Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [13C]DHA in phosphatidylcholine

Abstract

The amount and distribution of [13C]docosahexaenoic acid (DHA) in plasma, platelet, and erythrocyte lipid classes were followed as a function of time (1 to 72 h) in young adults after ingestion of a single dose of [13C]DHA esterified in a phosphatidylcholine (PC), in using gas chromatography combustion–isotope ratio mass spectrometry. [13C]DHA first appeared in plasma non-esterified fatty acids (NEFA) and triglycerides (TG), with a maximal appearance at 6 h and a further decline, then being delayed 3-fold compared to [13C]DHA ingested in triglycerides. Lysophosphatidylcholine (LPC) was also enriched in [13C]DHA, due mainly to earlier hepatic secretion, and plateaued at 6 h, whereas phosphatidylethanolamine (PE) and phosphatidylcholine (PC) containing [13C]DHA plateaued at 9 h. The labeling of erythrocyte and platelet phospholipids exhibited different kinetics, probably involving different metabolic pathways for [13C]DHA incorporation in cell membranes. Computation of the relative contribution of LPC and NEFA for delivery of [13C]DHA to blood cells showed that the supply to platelets occurred through NEFA. In contrast, [13C]DHA was carried by both LPC and NEFA to erythrocytes, which differs from what was previously been observed after intake of triglycerides labeled with [13C]DHA where LPC was the only source of [13C]DHA for erythrocytes. We conclude that the lipid form of ingested DHA affects markedly its kinetics and partly its metabolic fate.—Lemaitre-Delaunay, D., C. Pachiaudi, M. Laville, J. Pousin, M. Armstrong, and M. Lagarde. Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [13C]DHA in phosphatidylcholine. J. Lipid Res. 1999. 40: 1867–1874. The amount and distribution of [13C]docosahexaenoic acid (DHA) in plasma, platelet, and erythrocyte lipid classes were followed as a function of time (1 to 72 h) in young adults after ingestion of a single dose of [13C]DHA esterified in a phosphatidylcholine (PC), in using gas chromatography combustion–isotope ratio mass spectrometry. [13C]DHA first appeared in plasma non-esterified fatty acids (NEFA) and triglycerides (TG), with a maximal appearance at 6 h and a further decline, then being delayed 3-fold compared to [13C]DHA ingested in triglycerides. Lysophosphatidylcholine (LPC) was also enriched in [13C]DHA, due mainly to earlier hepatic secretion, and plateaued at 6 h, whereas phosphatidylethanolamine (PE) and phosphatidylcholine (PC) containing [13C]DHA plateaued at 9 h. The labeling of erythrocyte and platelet phospholipids exhibited different kinetics, probably involving different metabolic pathways for [13C]DHA incorporation in cell membranes. Computation of the relative contribution of LPC and NEFA for delivery of [13C]DHA to blood cells showed that the supply to platelets occurred through NEFA. In contrast, [13C]DHA was carried by both LPC and NEFA to erythrocytes, which differs from what was previously been observed after intake of triglycerides labeled with [13C]DHA where LPC was the only source of [13C]DHA for erythrocytes. We conclude that the lipid form of ingested DHA affects markedly its kinetics and partly its metabolic fate.—Lemaitre-Delaunay, D., C. Pachiaudi, M. Laville, J. Pousin, M. Armstrong, and M. Lagarde. Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [13C]DHA in phosphatidylcholine. J. Lipid Res. 1999. 40: 1867–1874. Marine oils, which contain high levels of polyunsaturated fatty acids (PUFA) of the n–3 family such as docosahexaenoic (DHA, 22:6n–3) and eicosapentaenoic (EPA, 20:5n–3) acids (1Bang H.O. Dyerberg J. Hjorne N. The composition of food consumed by greenland Eskimos.Acta Med. Scand. 