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Research Article| Volume 49, ISSUE 9, P1963-1980, September 2008

Effect of dietary docosahexaenoic acid on biosynthesis of docosahexaenoic acid from alpha-linolenic acid in young ratss⃞

Open AccessPublished:May 09, 2008DOI:https://doi.org/10.1194/jlr.M800117-JLR200
      Docosahexaenoic acid (DHA), a crucial nervous system n-3 PUFA, may be obtained in the diet or synthesized in vivo from dietary α-linolenic acid (LNA). We addressed whether DHA synthesis is regulated by the availability of dietary DHA in artificially reared rat pups, during p8 to p28 development. Over 20 days, one group of rat pups was continuously fed deuterium-labeled LNA (d5-LNA) and no other n-3 PUFA (d5-LNA diet), and a second group of rat pups was fed a d5-LNA diet with unlabeled DHA (d5-LNA + DHA diet). The rat pups were then euthanized, and the total amount of deuterium-labeled docosahexaenoic acid (d5-DHA) (synthesized DHA) as well as other n-3 fatty acids present in various body tissues, was quantified. In the d5-LNA + DHA group, the presence of dietary DHA led to a marked decrease (3- to 5-fold) in the total amount of d5-DHA that accumulated in all tissues that we examined, except in adipose. Overall, DHA accretion from d5-DHA was generally diminished by availability of dietary preformed DHA, inasmuch as this was found to be the predominant source of tissue DHA. When preformed DHA was unavailable, d5-DHA and unlabeled DHA were preferentially accreted in some tissues along with a net loss of unlabeled DHA from other organs.
      Docosahexaenoic acid (DHA; 22:6n-3) is an n-3 PUFA that is particularly enriched in the phospholipids of cells constituting the mammalian nervous system (
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      ). DHA is also the activating ligand for multiple transcriptional factors that control the expression of enzymes involved in fatty acid synthesis and β-oxidation (
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      Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome.
      ). DHA may be directly obtained in the diet, as preformed DHA, or synthesized in vivo from other common dietary n-3 PUFAs such as α-linolenic acid (LNA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), or docosapentaenoic acid (DPA, 22:5n-3) (
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      Incorporation of radioactive polyunsaturated fatty acids into liver and brain of developing rat.
      ). All of these n-3 PUFAs may be converted in vivo to DHA through sequential steps of elongation, desaturation, and peroxisomal β-oxidation (
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      ). Prolonged dietary deprivation of all n-3 PUFAs in rat pups, initiated prior to weaning, depletes up to 80% of their brain DHA (
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      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
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      ). Such depletion of brain DHA in rodents leads to distinct impairments in brain function (
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      N-3 fatty acid deficiency induced by a modified artificial rearing method leads to poorer performance in spatial learning tasks.
      ). Piglets and monkeys also show impaired neural function when deprived of n-3 PUFA for an extended period during infancy (
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      ). The essentiality of DHA for human infant nutrition in support of neuronal function has been shown by DHA supplementation, enhancing visual acuity and cognition-related test scores in human infants (
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      Visual acuity and fatty acid status of term infants fed human milk and formulas with and without docosahexaenoate and arachidonate from egg yolk lecithin.
      ,
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      • Neuringer M.
      Polyunsaturated fatty acid status and neurodevelopment: a summary and critical analysis of the literature.
      ). In adult humans, low DHA blood levels have been correlated with psychological disturbances such as alcoholism, major depression (non-psychotic), postpartum depression, and senile dementia (
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      Seafood consumption, the DHA content of mothers' milk and prevalence rates of postpartum depression: a cross-national, ecological analysis.
      ,
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      A double-blind, placebo-controlled study of the omega-3 fatty acid docosahexaenoic acid in the treatment of major depression.
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      Docosahexanoic acid and omega-3 fatty acids in depression.
      ,
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      Increased risk of postpartum depressive symptoms is associated with slower normalization after pregnancy of the functional docosahexaenoic acid status.
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      Effect of DHA oil supplementation on intelligence and visual acuity in the elderly.
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      • George D.T.
      • Salem Jr., N.
      Essential fatty acids predict metabolites of serotonin and dopamine in cerebrospinal fluid among healthy control subjects, and early- and late-onset alcoholics.
      ).
      Although dietary sources of LNA are often more readily available than that of DHA, feeding animals high levels of LNA does not produce identical tissue levels of DHA compared with animals that are provided lower levels of dietary DHA (
      • Abedin L.
      • Lien E.L.
      • Vingrys A.J.
      • Sinclair A.J.
      The effects of dietary alpha-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig.
      ,
      • Bowen R.A.
      • Clandinin M.T.
      High dietary 18:3n-3 increases the 18:3n-3 but not the 22:6n-3 content in the whole body, brain, skin, epididymal fat pads, and muscles of suckling rat pups.
      ,
      • Woods J.
      • Ward G.
      • Salem Jr., N.
      Is docosahexaenoic acid necessary in infant formula? Evaluation of high linolenate diets in the neonatal rat.
      ). Whereas dietary LNA can be converted to DHA in vivo, the efficiency of conversion is limited (
      • Salem Jr., N.
      • Wegher B.
      • Mena P.
      • Uauy R.
      Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.
      ). This has recently been investigated through kinetic intravenous infusion experiments in young rats using radiolabeled LNA to obtain the in vivo conversion rates in brain and liver (
      • DeMar 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.
      ,
      • Igarashi M.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      • DeMar Jr., J.C.
      Low liver conversion rate of alpha-linolenic to docosahexaenoic acid in awake rats on a high-docosahexaenoate-containing diet.
      ,
      • Igarashi M.
      • DeMar Jr., J.C.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      Docosahexaenoic acid synthesis from alpha-linolenic acid by rat brain is unaffected by dietary n-3 PUFA deprivation.
      ,
      • Igarashi M.
      • DeMar Jr., J.C.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      Upregulated liver conversion of alpha-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet.
      ). It was shown that the mature rat brain has little if any capacity to synthesize its own DHA, even when stimulated to do so under dietary conditions of feeding LNA alone or during n-3 PUFA dietary deprivation (
      • DeMar 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.
      ,
      • Igarashi M.
      • DeMar Jr., J.C.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      Docosahexaenoic acid synthesis from alpha-linolenic acid by rat brain is unaffected by dietary n-3 PUFA deprivation.
      ). In contrast, the adult rat liver does convert LNA to DHA, but at a rate sufficient to convert <2% of the total LNA that enters the liver per unit time, with the majority (>70%) of the entering LNA being lost to β-oxidation; and similar results were obtained in both the presence and the absence of dietary DHA (
      • Igarashi M.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      • DeMar Jr., J.C.
      Low liver conversion rate of alpha-linolenic to docosahexaenoic acid in awake rats on a high-docosahexaenoate-containing diet.
      ,
      • Igarashi M.
      • DeMar Jr., J.C.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      Upregulated liver conversion of alpha-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet.
      ). In these studies, adult rat liver production of DHA is elevated 3-fold in the absence of dietary DHA and the rate of liver-to-plasma secretion of synthesized DHA exceeds the replacement rate for brain DHA losses by 10-fold, suggesting that LNA metabolism is able to approach the DHA demands of the adult brain (
      • Igarashi M.
      • DeMar Jr., J.C.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      Upregulated liver conversion of alpha-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet.
      ,
      • DeMar 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.
      ).
      Although the above intravenous infusion studies provide kinetically accurate rates for DHA biosynthesis/accretion in adult rat tissues at a fixed time period and physiological state, they do not take into account the large changes over time in DHA degradative losses, biosynthesis, and the resulting net accretion found in growing neonatal animals (
      • DeMar 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.
      ,
      • Bourre J.M.
      • Piciotti M.
      • Dumont O.
      Delta 6 desaturase in brain and liver during development and aging.
      ,
      • Green P.
      • Glozman S.
      • Kamensky B.
      • Yavin E.
      Developmental changes in rat brain membrane lipids and fatty acids. The preferential prenatal accumulation of docosahexaenoic acid.
      ,
      • Maniongui C.
      • Blond J.P.
      • Ulmann L.
      • Durand G.
      • Poisson J.P.
      • Bezard J.
      Age-related changes in delta 6 and delta 5 desaturase activities in rat liver microsomes.
      ,
      • Yavin E.
      • Brand A.
      • Green P.
      Docosahexaenoic acid abundance in the brain: a biodevice to combat oxidative stress.
      ). This can only be truly examined using experiments that can follow the total accumulation of DHA over a prolonged period. We recently reported a study in which rat pups were continuously fed all of their dietary LNA as deuterium-labeled LNA (d5-LNA), over a 20 day postgestational time period (days p8–p28), to quantify the accumulation of newly biosynthesized deuterium-labeled docosahexaenoic acid (d5-DHA) in the developing brain and liver (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). These rat pups were provided a diet containing only d5-LNA (d5-LNA diet) or a diet containing d5-LNA plus unlabeled DHA at twice the level of the d5-LNA (d5-LNA + DHA diet), in order to test whether dietary preformed DHA decreases the net accretion of biosynthesized d5-DHA. In the rat pups receiving a d5-LNA + DHA versus d5-LNA diet, we found that brain and liver accretion of biosynthesized d5-DHA over 20 days were both decreased by 4-fold. This is consistent with findings by others that an absence of dietary DHA significantly upregulates the liver biosynthesis of DHA from LNA (
      • Abedin L.
      • Lien E.L.
      • Vingrys A.J.
      • Sinclair A.J.
      The effects of dietary alpha-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig.
      ,
      • Igarashi M.
      • DeMar Jr., J.C.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      Upregulated liver conversion of alpha-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet.
      ,
      • Poovaiah B.P.
      • Tinoco J.
      • Lyman R.L.
      Influence of diet on conversion of 14C1-linolenic acid to docosahexaenoic acid in the rat.
      ).
      The present study is an extension of our previous analysis (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ) of LNA metabolism in growing rat pups and its regulation by dietary DHA to a consideration of all the tissues in the body. Our hypothesis was that, as we had previously observed for brain and liver, preformed dietary DHA would decrease the amount of biosynthesized d5-DHA accretion in the major organs of the rat pups during development. We also hypothesized that the total body accumulation of preformed dietary DHA would exceed that of biosynthesized d5-DHA in the d5-LNA + DHA versus d5-LNA diet group, and we would demonstrate that dietary LNA is not nutritionally equivalent to dietary preformed DHA in developing animals. Overall, the underlying goal of this study is to generate an organ-based representation of whole-body n-3 PUFA metabolism to determine how it is regulated by input of dietary DHA. An abstract of this work has been presented (

