chromatography (HPLC) (Agilent, Palo Alto, CA) with a Symmetry® C18 column (9.2 × 250 mm, 5 μm, Agilent) before use. Purity was determined to be >95% by HPLC, and the concentration of purified [U-13C]LA was determined by gas chromatography (GC). Diheptadecanoate phosphatidylcholine (Di-17:0 PC), free heptadecanoic acid (17:0), n-6 PUFA standards (LA, γ-LNA, ETA, and AA), pentafluorobenzyl (PFB) bromide, and diisopropylamine were purchased from Sigma-Aldrich (St. Louis, MO). Solvents were HPLC-grade and were purchased from Fisher Scientific (Fair Lawn, NJ) or Sigma-Aldrich. This protocol was approved by the Animal Care and Use Committee of the Eunice Kennedy Schriver National Institute of Child Health and Human Development and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 80-23). Adult male Fischer-344 (CDF) rats (4 months old) were purchased from Charles River Laboratories (Portage, MI) and housed in a facility with regulated temperature, humidity, and a 12 h light/12 h dark cycle. They were acclimated for one week before surgery in this facility and had free access to water and rodent chow (NIH-31 Auto18-4). The chow contained soybean oil and fishmeal; it had 4% by weight crude fat. Its fatty acid composition has been reported (27DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. alpha-Linolenic acid does not contribute appreciably to docosahexaenoic acid within brain phospholipids of adult rats fed a diet enriched in docosahexaenoic acid.J. Neurochem. 2005; 94: 1063-1076Crossref PubMed Scopus (163) Google Scholar). Of n-3 PUFAs, α-LNA, EPA, and DHA contributed 5.1%, 2.0%, and 2.3% of total fatty acid, respectively, whereas n-6 PUFAs LA and AA contributed 47.9% and 0.02%, respectively. The surgery and infusion procedures have been described in detail (11Gao F. Kiesewetter D. Chang L. Ma K. Bell J.M. Rapoport S.I. Igarashi M. Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified {alpha}-linolenic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 749-758Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The rats were provided food the night before surgery. During the surgery, recovery from anesthesia (3–4 h), and the 2 h infusion period, they did not have access to food. A rat was anesthetized with 1–3% halothane, and polyethylene catheters (PE 50, Clay Adams, Becton Dickinson, Sparks, MD) filled with heparinized isotonic saline (100 IU/ml) were surgically implanted in the right femoral artery and vein. After it had recovered from anesthesia, the rat was infused via the femoral vein catheter with 3 μmol/100 g body weight [U-13C]LA, dissolved in 5 mM 4-(2-hydroxy-methyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) containing 50 mg/ml fatty acid-free bovine serum albumin, at a constant rate of 0.021 ml/min. During the 2 h, body temperature was maintained at 36–38°C using a feedback-heating element (YSI Indicating Temperature Controller, Yellow Springs Instruments, Yellow Springs, OH), and 2 ml of normal saline was injected subcutaneously to prevent dehydration. Arterial blood (130 μl) was collected in centrifuge tubes (polyethylene-heparin lithium fluoride-coated; Beckman) at 0, 0.5, 1.0, 2.0, 3.0, 5.0, 8.0, 10.0, 20, 30, 60, and 90 min of infusion. At 120 min, 500 μl blood was removed, and the rat was euthanized by an overdose of sodium pentobarbital (100 mg/kg iv). The blood samples were centrifuged at 13,000 rpm for 1 min, and plasma was collected and kept at −80°C until use. Plasma lipid extraction and pentafluorobenzyl (PFB) derivatization procedures have been reported (11Gao F. Kiesewetter D. Chang L. Ma K. Bell J.M. Rapoport S.I. Igarashi M. Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified {alpha}-linolenic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 749-758Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Appropriate amounts of internal standards (di-17:0 PC and free 17:0) were added to plasma, and then KOH solution was added. "Stable" lipids (phospholipids, triacylglycerol, and cholesteryl ester) containing esterified fatty acids were extracted with hexane twice. The remaining lower phase was acidified with HCl, and unesterified fatty acids were extracted with hexane twice. The extracted esterified fatty acids in stable lipids were hydrolyzed (10% KOH in methanol) at 70°C to release their free fatty acids, which were extracted with hexane and dried under N2. A freshly made PFB derivatizing reagent (PFB: diisopropylamine: acetonitrile, 10:100:1000) was added to the sample residue of hexane extraction and shaken for 15 min at room temperature. The sample was evaporated again to dryness under N2 and redissolved in 100 μl of hexane. The fatty acid PFB esters from the plasma samples were analyzed by GC/MS as described (11Gao F. Kiesewetter D. Chang L. Ma K. Bell J.M. Rapoport S.I. Igarashi M. Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified {alpha}-linolenic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 749-758Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Nonlabeled and labeled n-6 PUFAs were monitored by selected ion mode (SIM) of the base peak (M-PFB). The concentration of each n-6 PUFA was quantified by relating its peak area to the area of the internal standard, using an experimentally determined response factor for each fatty acid. Steady-state conversion rates of circulating unesterified LA to esterified AA and other elongated n-6 PUFAs were calculated by the method of Gao et al. (11Gao F. Kiesewetter D. Chang L. Ma K. Bell J.M. Rapoport S.I. Igarashi M. Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified {alpha}-linolenic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 749-758Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Gao F. Kiesewetter D. Chang L. Ma K. Rapoport S.I. Igarashi M. Whole-body synthesis secretion of docosahexaenoic acid from circulating eicosapentaenoic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 2463-2470Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). At steady-state secretion, the rate of change of labeled esterified n-6 PUFA i (i = LA, γ-LNA, ETA, or AA) in plasma is the sum of its rates of appearance and loss according to the following differential equation with constant coefficients,VplasmadCi,es∗dt=k1,iCLA,unes∗−k−1,iCi,es∗(Eq. 1) where Vplasma is plasma volume (ml) Ci,es∗; (nmol/ml) is plasma concentration of esterified stable isotope labeled i; CLA,unes∗ (nmol/ml) is plasma unesterified [U-13C]LA concentration; t is time (min); k1,i is the synthesis-secretion rate coefficient (ml/min) of conversion of unesterified labeled LA to esterified labeled PUFA i [which relates the esterified plasma PUFA concentration to the unesterified LA concentration times plasma volume]; and k−1,i is the disappearance rate coefficient (ml/min) of esterified labeled PUFA i from plasma. There is no isotope effect, so that k1,i and k−1,i are valid for unlabeled as well as labeled PUFAs. As reported (11Gao F. Kiesewetter D. Chang L. Ma K. Bell J.M. Rapoport S.I. Igarashi M. Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified {alpha}-linolenic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 749-758Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Gao F. Kiesewetter D. Chang L. Ma K. Rapoport S.I. Igarashi M. Whole-body synthesis secretion of docosahexaenoic acid from circulating eicosapentaenoic acid in unanesthetized rats.J. Lipid Res. 2009; 50: 2463-2470Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), esterified labeled PUFAs during intravenous labeled precursor infusion start to appear in rat plasma after a 30–60 min delay, after which their concentrations rapidly rise but later tend to rise more slowly due to the loss from plasma (see Fig. 2). To estimate their steady-state rates of secretion, we fit the following sigmoidal equation using nonlinear least squares (Origin 7.0 software, Originlab, Northhampton, MA) to esterified concentration × plasma volume data plotted against time, for each esterified n-6 PUFA i (see Fig. 2),VplasmaCi,es∗A1+exp[(t−t0)/B]+C(Eq. 2) where A, B, C are best-fit constants, and t0 = 0 is time (min) at the beginning of infusion and t is time of infusion. The first derivative of equation 2 was determined for each esterified PUFA i in each rat as a function of time. Its maximum value, Smax,i nmol/min, occurs when steady-state liver secretion is best approximated and was taken to represent the left-hand side of equation 1. A steady-state synthesis-secretion coefficient, (min−1), equal to k1,i/Vplasma. was calculated as,ki∗=Smax,iCLA,unes∗×Vplasma(Eq. 