1976; 200: 69-73Google Scholar), may have beneficial roles in the prevention of cardiovascular diseases (2Leaf A. Cardiovascular effects of fish oils. Beyond the platelet.Circulation. 1990; 82: 624-628Google Scholar, 3Dyerberg J. Bang H.O. Lipid metabolism, atherogenesis, and haemostasis in Eskimos: the role of prostaglandin–3 family.Haemostasis. 1979; 8: 227-233Google Scholar). N–3 PUFA exert antiatherothrombotic effects through modulation of blood lipids (4Harris W.E. Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review.J. Lipid Res. 1989; 30: 785-807Google Scholar) and lipoproteins (5Fisher W.R. Zech L.A. Stacpoole P.W. Apolipoprotein B metabolism in hypertriglyceridemic diabetic patients administrated either a fish oil- or vegetable oil-enriched diet.J. Lipid Res. 1998; 39: 388-401Google Scholar). Furthermore, it has been found that long chain polyunsaturated n–3 fatty acids could decrease plasma triglyceride levels (6Hansen J.B. Grimsgaard S. Nilsen H. Nordoy A. Bonna K.H. Effects of highly purified eicosapentaenoic acid and docosahexaenoic acid on fatty acid absorption, incorporation into serum phospholipids and postprandial triglyceridemia.Lipids. 1998; 33: 131-138Google Scholar) and cause a prolongation of bleeding time. The incorporation of n–3 PUFA in cell membranes also modifies eicosanoid production resulting in altered platelet and leukocyte reactivities (7Prescott S.M. The effect of eicosapentaenoic acid on leukotriene B production by human neutrophils.J. Biol. Chem. 1984; 259: 7615-7621Google Scholar, 8Leaf A. Weber P.C. Cardiovascular effects of n–3 fatty acids.N. Engl. J. Med. 1988; 318: 549-557Google Scholar). DHA, a minor component of human plasma lipids, is a major fatty acid of phospholipids in the brain and retina (9Neuringer M. Anderson G.J. Connor W.E. The essentiality of n–3 fatty acids for the development and function of the retina and brain.Annu. Rev. Nutr. 1988; 8: 517-541Google Scholar). This fatty acid is required for the development of visual acuity and learning in humans (10Makrides M. Neumann M.A. Gibson R.A. Is dietary docosahexaenoic acid essential for term infants?.Lipids. 1996; 31: 115-119Google Scholar, 11Werkman S.H. Carlson S.E. A randomized trial of visual attention of preterm infants fed docosahexaenoic acid until nine months.Lipids. 1996; 31: 91-97Google Scholar). Although the biological properties of DHA have been documented, the mechanisms by which this fatty acid is taken up by the brain and blood cells, the pathway of its absorption, and its transport and dynamic exchanges within blood lipid pools for the assimilation by human tissues remain largely unknown. Lysophosphatidylcholine (LPC), a second form of blood phospholipid (12Nelson G.J. The phospholipid composition of plasma in various mammalian species.Lipids. 1967; 2: 323-328Google Scholar), could be a transport system for PUFA (13Brindley D.N. Hepatic secretion of lysophosphatidylcholine: a novel transport system for polyunsaturated fatty acids and choline.J. Nutr. Biochem. 1993; 4: 442-449Google Scholar) when secreted by the liver and could enhance the PUFA absorption and distribution (14Viola G. Mietto L. Secchi F.E. Ping L. Bruni A. Absorption and distribution of arachidonate in rats receiving lysophospholipids by oral route.J. Lipid Res. 1993; 34: 1843-1852Google Scholar). The transport of DHA to target cells has been reported in humans after a single ingestion of this 13C-labeled fatty acid esterified in triglycerides (15Brossard N. Croset M. Normand S. Pousin J. Lecerf J. Laville M. Tayot J.L. Lagarde M. Human plasma albumin transports [13C]docosahexaenoic acid in two lipid forms to blood cells.J. Lipid