      DeMar, J. C., Jr., C. DiMartino, W. Lefkowitz, and N. Salem, Jr. 2007. Effect of dietary docosahexaenoic acid on the in vivo net biosynthesis of docosahexaenoic acid from alpha-linolenic acid in rat tissues. (Abstract in the Experimental Biology/American Society of Biochemistry and Molecular Biology meeting. Washington D.C., April 28–May 2, 2007).

      ).

      MATERIALS AND METHODS

      Materials

      2H5-17, 17, 18, 18, 18-18:3n-3 ethyl ester (d5-LNA) was purchased from Cambridge Isotope Labs (Andover, MA), and was previously shown by GC-MS to have a purity of >98% (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). 13, 16, 19-Docosatrienoic acid (22:3n-3), for use as an internal standard during fatty acid composition analyses by GC, was purchased from NuChek Prep (Elysian, MN). A standard mixture of fatty acid methyl esters (FAMEs) used for peak identifications during GC analyses was purchased from NuChek Prep (GLC reference mixture 462). PBS (pH 7.4) was from Invitrogen Corp. (Gibco™ brand; Grand Island, NY). Butylated hydroxytoluene (BHT; 2,6-di-tertbutyl-4-methylphenol) was purchased from Acros Organics, Inc. (Morris Plains, NJ). All solvents were high-purity HPLC/GC grade and were purchased from Burdick and Jackson (B and J brand®; VWR International, West Chester, PA) and EMD Chemicals (Gibbstown, NJ).

