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• W2108233320 abstract "We quantified the rates of incorporation of α-linolenic acid (α-LNA; 18:3n-3) into “stable” lipids (triacylglycerol, phospholipid, cholesteryl ester) and the rate of conversion of α-LNA to docosahexaenoic acid (DHA; 22: 6n-3) in the liver of awake male rats on a high-DHA-containing diet after a 5-min intravenous infusion of [1-14C]α-LNA. At 5 min, 72.7% of liver radioactivity (excluding unesterified fatty acid radioactivity) was in stable lipids, with the remainder in the aqueous compartment. Using our measured specific activity of liver α-LNA-CoA, in the form of the dilution coefficient λα-LNA-CoA, we calculated incorporation rates of unesterified α-LNA into liver triacylglycerol,phospholipid, and cholesteryl ester as 2,401, 749, and 9.6 nmol/s/g × 10−4, respectively, corresponding to turnover rates of 3.2, 8.7, and 2.9%/min and half-lives of 8–24 min. A lower limit for the DHA synthesis rate from α-LNA equaled 15.8 nmol/s/g × 10−4 (0.5% of the net in corporation rate). Thus, in rats on a high-DHA-containing diet, rates of β-oxidation and esterification of α-LNA into stable liver lipids are high, whereas its conversion to DHA is comparatively low and insufficient to supply significant DHA to the brain. High incorporation and turnover rates likely reflect a high secretion rate by liver of stable lipids within very low density lipoproteins. We quantified the rates of incorporation of α-linolenic acid (α-LNA; 18:3n-3) into “stable” lipids (triacylglycerol, phospholipid, cholesteryl ester) and the rate of conversion of α-LNA to docosahexaenoic acid (DHA; 22: 6n-3) in the liver of awake male rats on a high-DHA-containing diet after a 5-min intravenous infusion of [1-14C]α-LNA. At 5 min, 72.7% of liver radioactivity (excluding unesterified fatty acid radioactivity) was in stable lipids, with the remainder in the aqueous compartment. Using our measured specific activity of liver α-LNA-CoA, in the form of the dilution coefficient λα-LNA-CoA, we calculated incorporation rates of unesterified α-LNA into liver triacylglycerol,phospholipid, and cholesteryl ester as 2,401, 749, and 9.6 nmol/s/g × 10−4, respectively, corresponding to turnover rates of 3.2, 8.7, and 2.9%/min and half-lives of 8–24 min. A lower limit for the DHA synthesis rate from α-LNA equaled 15.8 nmol/s/g × 10−4 (0.5% of the net in corporation rate). Thus, in rats on a high-DHA-containing diet, rates of β-oxidation and esterification of α-LNA into stable liver lipids are high, whereas its conversion to DHA is comparatively low and insufficient to supply significant DHA to the brain. High incorporation and turnover rates likely reflect a high secretion rate by liver of stable lipids within very low density lipoproteins. Docosahexaenoic acid (DHA; 22:6n-3) is a nutritionally essential PUFA that must be obtained directly through the diet or be synthesized from its dietary essential precursor, α-linolenic acid (α-LNA; 18:3n-3). Mammalian tissues can convert α-LNA to DHA through serial steps of desaturation and elongation with final peroxisomal chain shortening (1Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim. Biophys. Acta. 2000; 1486: 219-231Crossref PubMed Scopus (662) Google Scholar, 2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar, 3DeMar Jr., J.C. Ma K. Bell J.M. Rapoport S.I. Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids.J. Neurochem. 2004; 91: 1125-1137Crossref PubMed Scopus (165) Google Scholar). Both Δ5 and Δ6 desaturases participate in this conversion, but the Δ6 desaturase is considered to be rate-limiting (1Sprecher H. Metabolism of highly unsaturated n-3 and n-6 fatty acids.Biochim. Biophys. Acta. 2000; 1486: 219-231Crossref PubMed Scopus (662) Google Scholar, 4Nakamura M.T. Nara T.Y. Essential fatty acid synthesis and its regulation in mammals.Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 145-150Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Human and rat Δ5 and Δ6 desaturases are expressed abundantly in brain, liver, and heart (5Cho H.P. Nakamura M. Clarke S.D. Cloning, expression, and fatty acid regulation of the human delta-5 desaturase.J. Biol. Chem. 1999; 274: 37335-37339Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 6Cho H.P. Nakamura M.T. Clarke S.D. Cloning, expression, and nutritional regulation of the mammalian delta-6 desaturase.J. Biol. Chem. 1999; 274: 471-477Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). Controversy remains regarding the extent of DHA synthesis in brain from α-LNA, compared with its delivery by blood to brain as DHA synthesized from α-LNA in the liver. In immature rats, Scott and Bazan (7Scott 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 (383) Google Scholar) concluded that the brain does not synthesize its own DHA to a significant extent but that DHA converted from α-LNA in the liver can contribute to brain DHA via the blood stream. In adult rats fed a high-DHA-containing diet [2.3% (w/w) of total fatty acid], we recently used an in vivo kinetic pulse-labeling model that confirmed a low synthesis rate of DHA from α-LNA in brain (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar), as proposed for immature rats (see above), and also showed that α-LNA was largely β-oxidized or esterified unchanged into brain phospholipid. We did not examine the rate of DHA synthesis from α-LNA in the liver of these rats, nor did we explore the kinetics of other pathways of liver α-LNA metabolism. We thought it important to try to do so here. A number of experimental procedures have been used to examine hepatic α-LNA metabolism, including studying isolated hepatocytes (8Jakobsson A. Ericsson J. Dallner G. Metabolism of fatty acids and their incorporation into phospholipids of the mitochondria and endoplasmic reticulum in isolated hepatocytes determined by isolation of fluorescence derivatives.Biochim. Biophys. Acta. 1990; 1046: 277-287Crossref PubMed Scopus (14) Google Scholar), infusing the liver in situ (9Bretillon L. Chardigny J.M. Noel J.P. Sebedio J.L. Desaturation and chain elongation of [1-14C]mono-trans isomers of linoleic and alpha-linolenic acids in perfused rat liver.J. Lipid Res. 1998; 39: 2228-2236Abstract Full Text Full Text PDF PubMed Google Scholar), and injecting labeled α-LNA intravenously in an animal and measuring its distribution in different liver compartments (10Sinclair A.J. Crawford M.A. The incorporation of linolenic aid and docosahexaenoic acid into liver and brain lipids of developing rats.FEBS Lett. 1972; 26: 127-129Crossref PubMed Scopus (66) Google Scholar). None of these studies measured the specific activity of liver α-LNA-CoA, the precursor pool for α-LNA esterification into stable lipids and for steps of DHA synthesis. This prevented calculating exact incorporation and synthesis rates as well as turnover rates and half-lives of α-LNA in stable liver lipids. In this study, we used controlled intravenous infusion of [1-14C]α-LNA in unanesthetized adult male rats to produce steady-state plasma and liver α-LNA-CoA specific activities, and measured the radioactive plasma exposure of the liver (input function) to calculate rate parameters as we did for the brain (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar, 11Rapoport S.I. In vivo approaches and rationale for quantifying kinetics and imaging brain lipid metabolic pathways.Prostaglandins Other Lipid Mediat. 