Res. 1997; 38: 1571-1582Google Scholar). It has been shown that plasma albumin carries DHA in two forms, as non-esterified fatty acids (NEFA) which supply this fatty acid to platelets, and as LPC which delivers it to erythrocytes. The mechanism of cellular uptake of fatty acids presumably involves a carrier-mediated and a passive transmembrane translocation (16Glatz J.F.C. Luiken J.J.P.F. vanNieuwenhoven F.A. VanderVusse G.J. Molecular mechanism of cellular uptake and intracellular translocation of fatty acids.Prostaglandins Leukot. Essent. Fatty Acids. 1997; 57: 3-9Google Scholar). On the other hand, the uptake of PUFA by red blood cells may be considered as a marker for their accretion into brain (17Innis S.M. Plasma and red blood cell fatty acid values as indexes of essential fatty acids in the developing organs of infants fed with milk or formulas.J. Pediatr. 1992; 120: S78-S86Google Scholar) and retina (18Simonelli F. Manna C. Romano N. Nunziata G. Voto O. Rinaldi E. Evaluation of fatty acids in membrane phospholipids of erythrocytes in retinitis pigmentosa patients.Ophthalmic. Res. 1996; 28: 93-98Google Scholar) lipids. It is now accepted that the fatty acid–albumin complex is a major route by which fatty acids reach the brain (19Dhopeshwarkar G.A. Uptake and transport of fatty acids into the brain and the role of the blood-brain barrier system.Adv. Lipid Res. 1973; 11: 109-142Google Scholar, 20Anderson G.J. Connor W.E. Uptake of fatty acids by the developing rat brain.Lipids. 1988; 23: 286-290Google Scholar, 21Anderson G.J. Tso P.S. Connor W.E. Incorporation of chylomicron fatty acids into the developing rat brain.J. Clin. Invest. 1994; 93: 2764-2767Google Scholar). More recently, it has been found that 2-acyl-LPC bound to albumin could be an efficient delivery form of unsaturated fatty acids to the developing rat brain (22Thies F. Delachambre M.C. Bentejac M. Lagarde M. Lecerf J. Unsaturated fatty acids esterified in 2-acyl-1-lysophosphatidylcholine bound to albumin are more efficiently taken up by the young rat brain than the unesterified form.J. Neurochem. 1992; 59: 1110-1116Google Scholar). The brain preferentially takes up DHA from lysoPC-DHA compared with non-esterified DHA (23Thies F. Pillon C. Moliere P. Lagarde M. Lecerf J. Preferential incorporation of sn-2 lysoPC DHA over unesterified DHA in young rat brain.Am. J. Physiol. 1994; 36: R1273-R1279Google Scholar). According to in vivo studies in humans and animals (24Galli C. Sirtori C.R. Mosconi C. Medini L. Gianfranceschi G. Vaccarino V. Scolastico C. Prolonged retention of doubly labeled phophatidylcholine in human plasma and erythrocytes after oral administration.Lipids. 1992; 27: 1005-1012Google Scholar), oral administration of labeled PC leads to the rapid appearance of labeled PC in plasma, with rather weak formation of labeled TG. The use of double-labeled PC, [3H]choline and a 14C-labeled fatty acid, showed that the 3H/14C ratio of plasma PC was only half of the absorbed PC (22Thies F. Delachambre M.C. Bentejac M. Lagarde M. Lecerf J. Unsaturated fatty acids esterified in 2-acyl-1-lysophosphatidylcholine bound to albumin are more efficiently taken up by the young rat brain than the unesterified form.J. Neurochem. 