      Animals

      All procedures for rearing animals, formulation of their respective experimental diets, and associated feeding regimes have been previously described in detail by Lefkowitz et al. (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). All animal experiments were performed under protocols approved by the Animal Care and Use Committee of the National Institute of Alcohol Abuse and Alcoholism, and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication 80-23). Animals were maintained in a conventional animal facility with controlled temperature, humidity, and illumination.
      In brief, Long Evans male rat pups were delivered and reared until 8 days of age (p8) by dams fed an AIN-93 diet modified to contain 3.1% of fatty acids as unlabeled LNA as the only source of n-3 fatty acids. Dams were provided the food and water on an ad libitum basis. Starting at p8 and continuing to p28, the rat pups were removed from the dams and placed on one of two artificial milk formulations. The first artificial milk formulation contained 1.0 ± 0.03% (n = 14) of its fatty acids as deuterium-labeled LNA (d5-LNA diet). Likewise, the second artificial milk formulation contained 1.1 ± 0.02% (n = 25) d5-LNA plus the addition of 2.0% of its fatty acids as unlabeled DHA (d5-LNA + DHA diet). The d5-LNA content of both diets, however, was not statistically different. Both diets contained essentially no unlabeled LNA (<0.01% of total fatty acids), as well as no other n-3 PUFA intermediates. In both diets, n-6 PUFA was entirely composed of unlabeled linoleic acid (LA; 18:2n-6) at 10.3–10.4% of total fatty acids, to give an n-6/n-3 PUFA ratio of 10.3:1 and 3.3:1 in the d5-LNA and d5-LNA + DHA diets, respectively.
      The rat pups were hand fed their respective d5-LNA- and d5-LNA + DHA-containing milk diets every 3–4 h for a total of seven feedings each day. Feeding volumes ranged from 0.07 ml/g body weight at p15 to 0.2 ml/g body weight by p28. In this manner, from p8 to p28, the rat pups in both dietary groups were provided a continuous oral input of dietary LNA that was solely in the form of d5-LNA. During this 21 day period of growth, any DHA that was derived from biosynthesis using the dietary d5-LNA as substrate and accreted in the rat pup tissues would also be labeled with five deuterium atoms, maintained at their original site in the fatty acyl chain, but now as incorporated into 2H-21, 21, 22, 22, 22-22:6n-3 (d5-DHA). Rat pups receiving the d5-LNA + DHA diet, unlike those on the d5-LNA diet, would accumulate new unlabeled DHA in their tissues, as derived from the preformed dietary DHA they were ingesting.
      At p28, rat pups taken from the d5-LNA and d5-LNA + DHA diet groups (n = 7 and n = 6, respectively) were fasted overnight, weighed, and euthanized by decapitation. Animals were fully eviscerated and skinned, with the whole carcass, skin, and subdivided internal organs wrapped separately in aluminum foil and immediately frozen on dry ice. During decapitation, a sample of blood was collected from the severed neck and centrifuged at 3,000 rpm for 10 min to collect plasma. In the same fashion, fully dam-reared reference groups of p8 and p28 rat pups (n = 11 and n = 4, respectively), with p8 representative of a starting baseline group, were euthanized, and whole tissues and plasma were collected for analysis of percent body composition as described below. All tissues were weighed and stored frozen at −80°C until utilized for analysis.

      Sampling of various animal tissues

      Individual organs taken for fatty acid compositional analysis were the brain, heart, kidneys, liver, lungs, retinas, spleen, and testes. The digestive tract was left intact and consisted of the esophagus, stomach, pancreas, and small and large intestines. Before use, the heart and kidneys were thawed on ice and cut laterally in half, and internal cavities were washed clean of blood and urine, respectively, using excess ice-cold PBS; they were then reweighed. All other organs, except the digestive tract, were thawed, and their external surfaces were picked clean of extraneous tissues then washed with PBS. The skin was thawed, the outside surface was shaved of hair with an electric razor, and the underside was cleaned of excess subcutaneous white adipose. Sections of skin, 1 cm2 in size, located at the mid-back region of the cleaned pelt, were cut out for analysis.
      The carcass was partially thawed, and samples of skeletal muscle, bone, and brown, white, and visceral adipose were taken. General skeletal muscle was sectioned from the right thigh region and included tissue from the gluteus maximus, vastus lateralis, and caput vertebralis muscle bundles. After collection of the thigh muscle, the right hip was further dissected to remove the intact femur bone (including posterior cartilage end cap), which was cleaned of adhering muscle, tendons, and fascia, then washed with PBS.
      To obtain muscle fibers representing fast-oxidative (type IIa-fast twitch), fast-glycolytic (type IIb-fast twitch), and slow-oxidative (type I-slow twitch) types, the red gastrocnemius, white gastrocnemius, and soleus muscles were dissected from the lower shank of the right-side rear leg, as described by Stark, Lim, and Salem (
      • Stark K.D.
      • Lim S.Y.
      • Salem Jr., N.
      Docosahexaenoic acid and n-6 docosapentaenoic acid supplementation alter rat skeletal muscle fatty acid composition.
      ). Whereas the soleus is found as a single muscle bundle, red and white gastrocnemius are actually zones of tissue located within the margin and center portions, respectively, of the medial and lateral heads of the gastrocnemius. Strips of red and white gastrocnemius were carefully cut from the middle of these regions to avoid any junctions of their intermixing. Samples of general thigh muscle and the three muscle fiber subtypes from the lower leg were cleaned of extraneous fascia and adipose and then rinsed with PBS before use.
      Brown adipose tissue was taken from the shoulder region of the carcass, inside the pocket formed between the scapula bones and attached surpraspinatus muscles. Significant deposits of brown adipose were also sampled from the body wall underneath the supraspinatus, infraspinatus, teres major, and serratus ventralis shoulder muscles. White adipose was gathered from large patches found to be loosely attached to the outer surface of the mid-back region and combined with fat that was attached to the underside of the opposing skin. Visceral fat was collected from deposits of whitish adipose lining the back wall of the abdominal cavity adjacent to both sides of the lumbar spinal column. At the end of the tissue collections, the remaining carcass, consisting of leftover muscle, bones, and adipose, was also saved for fatty acid composition analysis.

