Fetal baboons convert 18:3n-3 to 22:6n-3 in vivo: a stable isotope tracer study
2001; Elsevier BV; Volume: 42; Issue: 4 Linguagem: Inglês
10.1016/s0022-2275(20)31167-6
ISSN1539-7262
AutoresHui‐Min Su, Meng‐Chuan Huang, Nabil Saad, Peter W. Nathanielsz, J. Thomas Brenna,
Tópico(s)Metabolism and Genetic Disorders
ResumoUsing [13C]-tracers and direct fetal doses, we show for the first time that the fetal primate converts α-linolenic acid (18:3) to docosahexaenoic acid (22:6) in vivo, and we estimate the relative bioefficacy of the two substrates for brain 22:6 accretion. Pregnant female baboons consumed a diet free of long chain polyunsaturates (LCP), with n-6/n-3 ratio of 10/1. In the third trimester of pregnancy (normal gestation = 182 days), they were instrumented with chronic indwelling catheters in the maternal femoral artery and the fetal jugular artery. Doses of either [U-13C]-18:3 (18:3*, n = 3) or [U-13C]-22:6 (22:6*, n = 2) were administered directly to the fetus. Blood was collected from fetus and mother, and the fetus was taken by cesarean section when electromyographic activity indicated that parturition was imminent. Fetal liver, brain, retina, and retinal pigment epithelium (RPE) were collected, and 13C fatty acids determined. In 18:3*-dosed animals, labeled n-3 LCP were detected in fetal plasma at 1 day post-dose and peaked at 2–3 days; brain 22:6* was constant at 3, 5, and 9 days post-dose, at 0.57 ± 0.03 percent of dose (%Dose). In 22:6*-dosed animals, brain 22:6* was similar at 3 and 9 days post-dose (4.64 ± 0.43%Dose). From these data, we estimate that preformed 22:6 in the fetal bloodstream is 8-fold more efficacious for brain 22:6 accretion than is 18:3. Retina 22:6* was stable at about 0.0008%Dose from 3 to 9 days in 18:3-dosed animals, but RPE 22:6* dropped over the period; brain results were consistent with these observations. Liver showed about 0.5%Dose in 22:6* and in intermediary n-3 fatty acid metabolites 20:5* and 22:5* at 3 days post-dose, and declined afterward. Back-transfer of labeled fatty acids to the maternal bloodstream was measurable but not sufficient to compromise the quantitative conversion data in fetuses. We conclude 1) primate fetuses have the capacity to convert 18:3 to 22:6 in vivo; 2) fetal brain 22:6* as %Dose plateaus by 3 days post-dose; 3) fetal plasma 22:6 is about 8-fold more effective as a substrate for brain 22:6 accretion compared with 18:3; and 4) the fetal liver is likely to be an important site of 18:3 to 22:6 conversion. —Su, H-M., M-C. Huang, N. M. R. Saad, P. W. Nathanielsz, and J. T. Brenna. Fetal baboons convert 18:n-3 to 22:6n-3 in vivo: a stable isotope tracer study. J. Lipid Res. 2000. 42: 581–586. Using [13C]-tracers and direct fetal doses, we show for the first time that the fetal primate converts α-linolenic acid (18:3) to docosahexaenoic acid (22:6) in vivo, and we estimate the relative bioefficacy of the two substrates for brain 22:6 accretion. Pregnant female baboons consumed a diet free of long chain polyunsaturates (LCP), with n-6/n-3 ratio of 10/1. In the third trimester of pregnancy (normal gestation = 182 days), they were instrumented with chronic indwelling catheters in the maternal femoral artery and the fetal jugular artery. Doses of either [U-13C]-18:3 (18:3*, n = 3) or [U-13C]-22:6 (22:6*, n = 2) were administered directly to the fetus. Blood was collected from fetus and mother, and the fetus was taken by cesarean section when electromyographic activity indicated that parturition was imminent. Fetal liver, brain, retina, and retinal pigment epithelium (RPE) were collected, and 13C fatty acids determined. In 18:3*-dosed animals, labeled n-3 LCP were detected in fetal plasma at 1 day post-dose and peaked at 2–3 