2005; 77: 185-196Crossref PubMed Scopus (35) Google Scholar, 12Rapoport S.I. Chang M.C. Spector A.A. Delivery and turnover of plasma-derived essential PUFAs in mammalian brain.J. Lipid Res. 2001; 42: 678-685Abstract Full Text Full Text PDF PubMed Google Scholar). We also microwaved the liver to stop enzymatic activity and to prevent ischemia-related changes in liver unesterified fatty acid and acyl-CoA concentrations (13Rabin O. Deutsch J. Grange E. Pettigrew K.D. Chang M.C.J. Rapoport S.I. Purdon A.D. Changes in cerebral acyl-CoA concentrations following ischemia-reperfusion in awake gerbils.J. Neurochem. 1997; 68: 2111-2118Crossref PubMed Scopus (31) Google Scholar, 14Rabin O. Chang M.C. Grange E. Bell J. Rapoport S.I. Deutsch J. Purdon A.D. Selective acceleration of arachidonic acid reincorporation into brain membrane phospholipid following transient ischemia in awake gerbil.J. Neurochem. 1998; 70: 325-334Crossref PubMed Scopus (28) Google Scholar, 15Bazinet R.P. Lee H.J. Felder C.C. Porter A.C. Rapoport S.I. Rosenberger T.A. Rapid high-energy microwave fixation is required to determine the anandamide (N-arachidonoylethanolamine) concentration of rat brain.Neurochem. Res. 2005; 30: 597-601Crossref PubMed Scopus (63) Google Scholar). The rats were fed a diet containing a high DHA content [2.3% (w/w) of total fatty acid weight, contributing 0.23% of total food energy intake] compared with much lower fractional DHA weights and percentage energy intakes of current and recommended human diets in the United States (16Bourre J.M. Durand G. Pascal G. Youyou A. Brain cell and tissue recovery in rats made deficient in n-3 fatty acids by alteration of dietary fat.J. Nutr. 1989; 119: 15-22Crossref PubMed Scopus (131) Google Scholar, 17Van Aerde J.E. Clandinin M.T. Controversy in fatty acid balance.Can. J. Physiol. Pharmacol. 1993; 71: 707-712Crossref PubMed Scopus (20) Google Scholar, 18Kris-Etherton P.M. Taylor D.S. Yu-Poth S. Huth P. Moriarty K. Fishell V. Hargrove R.L. Zhao G. Etherton T.D. Polyunsaturated fatty acids in the food chain in the United States.Am. J. Clin. Nutr. 2000; 71: 179-188Crossref PubMed Google Scholar, 19Wijendran V. Hayes K.C. Dietary n-6 and n-3 fatty acid balance and cardiovascular health.Annu. Rev. Nutr. 2004; 24: 597-615Crossref PubMed Scopus (470) Google Scholar). They had been studied previously by us when we determined α-LNA kinetic parameters for their brains (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar). In this study, we used the labeled and unlabeled plasma α-LNA concentrations, plasma input functions, and specific activities that we had measured in that brain study (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar). An abstract of part of this work has been presented (20Igarashi, I., K. Ma, L. Chang, J. M. Bell, J. C. DeMar, and S. I. Rapoport. 2005. α-linolenic acid is minimally converted to docosahexaenoic acid in brain and liver of adult rats fed a DHA-containing diet. (Abstract in the 35th Neuroscience Annual Meeting. Washington D.C., November 12–16, 2005).Google Scholar). [1-14C]α-LNA in 100% ethanol was purchased from Perkin-Elmer Life Sciences, NEN Life Science Products (Boston, MA). Its specific activity was 54 mCi/mmol and its purity was 98% (determined by HPLC and scintillation counting). Di-heptadecanoate phosphatidylcholine (di-17:0 PC), free heptadecanoic acid (17:0), heptadecanoyl-CoA (17:0-CoA), and acyl-CoA standards for HPLC, as well as TLC standards for cholesterol, triglycerides,and cholesteryl esters, were purchased from Sigma-Aldrich (St. Louis, MO). Standards for general fatty acid methyl esters (FAMEs) for GC and HPLC were from NuChek Prep (Elysian, MN). FAMEs for unique n-3 PUFAs (20:4n-3, 22:5n-3, 24:5n-3, and 24:6n-3) were from Larodan Fine Chemicals (Malmoä, Sweden). 