1992; 59: 1110-1116Google Scholar). After oral administration, PC is more than 90% absorbed by the intestinal mucosa via conversion to LPC and reesterification (25Zierenberg O. Grundy S.M. Intestinal absorption of polyenephophatidylcholine in man.J. Lipid Res. 1982; 23: 1136-1142Google Scholar, 26Le Kim D. Betzing H. Intestinal absorption of polyunsaturated phophatidylcholine in the rat.Hoppe Seylers Z. Physiol. Chem. 1976; 357: 1321-1331Google Scholar). The absorbed PC is then incorporated into chylomicrons (26Le Kim D. Betzing H. Intestinal absorption of polyunsaturated phophatidylcholine in the rat.Hoppe Seylers Z. Physiol. Chem. 1976; 357: 1321-1331Google Scholar) and, after degradation to the TG-rich particles, taken up by the high density lipoprotein (HDL) fraction (25Zierenberg O. Grundy S.M. Intestinal absorption of polyenephophatidylcholine in man.J. Lipid Res. 1982; 23: 1136-1142Google Scholar). A small proportion of these PC is taken up without prior hydrolysis. Little information is available on PC in relation to the possible incorporation into human tissues and circulating cells. The aim of the present study was to assess whether circulating [13C]DHA-PC and subsequent [13C]DHA-LPC in plasma are preferential forms of DHA transport compared to [13C]DHA-TG and non-esterified [13C]DHA. This has been done by comparing the kinetics of [13C]DHA appearance in plasma and red cells after a single dose of DHA ingested either in PC (the present study) or in TG (our previous study). Docosahexaenoic acid and eicosapentaenoic acid methyl esters as well as internal standards for gas chromatography (heptadecaenoic acid, cholesteryl heptadecanoate, phosphatidylcholine diheptadecanoyl, phosphatidylethanolamine diheptadecanoyl, triheptadecanoyl glycerol, lysophosphatidylcholine heptadecanoyl) were purchased from Sigma-Chimie (L'Isle d'Abeau, France). All solvents were analytical or HPLC grade from SDS (Peypin, France) or Merck (Darmstadt, Germany). Silica gel 60 plates and Superspher HPLC column were purchased from Merck (Darmstadt, Germany). Phosphatidylcholine (PC) containing [13C]DHA was produced by growing a microalgae (Crypthecodinium cohnii) strain in a defined medium containing d-[1-13C]glucose. After harvesting the cells by centrifugation, the biomass was freeze-dried and lipids were extracted with hexane–isopropanol–water 5:5:1 (v/v/v). Phosphatidylcholine purification was carried out on a silica column (55 × 5 cm). The lipid extract (60 g) was first eluted with butanol and acetone. Then phosphatidylcholine was eluted by ethanol–water 85:15 (v/v) and ethanol–butanol 1:2 (v/v) mixtures and purified later by preparative liquid chromatography. The fatty acid composition of the PC is reported in Table 1. DHA represented 54.70% of the total fatty acids with a 13C abundance of 8 atom%. It was esterified mainly at the sn-2 position.TABLE 1.Fatty acid composition of phosphatidylcholine ingestedFatty Acidsmol%12:00.3914:015.5616:019.5718:01.3916:1n–70.1818:1n–98.3022:6n–3 (DHA)54.70Data are expressed as mol% of total fatty acids. Open table in a new tab Data are expressed as mol% of total fatty acids. Three healthy male volunteers (age: 35.6 ± 1.1; body mass index: 20.8 ± 0.5 kg/m2; glucose: 5.0 ± 0.5 mm; blood cholesterol: 5.2 ± 0.6 mm; triglycerides: 1.36 ± 0.44 g/L) signed a written consent form after being informed about the purpose and modalities of the study. The scientific protocol was approved by the institutional human ethical committee. Subjects were instructed to maintain their usual diet, but to exclude alcohol 24 h before the protocol as well as marine food a week before and during the experiment. Subjects, fasted overnight, consumed 250 mg of tracer PC mixed in yogurt (3.5% lipids, 3.7% proteins, 5% carbohydrates) and had a breakfast (120 g of bread and 30 g of jam) immediately after. At various periods of time (1 to 72 h) after ingestion, blood was taken by venipuncture on ACD as anti-coagulant (0.8% citric acid, 2% citrate, 2.45% dextrose, pH 4.5). A blood sample was also collected just before the tracer ingestion to measure the basal 13C abundance in various lipid pools. Blood samples