days; brain 22:6* was constant at 3, 5, and 9 days post-dose, at 0.57 ± 0.03 percent of dose (%Dose). In 22:6*-dosed animals, brain 22:6* was similar at 3 and 9 days post-dose (4.64 ± 0.43%Dose). From these data, we estimate that preformed 22:6 in the fetal bloodstream is 8-fold more efficacious for brain 22:6 accretion than is 18:3. Retina 22:6* was stable at about 0.0008%Dose from 3 to 9 days in 18:3-dosed animals, but RPE 22:6* dropped over the period; brain results were consistent with these observations. Liver showed about 0.5%Dose in 22:6* and in intermediary n-3 fatty acid metabolites 20:5* and 22:5* at 3 days post-dose, and declined afterward. Back-transfer of labeled fatty acids to the maternal bloodstream was measurable but not sufficient to compromise the quantitative conversion data in fetuses. We conclude 1) primate fetuses have the capacity to convert 18:3 to 22:6 in vivo; 2) fetal brain 22:6* as %Dose plateaus by 3 days post-dose; 3) fetal plasma 22:6 is about 8-fold more effective as a substrate for brain 22:6 accretion compared with 18:3; and 4) the fetal liver is likely to be an important site of 18:3 to 22:6 conversion. —Su, H-M., M-C. Huang, N. M. R. Saad, P. W. Nathanielsz, and J. T. Brenna. Fetal baboons convert 18:n-3 to 22:6n-3 in vivo: a stable isotope tracer study. J. Lipid Res. 2000. 42: 581–586. Docosahexaenoic acid (22:6n-3) is the major n-3 fatty acid of nervous tissues, particularly the brain and retina (1Innis S.M. Essential fatty acids in growth and development.Prog. Lipid Res. 1991; 30: 39-103Google Scholar, 2Sastry P.S. Lipids of nervous tissue: composition and metabolism.Prog. Lipid Res. 1985; 24: 69-176Google Scholar). Most brain 22:6 accumulates during the period of rapid development (3Martinez M. Tissue levels of polyunsaturated fatty acids during early human development.J. Pediatr. 1992; 120: S129-S138Google Scholar), which runs from the last trimester of gestation and continues up to 2 years of age in humans (4Dobbing J. Sands J. Comparative aspects of the brain growth spurts.Early Hum. Dev. 1979; 3: 79-83Google Scholar). The importance of brain 22:6 is related to its significant roles in maintaining neurological and visual development. Decreases in brain and retina 22:6 result in altered visual acuity (5Neuringer M. Connor W.E. Van Petten C. Barstad L. Dietary omega-3-fatty acid deficiency and visual loss in infant rhesus monkeys.J. Clin. Invest. 1984; 73: 272-276Google Scholar, 6Neuringer M. Connor W.E. Lin D.S. Barstad L. Luck S. Biochemical and functional effects of prenatal and postnatal omega 3 fatty acid deficiency on retina and brain in rhesus monkeys.Proc. Natl. Acad. Sci. USA. 1986; 83: 4021-4025Google Scholar), disturbance in electroretinographic measurements (7Wheeler T.G. Benolken R.M. Anderson R.E. Visual membranes: specificity of fatty acid precursors of the electrical response to illumination.Science. 1975; 188: 1312-1314Google Scholar, 8Bourre J.M. Francois M. Youyou A. Dumont O. Piciotti M. Pascal G. Durand G. The effects of dietary alpha-linolenic acid on the composition of nerve membranes, enzymatic-activity, amplitude of electrophysiological parameters, resistance to poisons and performance of learning-tasks in rats.J. Nutr. 1989; 119: 1880-1892Google Scholar) and learning impairment (9Yamamoto N. Saitoh M. Moriuchi A. Nomura M. Okuyama H. Effect of dietary alpha-linolenate/linoleate balance on brain lipid compositions and learning ability of rats.J. Lipid Res. 1987; 28: 144-151Google Scholar) in various species. A source of n-3 fatty acids is indispensable for the human diet, as mammals do not have the Δ12 and Δ15 desaturases required for de novo synthesis of this family of fatty acids. α-Linolenic acid (18:3n-3) is the predominant precursor