      Gross dissection of animals for percent body composition analysis

      Dam-reared reference animals 8 days and 28 days old (n = 4, each) were subjected to gross dissection to determine the total weights of hair, skin, skeletal muscle, bones, internal organs, and brown, white, and visceral adipose. The weights for each tissue were then converted to a percentage of the original body weight of the animal at time of euthanization (see supplementary Table I). All major internal organs were removed (brain, digestive tract, heart, kidneys, liver, lungs, retinas, spleen, and testes) and lumped together for weighing. Total hair, skin, and brown and visceral adipose present within the animals were collected for weighing as described above. Significant hair was not yet present on the 8 day-old animals, and thus their hair was not removed and measured. Total skeletal muscle was stripped from all bones then weighed. In the process of collecting total skeletal muscle, all substantial deposits of white adipose were dissected out and combined for weighing, along with loose white adipose removed from the underside of the skin. All bones, including tail and spinal vertebra, were carefully cleaned of adhering muscle, tendons, and fascia then polished with a soft tissue paper and weighed. Summed weights of all of the above tissues were subtracted from the original body weight of the animals and the remainder taken to represent blood and other body fluids such as urine. This arbitrary value for whole-body fluid content, however, was not applied to estimating the whole-body volumes for plasma and red blood cells (RBCs), as found in supplementary Table II.

      Homogenization of tissues

      Samples of all tissues collected for fatty acid composition analysis from the 8 day-old dam-reared reference, d5-LNA diet, and d5-LNA + DHA diet groups were first homogenized, using a motor-driven homogenizer equipped with a 5 or 10 mm sheer generator probe (Model TH115; Omni International, Marietta, GA), into 3 ml of ice-cold methanol containing 0.05 mg/ml BHT. Femur bone was first crushed, before homogenization into methanol-BHT, using a hammer-driven cryopluverization device (BioPulverizer, Model 59013N; Biospec Products Inc., Bartlesville, OK). Typically, a piece of each tissue ≤1.0 g in size was utilized per homogenate, except for the remaining carcass and intact digestive tract, as to be described below. Plasma was not subjected to homogenization; a 50–100 μl aliquot was added directly to the methanol-BHT for analysis. Prior to homogenization, docosatrienoic acid (22:3n-3), as the free fatty acid, in chloroform solution (<100 μl), was added to the samples as an internal standard for fatty acid composition analysis, such that the 22:3n-3 would constitute 5–10% of the total fatty acid concentration.
      To homogenize the carcasses and digestive tracts, they were frozen on dry ice, fragmented with surgical bone clippers, and crushed using the previously mentioned cryopluverization device. The crushed carcasses and digestive tracts were then homogenized in 10–100 ml of ice-cold methanol-BHT using a motor-driven homogenizer equipped with a 19 mm sheer generator probe (Ultra-Turrax, Model T18; VWR International). For both the carcass and digestive tract homogenates, an even dispersion of very fine particles in methanol was achieved. The tissue densities of the digestive tract and carcass homogenates were 0.24–0.39 g/ml and 0.07–0.14 g/ml, respectively. Out of each homogenate, duplicate aliquots for analysis were taken immediately after vortexing. For the carcass homogenates, this represented 2–8% of the total volume, whereas 5–25% of each digestive tract homogenate was sampled. This gave a final tissue sample size of 0.2–0.8 g per analysis. If required, homogenate samples were brought to 3 ml with methanol-BHT, then the appropriate amount of 22:3n-3 internal standard was added to all homogenates.

      Fatty acid compositional analysis

      Total lipids were extracted from tissue homogenates in 3 ml methanol-BHT (as described above), by the method of Folch, Lees, and Sloane-Stanley (
      • Folch A.C.
      • Lees M.
      • Sloane-Stanley G.M.
      A simple method for isolation and purification of total lipids from animal tissues.
      ), using appropriate portions of chloroform and an aqueous solution of 0.5 M KCl. All extraction procedures were done under nitrogen. At the end of the extraction procedure, the crude total lipid extracts in chloroform were washed using a theoretical aqueous phase of methanol-BHT and 0.5 M KCl (1:1; v/v) as described by Folch, Lees, and Sloane-Stanley (
      • Folch A.C.
      • Lees M.
      • Sloane-Stanley G.M.
      A simple method for isolation and purification of total lipids from animal tissues.
      ), dried under nitrogen, reconstituted in 1–3 ml of chloroform, and stored at −80°C. FAMEs were prepared from a portion of each total lipid extract using 14% wt/vol BF3-methanol at 100°C for 60 min (
      • Morrison W.R.
      • Smith L.M.
      Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride–methanol.
      ), followed by extraction into hexane using the method of Salem et al. (
      • Salem Jr., N.
      • Wegher B.
      • Mena P.
      • Uauy R.
      Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.
      ) as modified by Lefkowitz et al. (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). The extracts were subjected to fast-GC analysis as previously described by Masood, Stark, and Salem (
      • Masood A.
      • Stark K.D.
      • Salem Jr., N.
      A simplified and efficient method for the analysis of fatty acid methyl esters suitable for large clinical studies.
      ). Fast-GC analyses were carried out on an Agilent 6980 gas chromatograph in the split mode (200:1) and equipped with a DB-FFAP (J and W Scientific; LaPalma, CA) capillary column (15 m × 0.1 mm ID × 0.1 μm film thickness), a flame ionization detector, and hydrogen as the carrier gas at a linear velocity of 56 cm/s and constant head pressure of 344.7 kPa. The injector and detector temperatures were set to 250°C, and the temperature program was as follows: initial, 150°C with a 0.25 min hold; 150–200°C at 35°C/min; 200–225°C at 8°C/min with a 3.2 min hold; then 225–245°C at 80°C/min with a 2.75 min hold. Peaks were identified by comparison with a standard mixture (#462; NuChek Prep) and against chromatograms for tissues from the 8 day-old dam-reared reference group. Using the fast-GC method, complete identification and separation was achieved for the unlabeled (endogenous) and deuterium-labeled n-3 PUFA species of LNA, EPA, DPA, and DHA, as previously described for conventional capillary GC (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). Fatty acid concentrations in each tissue (mg fatty acid/g tissue) were calculated by comparison of the fatty acid peak areas in the GC chromatograms to that of the 22:3n-3 internal standard.