transferred to plastic tubes kept in ice were centrifuged at 100 g for 15 min at 4°C to obtain platelet-rich plasma. This was acidified to pH 6.4 with citric acid and centrifuged at 900 g for 12 min at 4°C to obtain the platelet pellet and platelet-poor plasma. This plasma was collected, centrifuged at 1850 g for 10 min at 4°C to eliminate the remaining platelets, and frozen at -20°C with 5 × 10-5 m butyl hydroxytoluene (BHT). The platelet pellet was resuspended into a Tyrode-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer (in mm: 135 NaCl, 2.68 KCl, 0.46 NaHPO4, 1 MgCl2, 5 HEPES, 5.5 glucose, 1 EDTA; pH 6.4) (27Lagarde M. Bryon P.A. Guichardant M. Dechavanne M. A simple and efficient method for platelet isolation from their plasma.Thromb. Res. 1980; 17: 581-588Google Scholar). This cell suspension was immediately centrifuged at 1000 g for 10 min at 4°C. Then the new platelet pellet was resuspended in 2 ml of Tyrode-HEPES buffer (pH 7.35) without EDTA and frozen at -20°C with BHT (5 × 10-5 m). For preparation of erythrocytes, the pellet resulting from centrifugation of whole blood at 100 g was diluted in Tyrode-HEPES buffer and centrifuged at 100 g for 10 min at 4°C. After removing the supernatant, red blood cells were diluted with 9% NaCl and centrifuged at 2000 g for 10 min at 4°C. This procedure was repeated twice and Tyrode-HEPES was added to red cells prior to freezing, in the presence of 5 × 10-5 m BHT. Total lipids were extracted from blood cells and plasma according to Bligh and Dyer (28Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). Lysophosphatidylcholine (LPC) from plasma and erythrocytes was prepared by spotting the lipid extracts on silica gel 60 plates which were developed, with chloroform–methanol–water 65:25:4 (v/v/v) as the mobile phase. Other lipids from plasma were first separated into neutral lipids and NEFA with the solvent mixture chloroform–methanol 80:8 (v/v). A second chromatography step was performed with chloroform–methanol–40% aqueous methylamine 60:20:5 (v/v/v) to separate phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Neutral lipids were extracted from the silica gel with chloroform–methanol 90:10 (v/v) and further fractionated into cholesteryl esters (CE) and triglycerides (TG) on thin-layer chromatography (TLC) plates developed with hexane–diethyl ether–acetic acid 80:20:1 (v/v/v). PE and PC from platelets and erythrocytes were purified by TLC using chloroform–methanol–40% aqueous methylamine as the mobile phase after predeveloping the plates with chloroform–methanol 80:8 (v/v). Lipid classes were scraped off the plate and treated for 90 min with 5% H2SO4 in methanol to obtain FA methyl esters (29Bowyer D.E. Leat W.M. Howard A.N. Gresham G.A. The determination of the fatty acid composition of serum lipids separated by thin-layer chromatography, and a comparison with column chromatography.Biochim. Biophys. Acta. 1963; 70: 423-431Google Scholar). Heptadecanoic acid, cholesteryl heptadecanoate, diheptadecanoyl phosphatidylcholine, diheptadecanoyl phosphatidylethanolamine, triheptadecanoyl glycerol, and heptadecanoyl lysophosphatidylcholine were added before lipid extraction, when appropriate, and the absolute amounts of fatty acid methyl esters were determined by GLC analysis, relative to the known amount of added 17:0. A Delsi chromatograph model DI200 equipped with a Ross injector and a SP2380 capillary column (30 m × 0.32 mm) (Supelco, Bellefonte, PA) was used for these analyses. The oven temperature was held at 145°C for 5 min and raised to 215°C at 2°C/min (30Croset M. Bayon Y. Lagarde M. Incorporation and turnover of eicosapentaenoic and docosahexaenoic acids in human blood platelets in vitro.Biochem. J. 1992; 281: 309-316Google Scholar). HPLC with a 5 μm Superspher 100 C18 column (4.6 mm internal diameter × 250 mm) and isocratic elution with acetonitrile–water 80:20 (v/v), at a flow rate of 2 ml/min was used to separate fatty acyl methyl esters (31Brossard N. Pachiaudi C. Croset M. Normand S. Lecerf J. Chirouze V. Riou J.P. Tayot J.L. Lagarde M. Stable isotope tracer and gas-chromatography combustion isotope ratio mass spectrometry to study the in vivo compartmental metabolism of docosahexaenoic acid.Anal. Biochem. 