of 22:6 in most human diets. Through a series of alternating desaturation and elongation reactions, 18:3 is converted to long chain polyunsaturates, particularly eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and 22:6. The relative efficiency with which this conversion is performed in part defines the dietary requirements. Human preterm and term infants fed formula with 18:3 as the only source of n-3 fatty acids display a drop in plasma 22:6 levels. Preterm infants consistently show compromised visual function compared with those fed 22:6-supplemented formula or reference groups fed breast milk containing a moderate amount of 22:6. Visual function was measured by acuity cards, forced preferential looking, or visually evoked potentials (10Uauy R.D. Birch D.G. Birch E.E. Tyson J.E. Hoffman D.R. Effect of dietary omega-3-fatty-acids on retinal function of very-low-birth-weight neonates.Ped. Res. 1990; 28: 485-492Google Scholar, 11Birch E.E. Birch D.G. Hoffman D.R. Uauy R. Dietary essential fatty acid supply and visual acuity development.Invest. Ophthamol. Vis. Sci. 1992; 33: 3242-3253Google Scholar, 12SanGiovanni J.P. Parra-Cabrera S. Colditz G.A. Berkey C.S. Dwyer J.T. Meta-analysis of dietary essential fatty acids and long-chain polyunsaturated fatty acids as they relate to visual resolution acuity in healthy preterm infants.Pediatrics. 2000; 105: 1292-1298Google Scholar). Infant rhesus monkey studies demonstrate that depletion of brain 22:6 in utero and neonatally causes functional deficiencies in vision that persist despite later biochemical repletion of brain and retinal 22:6 levels (5Neuringer M. Connor W.E. Van Petten C. Barstad L. Dietary omega-3-fatty acid deficiency and visual loss in infant rhesus monkeys.J. Clin. Invest. 1984; 73: 272-276Google Scholar, 6Neuringer M. Connor W.E. Lin D.S. Barstad L. Luck S. Biochemical and functional effects of prenatal and postnatal omega 3 fatty acid deficiency on retina and brain in rhesus monkeys.Proc. Natl. Acad. Sci. USA. 1986; 83: 4021-4025Google Scholar). The maternal circulation supplies n-3 fatty acids to the fetus in the form of 18:3 or preformed long chain polyunsaturates (LCP). Further metabolism by the fetal organs can convert 18:3 and intermediate LCP to 22:6 for incorporation into brain structural lipids, if the requisite enzymes are expressed in utero. Support for this possibility has been demonstrated by in vitro investigations showing significant Δ6 and Δ5 desaturase activities expressed in human fetal liver microsomes as early as 17–18 weeks of gestation (13Chambaz J. Ravel D. Manier M.C. Pepin D. Mulliez N. Bereziat G. Essential fatty acids interconversion in the human fetal liver.Biol. Neonate. 1985; 47: 136-140Google Scholar, 14Rodriguez A. Sarda P. Nessmann C. Boulot P. Leger C.L. Descomps B. Delta6- and delta5-desaturase activities in the human fetal liver: kinetic aspects.J. Lipid Res. 1998; 39: 1825-1832Google Scholar) and in prenatal rat liver and brain (15Bourre J.M. Piciotti M. Dumont O. Delta-6 desaturase in brain and liver during development and aging.Lipids. 1990; 25: 354-356Google Scholar). Furthermore, recent stable isotope tracer studies unambiguously demonstrate that human preterm infants are capable of synthesizing 22:6 in vivo subsequent to enteral administration of 13C-labeled 18:3 (16Carnielli V.P. Wattimena D.J.L. Luijendijk I.H.T. Boerlage A. Degenhart H.J. Sauer P.J.J. The very low birth weight premature infant is capable of synthesizing arachidonic and docosahexaenoic acids from linoleic and linolenic acids.Ped. Res. 1996; 40: 169-174Google Scholar, 17Salem J.N. Wegher B. Mena P. Uauy R. Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.Proc. Nat. Acad. Sci. USA. 1996; 93: 49-54Google Scholar, 18Sauerwald T.U. Hachey D.L. Jensen C.L. Chen H. Anderson R.E. Heird W.C. Intermediates in endogenous synthesis of C22:6 omega 3 and C20:4 omega 6 by term and preterm infants.Ped. Res. 1997; 41: 183-187Google Scholar). However, there are no reports showing whether a primate fetus expresses the full pathway required for synthesis of 22:6 from 18:3 in vivo. Recently, we have reported that the relative efficacy of 18:3 or 22:6 as a precursor for fetal brain 22:6 accumulation is about 20/1 following iv doses to the maternal bloodstream of pregnant baboons (19Greiner R.C. Winter J. Nathanielsz P.W. Brenna J.T. Brain docosahexaenoate accretion in fetal baboons: bioequivalence of dietary alpha-linolenic and docosahexaenoic acids.Ped. Res. 1997; 42: 826-834Google Scholar). These data do not directly demonstrate that the fetus is capable of converting 18:3 to 22:6, as reported in human neonates, because fetal 22:6 accretion can originate from maternal conversion prior to transport to the fetus. For this, direct doses of labeled 18:3 to the fetus are required. The purpose of this study is to establish whether non-human fetal primates are capable of synthesizing 22:6 from 18:3 in vivo and, secondarily, to estimate the relative efficacy of 18:3 and 22:6 as substrate for 22:6 accumulation in fetal baboon brain and associated organs using 13C stable isotope methodology, with analysis by high precision isotope ratio mass spectrometry (IRMS). Pregnant baboons (Papio cynocephalus) were bred at the South-west Foundation for Biomedical Research (San Antonio, TX) or at the University of Illinois at Chicago (Chicago, IL) and transported to the Laboratory for Pregnant and Newborn Research at Cornell University (Ithaca, NY). The Cornell Institutional Animal Care and Use Committee approved the care of the animals, and the American Association for Laboratory Animal Care (AALAC) approved the facility. A complete veterinary examination was performed on all pregnant baboons upon arrival, and they were housed individually in cages in sight of at least one other baboon and a video showing other baboons. The room temperature and humidity were maintained at 24°C and 70%, respectively, with a 14-h light cycle and a 10-h dark cycle. After an acclimation period, animals were jacketed with a flexible tether and swivel. After at least 1 week of acclimation to the jacket, pregnant baboons and their fetuses were instrumented with catheters to permit continuous access to the bloodstream via a tether, and electromyography leads were attached to the uterus. These procedures have been described in detail elsewhere (20Nathanielsz P.W. Figueroa J.P. Poore E.R. Methods of investigation of the chronically instrumented pregnant maintain on a tether and swivel system.in: Nathanielsz P.W. Animal Models in Fetal Medicine. Perinatology Press, Ithaca, NY1984: 110-160Google Scholar, 21Morgan M.A. Silavin S.L. Randolph M. Payne G.G. Sheldon R.E. Fishburne J.I. Wentworth R.A. Nathanielsz P.W. Effect of intravenous cocaine on uterine blood flow in the gravid baboon.Am. J. Obstet. Gynecol. 1991; 164: 1021-1030Google Scholar). Substrate and product feedbacks are common phenomena in a wide variety of physiological processes. Uncontrolled tracer dilution by dietary tracee (n-3 fatty acids) or other fatty acids would lead to unreliable kinetics as well as uncertain effects on conversion, possibly due to competition for desaturation and elongation enzymes with n-3 fatty acids (22Holman R.T. The slow discovery of the importance of omega 3 essential fatty acids in human health [see comments].J. Nutr. 1998; 128: 427S-433SGoogle Scholar). To minimize possible effects on 18:3 to 22:6 conversion, mother baboons were fed, for at least the last 8 weeks of pregnancy, an