      Calculations and statistics

      Tissue fatty acid concentrations (mg fatty acid/g tissue) were converted to the total amount of fatty acid per organ by multiplying these values by the whole-organ weight, as reported in supplementary Table II. Whole-body weights for skin, adipose, skeletal muscle, and bones were based on their determined percent body composition values (see above) multiplied by the total body weights of the animals, as found in supplementary Tables I and II, respectively. Total body plasma and RBC weights (1 g ≈ 1 ml) were estimated using the equations of Lee and Blaufox (
      • Lee H.B.
      • Blaufox M.D.
      Blood volume in the rat.
      ), where whole-body blood volume = 0.06 × body weight (g) + 0.77 and RBC/plasma = 0.45/0.55 (v/v). Retina, spleen, and testes fatty acid data are reported as micrograms of fatty acid per whole organ, whereas all other whole organs were reported in milligrams of fatty acid. Red gastrocnemius, white gastrocnemius, and soleus leg muscle types were expressed as fatty acid concentration values and reported as milligrams of fatty acid per gram tissue.
      All data were expressed as the mean ± SD. Statistical comparisons between means were carried out using the Student's t-test. Because this study involves independent and repetitive measurements of various fatty acids in multiple tissues from each diet group, to help reduce the likelihood of making type-2 statistical errors, a significant difference was taken as P < 0.010; however, differences near significance, where P = 0.010–0.050, were also separately noted in the tables and, where warranted, in the Results section. For the tissue percent body composition data (see supplementary Table I), comparisons were carried out between the means of the 8 versus the 28 day-old dam-reared reference groups (n = 4, each). For the organ weight (see supplementary Table II), tissue fatty acid composition (Tables 113 and supplementary Tables III–IX), and amount of d5-DHA accumulated in tissues data (Table 14), comparisons were only carried out between the d5-LNA versus d5-LNA + DHA diet groups (28 day-old animals; n = 7 and n = 6, respectively), and not against the 8 day-old dam-reared reference group (n = 7). For the amount of unlabeled DHA accumulated in tissues data (Table 15), however, comparisons were carried out between the 8 day-old dam-reared reference group versus the d5-LNA and d5-LNA + DHA diet groups.
      TABLE 1Plasma fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/whole-body plasma
      Saturates
       10:00.006 ± 0.006NDND
       12:00.08 ± 0.090.06 ± 0.020.07 ± 0.03
       14:00.13 ± 0.110.10 ± 0.030.13 ± 0.05
       16:00.59 ± 0.281.4 ± 0.32.0 ± 0.8
       18:00.33 ± 0.062.6 ± 0.72.7 ± 1.0
       20:00.004 ± 0.0010.01 ± 0.0040.01 ± 0.01
       22:00.004 ± 0.0010.02 ± 0.010.02 ± 0.01
       24:00.01 ± 0.0010.05 ± 0.020.03 ± 0.01
      Total saturates1.2 ± 0.54.2 ± 1.14.9 ± 1.8
      Monounsaturates
       16:ln-70.03 ± 0.010.05 ± 0.070.18 ± 0.06
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       18:ln-70.04 ± 0.010.09 ± 0.070.07 ± 0.03
       18:ln-90.21 ± 0.071.7 ± 0.42.4 ± 1.0
       20:ln-90.004 ± 0.0010.05 ± 0.010.05 ± 0.02
       22:ln-90.001 ± 0.00030.008 ± 0.0020.005 ± 0.006
       24:ln-90.005 ± 0.0040.21 ± 0.100.27 ± 0.25
      Total monounsaturates0.28 ± 0.092.2 ± 0.52.9 ± 1.3
      n-6 PUFA
       18:2n-60.38 ± 0.080.96 ± 0.202.1 ± 0.9
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       18:3n-60.006 ± 0.0020.02 ± 0.0050.03 ± 0.01
       20:2n-60.02 ± 0.010.03 ± 0.010.04 ± 0.01
       20:3n-60.03 ± 0.010.07 ± 0.030.20 ± 0.09
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:4n-60.37 ± 0.063.1 ± 0.94.2 ± 1.3
       22:2n-60.02 ± 0.0050.007 ± 0.006ND
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:4n-60.01 ± 0.0030.07 ± 0.040.03 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:5n-60.02 ± 0.0030.59 ± 0.170.03 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA0.85 ± 0.124.9 ± 1.36.6 ± 2.3
      n-3 PUFA
       d5-18:3n-30.02 ± 0.0040.03 ± 0.01
       18:3n-30.01 ± 0.01NDND
       d5-20:5n-30.01 ± 0.0030.04 ± 0.02
       20:5n-30.02 ± 0.002ND0.16 ± 0.07
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.04 ± 0.010.02 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-30.03 ± 0.010.005 ± 0.0030.06 ± 0.02
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-30.69 ± 0.150.24 ± 0.09
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:6n-30.11 ± 0.020.10 ± 0.032.1 ± 0.9
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA0.16 ± 0.020.85 ± 0.192.6 ± 1.1
      Total fatty acids2.5 ± 0.712.1 ± 3.017.1 ± 6.5
      ND, not detected (i.e., < 0.0001 mg/whole-body plasma). Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 13Liver fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total liver
      Saturates
       10:00.3 ± 0.50.08 ± 0.090.01 ± 0.03
       12:01.3 ± 1.30.40 ± 0.290.14 ± 0.04
       14:01.7 ± 1.51.7 ± 1.10.7 ± 0.3
       16:07.2 ± 4.023 ± 820 ± 2
       18:02.7 ± 1.028 ± 528 ± 2
       20:00.02 ± 0.010.08 ± 0.020.08 ± 0.01
       22:00.02 ± 0.0040.13 ± 0.020.14 ± 0.01
       24:00.05 ± 0.010.41 ± 0.090.33 ± 0.04
      Total saturates13 ± 853 ± 1449 ± 3
      Monounsaturates
       16:ln-70.31 ± 0.250.55 ± 0.500.22 ± 0.06
       18:ln-70.65 ± 0.381.4 ± 1.00.61 ± 0.13
       18:ln-93.9 ± 2.440 ± 2618 ± 7
       20:ln-90.07 ± 0.030.65 ± 0.260.51 ± 0.16
       22:ln-90.01 ± 0.0040.05 ± 0.010.05 ± 0.005
       24:ln-90.04 ± 0.0070.55 ± 0.080.68 ± 0.06
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total monounsaturates5.0 ± 3.143 ± 2820 ± 8
      n-6 PUFA
       18:2n-63.8 ± 2.08.5 ± 3.37.6 ± 1.8
       18:3n-60.08 ± 0.050.26 ± 0.140.09 ± 0.02
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:2n-6
       20:3n-60.36 ± 0.150.64 ± 0.201.2 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:4n-63.5 ± 1.126 ± 520 ± 2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:2n-6
       22:4n-60.67 ± 0.230.8 ± 0.20.21 ± 0.05
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-60.40 ± 0.146.7 ± 0.80.19 ± 0.03
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA8.8 ± 3.543 ± 930 ± 1
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      n-3 PUFA
       d5-18:3n-30.17 ± 0.080.16 ± 0.07
       18:3n-30.21 ± 0.17NDND
       d5-20:5n-30.05 ± 0.020.17 ± 0.04
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:5n-30.27 ± 0.140.01 ± 0.0070.71 ± 0.22