1994; 220: 192-199Google Scholar). The fatty acyl methyl esters were detected by UV absorbance at 210 nm. The DHA methyl ester was collected, taken to dryness under nitrogen, and redissolved into isooctane for GCC–IRMS analysis. Analyses were performed using an isotope ratio mass spectrometer (SIRA 12; VG Isogas, Middlewitch, UK) interfaced with a gas–liquid chromatograph (5890A; Hewlett-Packard, Evry, France) equipped with a Ross injector (240°C) and a capillary column (30 m × 0.32 mm, Supelco, Bellefonte, PA). The oven temperature was maintained at 180°C for 0.5 min and then raised at 25°C/min to 245°C where it was maintained for 5 min. Helium was used as a carrier gas (injector inlet pressure, 26 psi). The gas–liquid chromatograph effluent was diverted to a flame ionization detector until elution of DHA methyl ester. The effluent was then switched to a catalytic furnace filled with CuO and maintained at 800°C. The effluent from the furnace containing CO2 and H2O generated from DHA methyl ester, flowing in the continuous helium flux, was driven into a water trap at -100°C before its ionization by electron impact in the source of the GCC–IRMS. The different isotopomers were collected onto three different collectors at mass-to-charge ratio (m/z) 44 (main ion: 12C16O16O), 45 (13C16O16O, 12C16O17O), and 46 (12C17O17O, 12C16O18O, 13C16O17O). Ions at m/z 44, 45, and 46 were continuously recorded until the return of the 44 signal to the baseline value. Isotopomers at m/z 44 and 45 were measured, leading to the 13C/12C ratio. Before and after the CO2 peaks generated from DHA, a CO2 sample reference of known enrichment, calibrated against the international standard (Pee Dee Belemnite: PDB), was automatically injected into the mass spectrometer (32Burdge G.C. Kelly F.J. Postle A.D. Mechanisms of hepatic phophatidylcholine synthesis in the developing guinea pig: contributions of acyl remodelling of N-methylation of phosphatidylethanolamine.Biochem. J. 1993; 290: 67-73Google Scholar). The 13C/12C ratios of the sample and of the reference were used to calculate the δ per 1000 value (δ13C‰) of the sample using the following formula: δ13C%o=(13C/12C)sample-(13C/12C)reference(13C/12C)reference×103Eq. 1) where the reference is the international PDB standard, which is CO2 obtained from the carbonate shell of the cretaceous mollusc, Belemnitella americana, from the Pee Dee formation in South Carolina. The [13C]DHA appearance in biological samples is also expressed as the absolute amount of [13C]DHA in lipid pools by multiplying the [13C]DHA dilution by the endogenous DHA concentration quantitated by GLC. To determine the contribution of NEFA and LPC to [13C]DHA delivery to platelets and erythrocytes, a mathematical model, briefly presented here, was used. It should be noted that NEFA and LPC are the only providers for platelets and erythrocytes for the mathematical model considered here. As all possible transport phenomenon are not accounted for, the mathematical model is based mainly on the dynamic aspect of the total amount of [13C]DHA present in the providers. Two time-dependent functions, t1 and t2, express the total