LCP-free diet containing controlled levels of linoleic acid (18:2n-6) and 18:3. The diet had 2% of energy as 18:2, and 0.2% of energy as 18:3 (18:2/18:3 = 10) (Harlan Teklad, Madison, WI). The diet fatty acid composition was reported previously (19Greiner R.C. Winter J. Nathanielsz P.W. Brenna J.T. Brain docosahexaenoate accretion in fetal baboons: bioequivalence of dietary alpha-linolenic and docosahexaenoic acids.Ped. Res. 1997; 42: 826-834Google Scholar). Animals consumed this diet for at least 8 weeks before the administration of dose, and continued until cesarean section (CS). A tracer dose of 2.24 ± 0.42 mg [U-13C]-18:3 (18:3*) (n = 5) or 0.95 ± 0.11 mg [U-13C]22:6 (22:6*) (n = 2) was administered to the fetal jugular vein via a catheter in the third trimester. Experimental animal characteristics, including weights, and gestational ages, as well as dose administered, are presented in Table 1. The 18:3* or 22:6* was purified from an [U-13C]algal oil (Martek Biosciences, Columbia, MD) as described previously(23Sheaff R.C. Su H-M. Keswick L.A. Brenna J.T. Conversion of ?-linolenate to docosahexaenoate is not depressed by high dietary levels of linoleate in young rats: tracer evidence using high precision mass spectrometry.J. Lipid Res. 1995; 36: 998-1008Google Scholar). The dose was sonicated into 0.5 ml of 20% Intralipid (KaviVitrum, Franklin, OH), an intravenous emulsion consisting primarily of LCP-free soybean oil with trace LCP added incidentally as a component of egg lecithin emulsifier, and was diluted with 1.5 ml of sterile saline approximately 12 h before dosing.TABLE 1Characteristics of animals used in studyBaboon1234fNo tissue was available from these animals; plasma was collected from mother and fetus.5fNo tissue was available from these animals; plasma was collected from mother and fetus.67Fatty acid dose18:3n-318:3n-318:3n-318:3n-318:3n-322:6n-322:6n-3Dosea[U-13C]-linolenic acid (18:3n-3*) was in a free fatty acid form, blended with Intralipid. (mg)2.552.411.772.251.931.020.83dGA at dosingbAge of fetus (days of gestation) at dose administration.139136137135146130131dGA at CScAge of fetus (days of gestation) at cesarean section.142141146133140Dosing period (d)35939Maternal weightdMaternal weight at the time of maternal catheterization near or at the cesarean section. (kg)16141816191318Samples collectedeT, fetal tissues; F, fetal plasma; M, maternal plasma.T,F,MT,MT,FF,MF,MT,MTa [U-13C]-linolenic acid (18:3n-3*) was in a free fatty acid form, blended with Intralipid.b Age of fetus (days of gestation) at dose administration.c Age of fetus (days of gestation) at cesarean section.d Maternal weight at the time of maternal catheterization near or at the cesarean section.e T, fetal tissues; F, fetal plasma; M, maternal plasma.f No tissue was available from these animals; plasma was collected from mother and fetus. Open table in a new tab For fetal plasma, baseline samples were drawn prior to dosing via a fetal carotid artery catheter, then after dosing once per day as available. Maternal baseline plasma was initially drawn from maternal femoral artery catheters, then drawn daily after dosing. Pregnancies were allowed to continue until electromyographic activity indicated that labor was imminent. At that time, CS was performed and the time recorded between dose administration and CS, as shown in Table 1. The fetus, continuously under halothane general anesthesia from the time of CS, was euthanized by exsanguination and fetal tissues collected immediately. The brain (occipital lobes) and liver were removed quickly, weighed, wrapped in aluminum foil, and frozen in liquid N2. Retina and retinal pigment epithelium (RPE) were immediately dissected from the eyes, separated, collected, and