6-p-Toluidine-2-naphthalene sulfonic acid was from Acros Organics (Fair Lawn, NJ). Liquid scintillation cocktail (Ready Safe™) was purchased from Beckman Coulter (Fullerton, CA). Solvents were HPLC-grade and were purchased from Fisher Scientific (Fair Lawn, NJ) or EMD Chemicals (Gibbstown, NJ). Other chemicals and reagents, unless noted otherwise, were purchased from Sigma-Aldrich or Fisher Scientific. The protocol was approved by the Animal Care and Use Committee of the National Institute of Child Health and Human Development and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication 80-23). Adult (2 months old) Fischer-344 (CDF) male rats were purchased from Charles River Laboratories (Portage, MI) and were housed for 4 weeks before study in an animal facility with regulated temperature, humidity, and 12 h light/12 h dark cycle. They had free access to water and to rodent chow formulation NIH-31 which contains 4% (w/w) crude fat (Zeigler Bros., Gardners, PA). The fatty acid composition of this chow is described in a prior report (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar). Saturated and monounsaturated fatty acids contributed 20.1% and 22.5%, respectively, to its total fatty acid content. The n-3 PUFAs α-LNA, eicosapentaenoic acid (EPA; 20:5n-3), and DHA contributed 5.1%, 2.0%, and 2.3%, respectively, whereas the n-6 PUFAs linoleic acid (LA; 18:2n-6) and arachidonic acid contributed 47.9% and 0.02%, respectively. The 9:1 ratio of LA to α-LNA is close to the recommended dietary balance, but the energy contribution of DHA (0.23% of energy consumed) and of EPA plus DHA (0.45% of energy) in the diet were high compared with the EPA plus DHA contributions in the average diet in the United States (<0.1%) as well as in the recommended diet (0.25%) (16Bourre J.M. Durand G. Pascal G. Youyou A. Brain cell and tissue recovery in rats made deficient in n-3 fatty acids by alteration of dietary fat.J. Nutr. 1989; 119: 15-22Crossref PubMed Scopus (131) Google Scholar, 17Van Aerde J.E. Clandinin M.T. Controversy in fatty acid balance.Can. J. Physiol. Pharmacol. 1993; 71: 707-712Crossref PubMed Scopus (20) Google Scholar, 18Kris-Etherton P.M. Taylor D.S. Yu-Poth S. Huth P. Moriarty K. Fishell V. Hargrove R.L. Zhao G. Etherton T.D. Polyunsaturated fatty acids in the food chain in the United States.Am. J. Clin. Nutr. 2000; 71: 179-188Crossref PubMed Google Scholar, 19Wijendran V. Hayes K.C. Dietary n-6 and n-3 fatty acid balance and cardiovascular health.Annu. Rev. Nutr. 2004; 24: 597-615Crossref PubMed Scopus (470) Google Scholar). A rat weighing 300 ± 39 g (SD) was anesthetized with 1–3% halothane. Polyethylene catheters filled with heparinized saline (100 IU/ml) were surgically implanted into the right femoral artery and vein, after which the skin was closed and treated with 1% lidocaine for pain control, as described previously (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar). The rat was loosely wrapped in a fast-setting plaster cast that was taped to a wooden block, then allowed to recover from anesthesia for 3–4 h. Body temperature was maintained at 36–38°C using a feedback-heating device. Surgery, which took ∼20 min, was performed between 10:00 AM and noon. Animals were provided with food the night before surgery. Each rat was infused via the femoral vein catheter with 500 μCi/kg [1-14C]α-LNA (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar). An aliquot of [1-14C]α-LNA in ethanol was dried under nitrogen gas, and the residue was dissolved in 5 mM HEPES buffer (pH 7.4) containing 50 mg/ml fatty acid-free BSA to a final volume of 1.3 ml. The mixture was sonicated at 40°C for 20 min and mixed by vortexing. A computer-controlled variable-speed pump (No. 22; Harvard Apparatus, South Natick, MA) was used to infuse the 1.3 ml solution at a rate of 0.223 (1 − e−1.92t) ml/min (t in min), which was designed to rapidly establish a steady-state plasma radioactivity (21Washizaki K. Smith Q.R. Rapoport S.I. Purdon A.D. Brain arachidonic acid incorporation and precursor pool specific activity during intravenous infusion of unesterified [3H]arachidonate in the anesthetized rat.J. Neurochem. 