amount of [13C]DHA carried by NEFA and LPC. C1 and C2, also time-dependent functions, represent the total amount of [13C]DHA in platelets and erythrocytes, verifying the relationships: ddtC1(t)=αγ1[t1(t)-t1as]+βγ2[t2(t)-t2as]Eq. 2) ddtC2(t)=(1-α)γ1[t1(t)-t1as]+(1-β)γ2[t2(t)-t2as]Eq. 3) The constants α, β, γ1, γ2 represent the relative contribution of NEFA to platelets, the relative contribution of LPC to erythrocytes, the fractions of NEFA and LPC involved in our process, respectively. These constants were identified by means of experimental data. The constants t1as and t2as stand for the asymptotic values of functions t1 and t2 reached when the process does not evolve anymore. These constants were obtained from experimental data. By integrating equations (2) and (3) from time ti to time ti+1 the following expressions are obtained for functions C1 and C2: C1(ti+1)-C1(ti)=αγ1∫titi+1[t1(s)-t1as]ds++βγ2∫titi+1[t2(s)-t2as]dsEq. 4) C2(ti+1)-C2(ti)=(1−α)γ1∫titi+1[t1(s)-t1as]ds++(1−β)γ2∫titi+1[t2(s)-t2as]dsEq. 5) By choosing the collection of times {ti}i=1i=9 as the times where measurements took place (t1 = 1, t2 = 2, t3 = 4, t4 = 6, t5 = 9, t6 = 12, t7 = 24, t8 = 48, t9 = 72) and by assuming that function t1 and t2 vary linearly in [ti, ti+1], numerical values for functions C1 and C2 depending on α, β, γ1, γ2 at times tj are obtained. If Ĉ1(ti), Ĉ2(t1) denote the measured values of total amounts of [13C]DHA in platelets and in erythrocytes, respectively, then the constants α, β, γ1, γ2 are calculated according to a least square minimizing procedure between these values and the ones provided by numerical simulations equations Eq. 4), Eq. 5): α,β,γ1,γ2inf−∑i=1i=9[C^1(ti)−C1(ti)]2+[C^2(ti)−C2(ti)]2Eq. 6) Natural 13C abundance was measured on pure, commercially available DHA methyl ester. Analyses were performed with 300 ng of the methyl ester, which gave a similar intensity to the standard CO2. δ13C‰ values were -26.6 ± 0.2 (mean of 10 determinations), with less than 1% variation. This reproducibility ensured precise determinations of low 13C abundance after intake. [13C]DHA appeared rapidly in TG and NEFA. Six hours after ingestion of the tracer, the labeling was maximal in both fractions (Fig. 1A), with δ13C‰ values of +1329 ± 313 and +538 ± 110, respectively. The abundance at 6 h was 2.47-fold higher in the TG than in NEFA. Between 6 and 24 h after ingestion, the [13C]DHA abundance decreased in both fractions to reach a plateau, with minimal values of +76.1 ± 39.4 in TG and +26.2 ± 19.4 in NEFA observed at 72 h post-ingestion. In contrast (Fig. 1B), the labeling of PE and PC was more progressive until 9 h and plateaued from 9 to 72 h where the [13C]DHA abundance was higher in PE than in PC. For example, the δ13C‰ values were +197.9 ± 121.5 and +97.2 ± 43.6, respectively, at 24 h and +180.7 ± 89.3 and 100.2 ± 43.9, respectively, at 48 h. In PE, the abundance tended to decrease at the later points of the kinetics with δ13C‰ values of +197.9 ± 121 at 24 h and +123.6 ± 63.1 at 72 h. For LPC, a plateau was reached from 6h with δ13C‰ values slightly lower than those of PC between 9 and 72 h. The labeling of CE was lower and increased in function of time. [13C]DHA appearance in these lipid species was also expressed as the absolute amount of [13C]DHA at various time points (Fig. 2). [13C]DHA accumulation in NEFA and LPC (Fig. 2A) was lower than in other lipid classes (Fig. 2B). Six hours after ingestion, the [13C]DHA concentration in NEFA was 33.7-fold lower than in TG but 7.3-fold higher than in LPC. In contrast, the [13C]DHA concentration was slightly higher in LPC than in NEFA after 24 h, reaching 1.48-fold at 72 h. Finally, the [13C]DHA accumulation in