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.47 ± 0.090.15 ± 0.04
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-31.1 ± 0.40.04 ± 0.020.53 ± 0.12
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-39.9 ± 1.62.5 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:6n-33.6 ± 1.01.5 ± 0.322 ± 2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA5.2 ± 1.712 ± 226 ± 2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids32 ± 16145 ± 51124 ± 11
      ND, not detected(i.e., < 0.0001 mg/total liver). Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet). 20:2n-6 and 22:2n-6 were not reported; and this table was originally found in Ref.
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      .
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 14d5-DHA accumulated in rat tissues expressed as percentage of total-body d5-DHA
      28 Day-old
      Tissuesd5-LNA Dietd5-LNA + DHA Diet
      % mg/total body d5-DHA
      Plasma2.1 ± 0.41.7 ± 0.6
      Brain7.5 ± 1.24.4 ± 0.4
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Retina0.15 ± 0.030.07 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Heart1.2 ± 0.11.0 ± 0.2
      Lung0.45 ± 0.060.44 ± 0.05
      Spleen0.15 ± 0.030.13 ± 0.02
      Digestive tract
      Digestive tract includes the stomach, small and large intestines, and pancreas.
      5.6 ± 0.34.3 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Liver30 ± 317 ± 1
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Kidney1.1 ± 0.10.66 ± 0.11
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Testes0.35 ± 0.060.24 ± 0.03
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Skin5.0 ± 0.46.6 ± 0.8
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Brown adipose3.8 ± 0.98.2 ± 1.0
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      White adipose6.7 ± 1.320 ± 2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Visceral adipose1.1 ± 0.12.9 ± 0.4
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Skeletal muscle
      Skeletal muscle was determined from fatty acid concentration data derived from thigh muscle samples.
      32 ± 230 ± 4
      Bones
      Bone was determined from fatty acid concentration data derived from femur bone samples.
      2.7 ± 0.32.4 ± 1.0
      Total-body d5-DHA, mg33 ± 514 ± 2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      Values were calculated by dividing the d5-DHA content of each tissue (Tables 113 and supplementary Tables III–IX) by the total-body d5-DHA as shown here. Data are given as percent total-body d5-DHA ± SD (n = 7 for d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Digestive tract includes the stomach, small and large intestines, and pancreas.
      b Skeletal muscle was determined from fatty acid concentration data derived from thigh muscle samples.
      c Bone was determined from fatty acid concentration data derived from femur bone samples.
      d Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.01.
      TABLE 15Unlabeled DHA present in dam-reared rat tissues at 8 days and after 28 days on d5-LNA and d5-LNA + DHA diets
      28 Day-old
      Tissues8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total tissue
      Plasma0.11 ± 0.020.10 ± 0.032.1 ± 0.9
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Brain1.6 ± 0.13.1 ± 0.4
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      6.3 ± 0.3
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Retina0.022 ± 0.0040.053 ± 0.005
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      0.091 ± 0.003
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Heart0.16 ± 0.030.12 ± 0.02
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P = 0.010–0.050.
      1.5 ± 0.2
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Lung0.27 ± 0.030.04 ± 0.01
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      0.70 ± 0.08
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Spleen0.020 ± 0.0070.010 ± 0.003
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      0.20 ± 0.05
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Digestive tract
      Digestive tract includes the stomach, small and large intestines, and pancreas.
      0.60 ± 0.130.34 ± 0.06
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      7.6 ± 0.8
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Liver3.6 ± 1.01.5 ± 0.3
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      22 ± 2
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Kidney0.13 ± 0.030.07 ± 0.01
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      1.0 ± 0.2
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Testes0.009 ± 0.0020.024 ± 0.01
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      0.29 ± 0.04
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Skin2.4 ± 0.50.50 ± 0.10
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      12 ± 3
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Brown adipose0.89 ± 0.191.8 ± 0.5
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      15 ± 1
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      White adipose3.4 ± 1.30.86 ± 0.28
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P = 0.010–0.050.
      34 ± 7
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Visceral adipose0.19 ± 0.030.11 ± 0.02
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      4.5 ± 1.1
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Skeletal muscle
      Sketetal muscle was determined from fatty acid concentration data derived from thigh muscle samples.
      1.1 ± 0.24.3 ± 0.6
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      46 ± 9
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Bones
      Bone was determined from fatty acid concentration data derived for femur bone samples.
      0.40 ± 0.080.21 ± 0.04
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      3.8 ± 1.1
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
       Total body unlabeled DHA, mg15 ± 413 ± 2156 ± 26
      Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Digestive tract includes the stomach, small and large intestines, and pancreas.
      b Sketetal muscle was determined from fatty acid concentration data derived from thigh muscle samples.
      c Bone was determined from fatty acid concentration data derived for femur bone samples.
      d Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P = 0.010–0.050.
      e Statistically different values between the 8 day dam-reared versus the d5-LNA or d5-LNA + DHA diet group of P < 0.010.