1994; 63: 727-736Crossref PubMed Scopus (93) Google Scholar). Arterial blood was collected in centrifuge tubes at 0, 0.25, 0.5, 0.75, 1.5, 3, 4, and 5 min after starting the infusion. At 5 min, the rat was euthanized by an overdose of sodium pentobarbital (100 mg/kg i.v), and its head and torso were immediately subjected to high-energy focused beam microwave irradiation (5.5 kW, 3.4 s) (model S6F; Cober Electronics; Stamford, CT). Liver weight was recorded, and tissue samples that were confirmed visually to be browned or “cooked” were removed and stored at −80°C until assay. The arterial blood samples were centrifuged at 13,000 rpm for 5 min, and plasma was collected and frozen at −80°C. Total lipids from liver and plasma were extracted by the procedure of Folch, Lees, and Sloane Stanley (22Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Total lipid extracts were separated into neutral lipid subclasses by TLC on silica gel 60 plates (EM Separation Technologies; Gibbstown, NJ), as described previously (3DeMar Jr., J.C. Ma K. Bell J.M. Rapoport S.I. Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids.J. Neurochem. 2004; 91: 1125-1137Crossref PubMed Scopus (165) Google Scholar). The bands were scraped, and the silica gel was used directly to quantify radioactivity by scintillation counting and to prepare FAMEs (described below). In addition to measuring unlabeled total phospholipid concentrations, an aliquot of total lipid extract was added to a tube and dried using a SpeedVac. To measure individual phospholipids, total lipid extracts were separated into phospholipid classes by TLC on silica gel 60 plates (23Skipski V.P. Good J.J. Barclay M. Reggio R.B. Quantitative analysis of simple lipid classes by thin-layer chromatography.Biochim. Biophys. Acta. 1968; 152: 10-19Crossref PubMed Scopus (146) Google Scholar). The bands were scraped and added to the tube. The silica gel was used directly to analyze phospholipid concentrations. The phosphorous assay followed the method of Rouser, Fkeischer, and Yamamoto (24Rouser G. Fkeischer S. Yamamoto A. Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots.Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2880) Google Scholar). To quantify total and free cholesterol and triacylglycerol concentrations, lipid extracts were dried using a SpeedVac, and the residue was dissolved in isopropanol. Total cholesterol and free cholesterol concentrations were determined with a commercial kit (BioVision Research Products, Mountain View, CA), as was the triacylglycerol concentration (Sigma-Aldrich). The FAMEs were used for the GC and HPLC analyses. Lipid and aqueous extracts were methylated with 1% H2SO4-methanol for 3 h at 70°C (3DeMar Jr., J.C. Ma K. Bell J.M. Rapoport S.I. Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids.J. Neurochem. 2004; 91: 1125-1137Crossref PubMed Scopus (165) Google Scholar, 25Makrides M. Neumann M.A. Byard R.W. Simmer K. Gibson R.A. Fatty acid composition of brain, retina, and erythrocytes in breast- and formula-fed infants.Am. J. Clin. Nutr. 1994; 60: 189-194Crossref PubMed Scopus (694) Google Scholar). Before methylation, an appropriate quantity of di-17:0 PC (for triacylglycerol, phospholipid, and cholesteryl ester) or of 17:0 fatty acid (for unesterified fatty acid) was added to the sample as an internal standard. Samples for measuring radioactivity were placed in scintillation vials and dissolved with liquid scintillation cocktail (Ready Safe™ plus 1% glacial acetic acid), and their radioactivity was determined using a liquid scintillation analyzer (2200CA, TRI-CARB®; Packard Instruments, Meriden, CT). Fatty acid concentrations of liver lipids and plasma unesterified fatty acids were determined by GC. GC separation and analysis were performed as described (3DeMar Jr., J.C. Ma K. Bell J.M. Rapoport S.I. Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids.J. Neurochem. 2004; 91: 1125-1137Crossref PubMed Scopus (165) Google Scholar), with fatty acid concentrations (nmol/g liver) calculated by proportional comparison of GC peak areas with the area of the 17:0 internal standard. The concentration of unesterified α-LNA in plasma also was determined this way as 41 ± 13 nmol/ml (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar). FAMEs from liver lipids were analyzed by HPLC by the method of Aveldano, VanRollins, and Horrocks (26Aveldano M.I. VanRollins M. Horrocks L.A. Separation and quantitation of free fatty acids and fatty acid methyl esters by reverse phase high pressure liquid chromatography.J. Lipid Res. 1983; 24: 83-93Abstract Full Text PDF PubMed Google Scholar) with modifications. The FAMEs were dissolved in acetonitrile, and the solution was fractionated by reverse-phase column HPLC using a pump (System GOLD 126; Beckman Coulter) outfitted with an ultraviolet light detector (UV/VIS-151; Gilson, Middleton, WI) and an on-line continuous scintillation counter β-RAM detector (β-RAM model 2; IN/US Systems). The reverse-phase column, Luna 5 μ C18 (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar) (5 μM particle size, 4.6 × 250 mm), was from Phonomenex (Torrance, CA). Chromatography was performed using a linear gradient system of water and acetonitrile. The acetonitrile was held at 85% for 30 min, increased to 100% over 10 min, and held again at 100% for 20 min. The flow rate was 1.0 ml/min. The ultraviolet light detector was set at 205 nm. Long-chain acyl-CoAs were extracted from microwaved liver using an affinity chromatography method with slight modifications (27Deutsch J. Grange E. Rapoport S.I. Purdon A.D. Isolation and quantitation of long-chain acyl-coenzyme A esters in brain tissue by solid-phase extraction.Anal. Biochem. 1994; 220: 321-323Crossref PubMed Scopus (101) Google Scholar). After 5 nmol of heptadecanoyl-CoA (17:0-CoA) was added as an internal standard to ∼1 g of liver, the sample was homogenized in 25 mM KH2PO4 (Tissuemizer; Tekmar, Cincinnati, OH). The homogenate was adjusted with isopropanol and acetonitrile to isopropanol /25 mM KH2PO4/acetonitrile (1:1:2, v/v/v), then sonicated using a probe sonicator (model W-225; Misonix, Farmingdale, NY). A small volume (∼3% of total) of saturated (NH4)2SO4 solution was added to the homogenate to precipitate proteins, after which the sample was mixed vigorously for 5 min and centrifuged. The supernatant was washed with hexane (equal volume) to remove nonpolar lipids and then diluted with a 1.25-fold volume of 25 mM KH2PO4. Extracting nonpolar lipids with hexane was important, as it markedly improved HPLC separation of acyl-CoAs compared with samples that were not extracted (data not shown). The solution was passed three times through an oligonucleotide purification cartridge (ABI Masterpiece™, OPC®; Applied Biosystems, Foster City, CA), and then the cartridge was washed with 25 mM KH2PO4. Acyl-CoA species were eluted with a small volume of isopropanol-1 mM glacial acetic acid (75:25, v/v). HPLC separation and analysis for acyl-CoA were performed as described (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar). Under the HPLC system, 14:0-CoA, EPA-CoA, and α-LNA-CoA coeluted as a single peak (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar). This peak was collected and saponified with 2% (w/v) KOH/ethanol