PC was more than 3.4-fold higher when compared to PE after 9 h. An increase in the labeling in platelet PC (Fig. 3A) occurred till 24 h post-ingestion, followed by a plateau between 24 and 72 h. The [13C]DHA abundance increased also in platelet PE but the labeling in PC was higher than in PE from 6 to 24 h, and very close after 48 and 72 h. The amount of [13C]DHA stored in platelet phospholipids was also calculated (Fig. 3B). [13C]DHA was preferentially incorporated in PE, being 3-fold higher than in PC, after 72 h. The uptake of [13C]DHA by erythrocytes was different. A slow and progressive increase of PC labeling was observed, as a function of time (Fig. 4A). Values ranged from -27.3 ± 0.8 (baseline) to -9.7 ± 6.8 (12 h) and +51.9 ± 26.5 (72 h). The labeling of erythrocyte PE was lower, attaining -19.5 ± 2.2 at 72 h. When the results are expressed in the amount of [13C]DHA found in these two compartments (Fig. 4B), a preferential accumulation occurred in erythrocyte PC. On the other hand, the incorporation of [13C]DHA into erythrocyte PE was weak. A small amount of [13C]DHA appeared in erythrocyte LPC (Fig. 5), with an increase until 9 h and a maximal amount between 9 h and 72 h. This plateau was attained when the incorporation of [13C]DHA started to rise into PC. At the latter time point, the LPC content of [13C]DHA was +0.06 ± 0.02 ng/ml of whole blood.Fig. 5.Concentration of [13C]DHA, expressed in ng/ml whole blood, in human erythrocyte lysophosphatidylcholine (LPC) as a function of time after the ingestion of a single dose of [13C]DHA-PC. The [13C]DHA amount in each lipid class was calculated from the δ13C‰ abundance and the DHA endogenous concentrations evaluated by GC. Each point represents the mean ± SD of determinations from three human subjects.View Large Image Figure ViewerDownload (PPT) The contribution of NEFA and LPC to supply [13C]DHA to blood cells was investigated. We calculated that the relative contribution factor of NEFA involved in the [13C]DHA supply to erythrocytes and platelets was γ1 = 0.32 and that the relative contribution factor of LPC to this supply was γ2 = 1.0. The computed relative contribution factors of NEFA and LPC to platelets were α = 0.069 and β = 0, respectively. The computed relative contribution factors of NEFA and LPC to erythrocytes were 1 - α = 0.931 and 1 - β = 1.0, respectively. It appears that the DHA supply to platelets occurred through NEFA, α(γ1) = 0.022, and not through LPC, β(γ2) = 0, due to the isotope enrichment pattern in LPC which does not fit with that in platelets. In contrast, these two lipid compartments are involved in DHA supply to erythrocytes. The value of coefficient (1 - β)(γ2) = 1 relates to the amount of DHA available through LPC, with NEFA being another provider according to the following equation: (1−α)γ1=0.299Eq. 7) Previous studies (15Brossard N. Croset M. Normand S. Pousin J. Lecerf J. Laville M. Tayot J.L. Lagarde M. Human plasma albumin transports [13C]docosahexaenoic acid in two lipid forms to blood cells.J. Lipid Res. 1997; 38: 1571-1582Google Scholar) under the same conditions, except for the form of DHA ingested, have shown that ingestion of a single dose of [13C]DHA-TG leads to the appearance of a [13C]DHA peak in plasma TG and NEFA after 2 h. In plasma LPC and PC, the incorporation of [13C]DHA was more progressive until 6 and 12 h, respectively, and a plateau was observed in both compartments thereafter. LPC was found to be the main vehicle for DHA to erythrocytes and NEFA the major one to leukocytes and platelets. In the present study, after ingestion of a single dose of [13C]DHA-PC corresponding to the same dose of [13C]DHA, it appears that [13C]DHA peaked i