      RESULTS

      Percent body composition analysis

      Supplementary Table I shows the total body weights and the weight percentages of the whole body represented by the combined major internal organs (brain, digestive tract, heart, kidneys, liver, lungs, retinas, spleen, and testes) and various extraneous tissues for the 8 and 28 day-old dam-reared reference group animals (n = 4, each). For the two dam-reared reference groups, significant differences were detected between the mean total body weights and the related mean weight percentages of hair, skin, skeletal muscle, brown adipose, visceral adipose, and blood/other fluids. The total body weight of the 28 day-old dam-reared reference group was found to have increased 6-fold over that of the 8 day-old group. In the 28 day-old rat pups, the weight percentages representing the skeletal muscle and visceral fat were increased compared with the 8 day-old animals, at 1.9- and 2.4-fold, respectively; whereas for brown adipose and blood/other fluids, the weight percentages were decreased by 2.2- and 1.6-fold, respectively. Significant hair was not present on the skin of the 8 day-old rat pups, but it was found on the 28 day-old animals. There were no significant differences detected between the two age groups in the mean weight percentages of total bones, white adipose, and combined internal organs, but the skin approached a significantly lower percentage of the body weight in the 28 day-old animals (1.3-fold; P = 0.011). The percent body composition data (see supplementary Table I) for skin, skeletal muscle, bones, and brown, white, and visceral adipose were utilized in supplementary Table II to derive the whole-body masses of these corresponding tissues using the body weights of the d5-LNA and d5-LNA + DHA diet groups (28 day-old animals) and the 8 day-old dam-reared reference group.

      Body and organ/tissue weights

      Supplementary Table II shows the baseline body and whole-organ/tissue weights for the 8 day-old dam-reared reference, d5-LNA diet, and d5-LNA + DHA groups (n = 7, 7, and 6, respectively). After 21 days (p8–p28) on their respective diets, the final mean body weights of the rat pups in the d5-LNA and d5-LNA + DHA diet groups did not differ significantly from each other, but they both had increased ∼6-fold in size over the 8 day-old group. Growth curves showing the weight gain per day for these animals were previously reported by Lefkowitz et al. (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). Likewise, the weights for the whole organs/tissues did not significantly differ between the two diet groups, but the retinas approached a significantly lower weight in the d5-LNA + DHA diet group (1.2-fold; P = 0.011). As anticipated, the mean organ/tissue weights recorded for 8 day-old dam-reared reference group all underwent a dramatic increase in size in the 28 day-old animals in both dietary groups.