at 100°C for 45 min and acidified with HCl, and then fatty acids were extracted with hexane. The unesterified fatty acids were converted to FAMEs and separated by HPLC to measure radioactivity. The concentrations of the FAMEs that came from the acyl-CoA species also were determined by GC. Thus, the concentrations of 14:0, EPA, and α-LNA in the original acyl-CoA peak were determined by proportional comparison of their GC peak areas with each other. The general pulse-labeling equations for determining the in vivo kinetics of a fatty acid in any organ, after the intravenous infusion of a radiolabeled fatty acid to produce a steady-state plasma radioactivity, have been described elsewhere (2DeMar Jr., J.C. Ma K. Chang L. Bell J.M. Rapoport S.I. α-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 (167) Google Scholar, 11Rapoport S.I. In vivo approaches and rationale for quantifying kinetics and imaging brain lipid metabolic pathways.Prostaglandins Other Lipid Mediat. 2005; 77: 185-196Crossref PubMed Scopus (35) Google Scholar, 12Rapoport S.I. Chang M.C. Spector A.A. Delivery and turnover of plasma-derived essential PUFAs in mammalian brain.J. Lipid Res. 2001; 42: 678-685Abstract Full Text Full Text PDF PubMed Google Scholar, 21Washizaki K. Smith Q.R. Rapoport S.I. Purdon A.D. Brain arachidonic acid incorporation and precursor pool specific activity during intravenous infusion of unesterified [3H]arachidonate in the anesthetized rat.J. Neurochem. 1994; 63: 727-736Crossref PubMed Scopus (93) Google Scholar, 28Robinson P.J. Noronha J. DeGeorge J.J. Freed L.M. Nariai T. Rapoport S.I. A quantitative method for measuring regional in vivo fatty-acid incorporation into and turnover within brain phospholipids: review and critical analysis.Brain Res. Brain Res. Rev. 1992; 17: 187-214Crossref PubMed Scopus (229) Google Scholar). These equations were applied to a model for liver α-LNA metabolism (see Fig. 3 below). In this model, unesterified plasma α-LNA enters the liver unesterified α-LNA pool (not shown in Fig. 3 below), from where it is delivered to the α-LNA-CoA pool through the action of an acyl-CoA synthetase. From there, it can be converted to DHA-CoA by elongation and desaturation enzymes. Both α-LNA and DHA (as well as n-3 PUFA conversion intermediates) can be transacylated from their acyl-CoA forms into phospholipid, triacylglycerol, or cholesteryl ester (called “stable” lipids). The esterified fatty acids can be released from these stable lipids back to the unesterified liver fatty acid pool (data not shown) and then activated again to acyl-CoA by an acyl-CoA synthetase, or they can be secreted into blood while esterified within the stable lipids as packaged in VLDLs. A fatty acid in the liver acyl-CoA pool also can be transferred by carnitine O-palmitoyl transferase to mitochondria for β-oxidation (29Gavino G.R. Gavino V.C. Rat liver outer mitochondrial carnitine palmitoyltransferase activity towards long-chain polyunsaturated fatty acids and their CoA esters.Lipids. 1991; 26: 266-270Crossref PubMed Scopus (101) Google Scholar). Aqueous radiolabeled β-oxidation fragments that are formed (predominantly acetyl-CoA) then can be recycled into cholesterol, saturated long-chain fatty acids, or any number of other products. Incorporation coefficients k*i(α-LNA) (ml/s/g liver), representing the transfer of unesterified [1-14C]α-LNA from plasma into stable liver lipid i, were calculated as follows: ki(α-LNA)*=cliver,i*(α-LNA)(T)∫0TCplasma(α-LNA)dt*(Eq. 1)" @default.
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• W2108233320 title "Low liver conversion rate of α-linolenic to docosahexaenoic acid in awake rats on a high-docosahexaenoate-containing diet" @default.
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