stored in saline. All samples were kept in a freezer at −80°C until analysis. Total lipids were extracted from tissue homogenates by the method of Bligh and Dyer (24Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar), and fatty acid methyl esters (FAME) prepared using 14% BF3 in methanol. A known amount of fresh heptadecanoic acid (17:0; 99+% pure, Sigma Chemicals, St. Louis, MO) was added as an internal standard to the tissue homogenate prior to extraction. The purified FAME were dissolved in hexane with butylated hydroxytoluene (BHT) as an antioxidant, flushed with N2, and stored in a −20°C freezer until analysis. FAME were analyzed with a Hewlett Packard 5890 Series II gas chromatograph with flame ionization detector (GC-FID) using H2 carrier gas. Quantitative profiles were calculated using the internal standard and an equal weight mixture to derive response factors for each fatty acid. GC conditions and calibration details are reported elsewhere (25Su H-M. Brenna J.T. Simultaneous measurement of desaturase activities using stable isotope tracers or a non-tracer method.Anal. Biochem. 1998; 261: 43-50Google Scholar). Tracer analysis was performed with a high precision gas chromatography-combustion-isotope ratio mass spectrometer (GCC-IRMS), described in detail previously (26Brenna J.T. Corso T.N. Tobias H.J. Caimi R.J. High-precision continuous-flow isotope ratio mass spectrometry.Mass Spectrom. Rev. 1997; 16: 227-258Google Scholar, 27Goodman K.J. Brenna J.T. High sensitivity tracer detection using high precision gas chromatography combustion isotope ratio mass spectrometry and highly enriched [U-13C]-labeled precursors.Anal. Chem. 1992; 64: 1088-1095Google Scholar). The concentration of tracer in tissues was calculated from the isotopic enrichment measured by GCC-IRMS and the quantity of fatty acids determined by GC-FID, as has been described in detail elsewhere (26Brenna J.T. Corso T.N. Tobias H.J. Caimi R.J. High-precision continuous-flow isotope ratio mass spectrometry.Mass Spectrom. Rev. 1997; 16: 227-258Google Scholar, 27Goodman K.J. Brenna J.T. High sensitivity tracer detection using high precision gas chromatography combustion isotope ratio mass spectrometry and highly enriched [U-13C]-labeled precursors.Anal. Chem. 1992; 64: 1088-1095Google Scholar). The molar amount of 18:3* present in any fatty acid pool is calculated as the product of the total fatty acid per unit of tissue and the atom fraction excess (AFE), corrected for the ratio of carbon in analyte fatty acid to that in 18:3* (28Su H-M. Bernardo L. Mirmiran M. Ma X.H. Corso T.N. Nathanielsz P.W. Brenna J.T. Bioequivalence of dietary alpha-linolenic and docosahexaenoic acids as sources of docosahexaenoate accretion in brain and associated organs of neonatal baboons.Pediatr. Res. 1999; 45: 87-93Scopus (123) Google Scholar). For 18:3 this factor is 1; for C20 and C22 fatty acids it is 20/18 and 22/18, respectively. The final reported tracer concentration, therefore, refers to the mol of nascent (dose) 18:3* that have entered the particular pool. Percent of dose (%Dose) was calculated from each labeled n-3 fatty acid (FA*) in each pool, divided by the total dose to each animal, multiplied by 100 and normalized to pool units (per liter plasma or per g whole organ). %Dose adjusts for different dose sizes among the animals and ensures that the differences are related to function rather than dose size. The fetal and maternal plasma kinetic curves were evaluated as mol %Dose per 100 ml of plasma at each sampling time point. Data are reported as %Dose found in whole fetal brain or liver, or per single retina, per single RPE. Least-squares fits through the data are presented to aid the eye, using Excel 2000 for Windows98 (Microsoft, Seattle, WA). Fetal plasma kinetic curves for n-3 labeled fatty acids after a 18:3* dose to fetal baboons from 1 to 9 days post-dose are illustrated in Fig. 1A and B. Figure 1A shows that 18:3* dropped to 50% of its initial concentration at 2 days post-dose; 99% of 18:3* had disappeared by 8 days post-dose. About 10%Dose as 18:3* per deciliter fetal plasma was detected at 1 day post-dose. Figure 1B shows that 20:5* and 22:5* peaked at 2 days post-dose; in comparison, 22:6 appears slightly delayed, with its maximal mean level observed at 3 days post-dose. Maternal plasma kinetic curves for n-3 labeled metabolites from 3 h to 4 days post-dose are presented in Fig. 2. 18:3* was detected as early as 3 h post-dose and increased at 5 h. Half the 18:3* disappeared by 1 day post-dose, and it continued to drop gradually. LCP* were detected at 3 h post-dose. By 1 day post-dose, 20:5* showed a mild peak and then gently dropped. 22:5* plateaued up to day 4 post-dose; 22:6* was not detected at 3 h but then gradually increased and had plateaued by 4 days. The time course of incorporation of n-3 fatty acids following an 18:3* or 22:6* iv dose to 3 fetuses is shown in Fig. 3A and B. Each point represents a single animal, for which the time point is plotted as the period between dosing and CS, at 3, 5, or 9 days. For 18:3*-dosed animals, 18:3* and its metabolites, 20:5*, 22:5*, and 22:6* were all detected at 3 days post-dose, and all labeled n-3 FA* gradually decreased at longer times post-dose. A similar pattern was observed for two 22:6*-dosed groups. Three days after the fetal animal was injected with22:6*, approximately 15%Dose was recovered as liver 22:6*. This amount dropped to 2.4% at 9 days post-dose. Liver 22:6* comprised 90–97% of all n-3 FA* measured in 22:6* dosed animals. Neural tissue 22:6* resulting from 18:3* or 22:6* fetal dosing in the whole brain, retina, and RPE is presented in Table 2. The incorporation of 22:6* derived from preformed 22:6* was greater than that from the 18:3* dose. For both groups, brain 22:6* reached a plateau by the first time point investigated, 3 days post-dose. The average brain 22:6* plateau levels were 0.57 ± 0.03% and 4.64 ± 0.43% of the 18:3* and preformed 22:6* doses, respectively. From these data, we construct a ratio that indicates that preformed 22:6* was 8-fold more efficiently incorporated as brain lipids as compared with 18:3-derived 22:6*.TABLE 222:6* accretion (%Dose) in fetal baboons following a fetal iv dose of 18:3* ([U-13C]-18:3n-3) or 22:6* ([U-13C]-22:6n-3)aWhole organ 22:6* (22:6n-3*) accretion due to doses, or either 18:3* ([U-13C]-linolenic acid; 18:3n-3*) or 22:6* ([U-13C]-docosahexaenoic acid; 22:6n-3*), expressed as percent of dose (%Dose). Each time point (day) represents separate animal; errors average 10%CV, and are associated only with the workup and chemical analyses.%Dose as 22:6*OrganTime Day*18:3 dosedb18:3* dose was administered iv to fetal baboons; organs were collected at 3, 5, and 9 days post-dose.*22:6 dosedc22:6* dose was administered iv to fetal baboons; organs were collected at 3 and 9 days post-dose.RatiodRatio: (18:3-derived 22:6)/(preformed 22:6-derived 22:6). In the brain, 22:6 derived from 18:3 (18:3* dose) was approximately 8-fold lower compared with that from preformed 22:6 (22:6*dose). Since brain 22:6* plateaus, we take this ratio as the bioequivalence. In the retina, 22:6 derived from 18:3 (18:3* dose) was approximately 13-fold lower compared with that from preformed 22:6 (22:6*dose). The ratio for the retinal pigment epithelium (RPE) is not calculated because 22:6* accretion did not plateau over the measurement period.Brain30.564.94850.60—90.544.33Retina30.00070.00981350.0007—90.00100.0112RPE30.00060.00550.0002—90.00010.002a