3) is proportional to the net rate of the elongation and desaturation reactions, and unlike k1,I, is independent of plasma volume; thus, it is a marker for evaluating net efficiency of synthesis. The synthesis-secretion rate (nmol/min) Ji of unlabeled esterified PUFA i from unlabeled unesterified LA equals,Ji=Smax,i×CLA,unesCLA,unes∗(Eq. 4) where CLA,unes is the unesterified total (labeled and unlabeled) plasma LA concentration. Because plasma concentration Ci,es (nmol/ml) of esterified PUFA i was constant during the study, turnover Fi (min−1) and half-life t1/2,i (min) of esterified plasma PUFA i due explicitly to conversion from unesterified LA equal, respectively,Fi=JiCi,es×Vplasma(Eq. 5) andt1/2,i=0.693Fi(Eq. 6) Steady-state synthesis-secretion rates Ji and plasma turnover and half-lives were calculated for i = LA, γ-LNA, ETA, and AA by (Eq. 1), (Eq. 2), (Eq. 3), (Eq. 4), (Eq. 5), (Eq. 6). Data are given as means ± SD. Concentrations and ratios of unlabeled esterified and unesterified n-6 PUFAs in arterial plasma were determined before the 2 h [U-13C]LA infusion (Table 1) and agree with reported values in adult male rats fed the same diet (27DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. alpha-Linolenic acid does not contribute appreciably to docosahexaenoic acid within brain phospholipids of adult rats fed a diet enriched in docosahexaenoic acid.J. Neurochem. 2005; 94: 1063-1076Crossref PubMed Scopus (163) Google Scholar). The concentrations of unesterified LA, γ-LNA, ETA, and AA equaled 219 ± 25, 3.4 ± 1.6, 8.7 ± 4.2, and 27.8 ± 6.9 nmol/ml plasma, respectively, whereas their respective esterified concentrations equaled 961 ± 80, 25.3 ± 4.3, 60.6 ± 9.8, and 809 ± 51 nmol/ml plasma. Mean unesterified to esterified concentration ratios equaled 0.23, 0.14, 0.20, and 0.034 for LA, γ-LNA, ETA, and AA, respectively (Table 1). The unesterified LA concentration was used to calculate Ji for each esterified PUFA i by equation 4, and Ji was used to calculate respective plasma half-lives and turnovers by equations 5 and 6.TABLE 1Unlabeled concentrations and concentration ratios of unesterified and esterified PUFAs in arterial plasma before [U-13C]LA infusionn-6 PUFAUnesterified PUFAEsterified PUFARatio of unesterified to esterified PUFAnmol/ml plasmanmol/ml plasmaLA (18:2 n-6)219 ± 25961 ± 800.23 ± 0.02γ-LNA (18:3 n-6)3.4 ± 1.625.3 ± 4.30.14 ± 0.04ETA (20:3 n-6)8.7 ± 4.260.6 ± 9.80.20 ± 0.06AA (20:4 n-6)27.8 ± 6.9809 ± 510.034 ± 0.011DTA (22:4 n-6)ND16.1 ± 5.9–DPA (22:5 n-6)ND10.8 ± 3.2–Data are mean ± SD (n = 5). AA, arachidonic acid; DPA, docosapentaenoic acid; DTA, docosatetraenoic acid; ETA, eicosatrienoic acid; LA, linoleic acid; γ-LNA, γ-linolenic acid; ND, not detected. Open table in a new tab Data are mean ± SD (n = 5). AA, arachidonic acid; DPA, docosapentaenoic acid; DTA, docosatetraenoic acid; ETA, eicosatrienoic acid; LA, linoleic acid; γ-LNA, γ-linolenic acid; ND, not detected. A constant unesterified arterial plasma concentration was achieved within 10 min after the start of intravenous [U-13C]LA infusion (Fig. 1). No other labeled unesterified fatty acid was detected in plasma during the 2 h infusion. The mean concentration of unesterified [U-13C]LA, CLA, unes∗, equaled 1.10 ± 0.10 nmol/ml. Esterified [13C]LA was detected in plasma 30 min after infusion had started. Esterified longer-chain n-6 PUFAs could be detected at 30–60 min, after which their concentrations increased rapidly with time but then began to plateau. At all times, esterified plasma [13C]LA was higher than the concentration of each of its three esterified labeled elongation products (Table 2).TABLE 2.[13C]labeled esterified n-6 PUFA concentrations in arterial plasma during intravenous infusion of [U-13C]LAInfusion Timen-6 PUFAaIndicates the 13C-labeled n-6 PUFA.30 min60 min90 min120 minConcentration (nmol/ml plasma)LA (18:2 n-6)5.66 ± 1.007.05 ± 0.558.87 ± 1.0311.1 ± 1.8γ-LNA (18:3 n-6)ND0.60 ± 0.070.84 ± 0.131.13 ± 0.26ETA (20:3 n-6)ND0.08 ± 0.060.36 ± 0.050.45 ± 0.10AA (20:4 n-6)ND0.08 ± 0.020.23 ± 0.040.30 ± 0.04Data are mean ± SD (n = 5). AA, arachidonic acid; ETA, eicosatrienoic acid; LA, linoleic acid; γ-LNA, γ-linolenic acid; ND, not detect

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