Whole organ 22:6* (22:6n-3*) accretion due to doses, or either 18:3* ([U-13C]-linolenic acid; 18:3n-3*) or 22:6* ([U-13C]-docosahexaenoic acid; 22:6n-3*), expressed as percent of dose (%Dose). Each time point (day) represents separate animal; errors average 10%CV, and are associated only with the workup and chemical analyses.b 18:3* dose was administered iv to fetal baboons; organs were collected at 3, 5, and 9 days post-dose.c 22:6* dose was administered iv to fetal baboons; organs were collected at 3 and 9 days post-dose.d Ratio: (18:3-derived 22:6)/(preformed 22:6-derived 22:6). In the brain, 22:6 derived from 18:3 (18:3* dose) was approximately 8-fold lower compared with that from preformed 22:6 (22:6*dose). Since brain 22:6* plateaus, we take this ratio as the bioequivalence. In the retina, 22:6 derived from 18:3 (18:3* dose) was approximately 13-fold lower compared with that from preformed 22:6 (22:6*dose). The ratio for the retinal pigment epithelium (RPE) is not calculated because 22:6* accretion did not plateau over the measurement period. Open table in a new tab As observed in the brain, preformed 22:6* was preferentially incorporated as retina and RPE 22:6* as comparedwith 18:3-derived 22:6*. The range of 22:6* incorporation resulting from the 18:3* dose was 0.0007–0.0010%Dose per retina, whereas that from the 22:6* dose was 0.010–0.012%. No time-dependent changes can be discerned from these modest differences, therefore we pooled the data from the animals in each group to arrive at a relative efficacy of 13:1 in favor of 22:6 over 18:3 for retinal 22:6. In contrast to retina, RPE 22:6* content declined sharply during the 3–9 days post-dose in both groups. For 18:3*-dosed animals, 18:3-derived* 22:6* dropped from 0.0006–0.0001%, whereas a drop from 0.005–0.002%Dose was observed for the two 22:6*-dosed animals. In animals dosed with 18:3*, 22:6* dropped for 3–5 days post-dose and then remained constant. The goal of this study was to determine whether the fetal primate is capable of synthesizing 22:6 from its precursor, 18:3, in vivo. Back-transfer of the fetal dose to the maternal circulation was sufficient to induce detectable levels of labeled 18:3* in the maternal plasma for 1 day. Since the maternal liver is known to be capable of biosynthesizing 22:6 from 18:3, the possibility arises that the 18:3* entering the mother might have been taken up by her liver, converted to 22:6*, transferred back to the fetus, then incorporated into fetal organs. In this study, the data presented in Fig. 2 show that n-3 LCP* appear in the maternal bloodstream within 5 h of dosing, with total labeled n-3 metabolites at 0.12%Dose/dl. Compelling evidence shows that this is not the sole source of the fetal 22:6*. In previous work, 18:3* doses to the mother accumulated in fetal brain 22:6* at their maximal level of 0.075%Dose after about 10 days (19Greiner R.C. Winter J. Nathanielsz P.W. Brenna J.T. Brain docosahexaenoate accretion in fetal baboons: bioequivalence of dietary alpha-linolenic and docosahexaenoic acids.Ped. Res. 1997; 42: 826-834Google Scholar). This accretion level represents an upper bound for 18:3-derived-22:6 accretion in the present fetuses because, in the present experiment, the dose must be transferred from fetus to mother prior to maternal metabolism. Our measurements for fetal doses, shown in Table 2, indicate a plateau level of 0.6%Dose as 18:3-derived 22:6*. We conclude that maternal metabolism of the 18:3* dose accounts for a negligible fraction of the 22:6* found in the fetus, and that the primate fetus does have the full capacity to convert 18:3 to 22:6. In this study, 18:3 or 22:6 as nonesterified fatty acid was admini
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