      Fatty acid compositional analyses

      Tables 113 present the fatty acid compositional data, expressed as micrograms (testes only) or milligrams fatty acid/whole organ or tissue, for the plasma (Table 1), heart (Table 2), lungs (Table 3), kidneys (Table 4), testes (Table 5), skin (Table 6), skeletal muscle (Table 7), bones (Table 8), brown adipose (Table 9), white adipose (Table 10), visceral adipose (Table 11), brain (Table 12), and liver (Table 13) of the two 28 day dietary (d5-LNA and d5-LNA + DHA) groups and the 8 day-old dam-reared reference group (n = 7, 6, and 7, respectively). Likewise, located in supplementary Tables III–IX are the corresponding data for retina (see supplementary Table III), spleen (see supplementary Table IV), digestive tract (see supplementary Table V), carcass (see supplementary Table VI), and the lower rear leg muscle subtypes, red gastrocnemius (see supplementary Table VII), white gastrocnemius (see supplementary Table VIII), and soleus (see supplementary Table IX), which are also expressed as milligrams (digestive tract and carcass), micrograms (retina and spleen only) or milligrams fatty acid/gram tissue (muscle subtypes). The fatty acid composition data shown here for brain and liver (Tables 12 and 13, respectively) has been previously reported by Lefkowitz et al. (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). In this paper, the fatty acid compositional data for liver has undergone mathematical correction to account for a mistaken repeat in multiplication by the liver total weights in the previous paper (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). Brain values did not merit any changes. In order to condense the presentation of this enormous volume of data, the whole-organ/tissue fatty acid composition results will be generalized under saturated, monounsaturated, n-6 PUFA, unlabeled LNA and d5-LNA, unlabeled EPA and deuterium-labeled EPA (d5-EPA), unlabeled DPA and deuterium-labeled DPA (d5-DPA), and unlabeled DHA and d5-DHA fatty acid sections to follow below. Data only for significant (P < 0.010) and near-significant (P = 0.010–0.050) differences between the d5-LNA and d5-LNA + DHA diet groups will be described in these Result sections, and all comparisons of these two diet groups to the 8 day-old dam-reared reference group will be reserved for the Discussion section. The units of data expression for red gastrocnemius, white gastrocnemius, and soleus leg muscle (see supplementary Tables VII–IX) are on a per gram tissue basis and their data will be presented separately at the end of the Results section.
      TABLE 2Heart fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/whole heart
      Saturates
       10:00.001 ± 0.0010.003 ± 0.0030.006 ± 0.005
       12:00.01 ± 0.010.07 ± 0.050.05 ± 0.03
       14:00.03 ± 0.020.08 ± 0.040.06 ± 0.02
       16:00.30 ± 0.070.80 ± 0.100.75 ± 0.034
       18:00.41 ± 0.061.8 ± 0.11.7 ± 0.1
       20:00.008 ± 0.0010.03 ± 0.0040.03 ± 0.002
       22:00.005 ± 0.0010.02 ± 0.0030.02 ± 0.002
       24:00.007 ± 0.0010.02 ± 0.0030.02 ± 0.005
      Total saturates0.8 ± 0.22.8 ± 0.32.6 ± 0.2
      Monounsaturates
       16:ln-70.007 ± 0.0020.01 ± 0.0050.01 ± 0.002
       18:ln-70.07 ± 0.010.15 ± 0.030.11 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       18:ln-90.12 ± 0.031.4 ± 0.41.1 ± 0.3
       20:ln-90.004 ± 0.0010.03 ± 0.010.02 ± 0.003
       22:ln-90.002 ± 0.00040.008 ± 0.0010.007 ± 0.0005
       24:ln-90.007 ± 0.0010.04 ± 0.010.05 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total monounsaturates0.21 ± 0.051.6 ± 0.51.3 ± 0.3
      n-6 PUFA
       18:2n-60.11 ± 0.041.0 ± 0.10.87 ± 0.10
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       18:3n-60.001 ± 0.00010.006 ± 0.0010.005 ± 0.002
       20:2n-60.01 ± 0.0020.04 ± 0.0040.04 ± 0.002
       20:3n-60.02 ± 0.010.06 ± 0.010.07 ± 0.004
       20:4n-60.48 ± 0.071.6 ± 0.21.1 ± 0.04
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:2n-60.001 ± 0.00030.003 ± 0.0010.004 ± 0.002
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:4n-60.05 ± 0.010.13 ± 0.020.04 ± 0.004
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-60.02 ± 0.0040.54 ± 0.040.04 ± 0.002
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA0.70 ± 0.123.4 ± 0.32.1 ± 0.1
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      n-3 PUFA
       d5-18:3n-30.01 ± 0.0050.01 ± 0.003
       18:3n-30.002 ± 0.0010.001 ± 0.00050.001 ± 0.001
       d5-20:5n-30.004 ± 0.0010.005 ± 0.001
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:5n-30.005 ± 0.0020.001 ± 0.00070.01 ± 0.002
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.06 ± 0.010.02 ± 0.001
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-30.06 ± 0.010.02 ± 0.0050.04 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-30.38 ± 0.020.15 ± 0.02
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:6n-30.16 ± 0.030.12 ± 0.021.5 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA0.23 ± 0.040.61 ± 0.041.8 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids1.9 ± 0.48.4 ± 1.07.8 ± 0.6
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 3Lung fatty acid compositions of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/both whole lungs
      Saturates
       10:00.004 ± 0.0020.03 ± 0.020.02 ± 0.01
       12:00.06 ± 0.020.28 ± 0.120.20 ± 0.14
       14:00.22 ± 0.060.42 ± 0.090.33 ± 0.13
       16:01.4 ± 0.22.8 ± 0.42.5 ± 0.5
       18:00.69 ± 0.081.6 ± 0.21.5 ± 0.1
       20:00.02 ± 0.0020.05 ± 0.010.05 ± 0.01
       22:00.02 ± 0.0040.05 ± 0.010.06 ± 0.01
       24:00.03 ± 0.0060.08 ± 0.010.10 ± 0.02
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      Total saturates2.5 ± 0.45.3 ± 0.84.7 ± 0.7
      Monounsaturates
       16:ln-70.06 ± 0.020.06 ± 0.020.07 ± 0.04
       18:ln-70.14 ± 0.020.08 ± 0.020.07 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       18:ln-90.79 ± 0.086.2 ± 1.64.9 ± 1.3
       20:ln-90.02 ± 0.0010.13 ± 0.020.13 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:ln-90.007 ± 0.0010.05 ± 0.010.06 ± 0.01
       24:ln-90.03 ± 0.0040.24 ± 0.030.28 ± 0.04
      Total monounsaturates1.0 ± 0.16.8 ± 1.65.5 ± 1.3
      n-6 PUFA
       18:2n-60.41 ± 0.061.2 ± 0.31.1 ± 0.3
       18:3n-60.008 ± 0.0010.01 ± 0.0030.01 ± 0.001
       20:2n-60.04 ± 0.0040.05 ± 0.010.05 ± 0.004
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:3n-60.07 ± 0.030.08 ± 0.010.10 ± 0.02
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:4n-60.64 ± 0.081.2 ± 0.11.0 ± 0.1
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:2n-60.009 ± 0.0060.007 ± 0.0010.009 ± 0.002
       22:4n-60.18 ± 0.010.25 ± 0.030.13 ± 0.02
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-60.05 ± 0.0060.26 ± 0.030.03 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA1.4 ± 0.13.1 ± 0.52.4 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      n-3 PUFA
       d5-18:3n-30.05 ± 0.010.04 ± 0.02
       18:3n-30.02 ± 0.0040.007 ± 0.0030.004 ± 0.002
       d5-20:5n-30.009 ± 0.0010.01 ± 0.003
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:5n-30.03 ± 0.010.002 ± 0.0010.04 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.05 ± 0.010.03 ± 0.004
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-30.14 ± 0.020.01 ± 0.0020.05 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-30.15 ± 0.020.06 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:6n-30.27 ± 0.030.04 ± 0.010.70 ± 0.08
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA0.46 ± 0.060.31 ± 0.040.94 ± 0.10
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids5.4 ± 0.615.5 ± 2.913.5 ± 2.1
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 4Kidney fatty acid compositions of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/both whole kidneys
      Saturates
       10:00.001 ± 0.00050.003 ± 0.0030.001 ± 0.001
       12:00.01 ± 0.010.04 ± 0.030.04 ± 0.01
       14:00.05 ± 0.020.14 ± 0.030.13 ± 0.02
       16:00.50 ± 0.112.4 ± 0.32.6 ± 0.4
       18:00.40 ± 0.082.9 ± 0.23.0 ± 0.4
       20:00.01 ± 0.0020.06 ± 0.010.07 ± 0.01
       22:00.02 ± 0.0040.08 ± 0.010.08 ± 0.01
       24:00.02 ± 0.0040.29 ± 0.030.33 ± 0.06
      Total saturates1.0 ± 0.25.9 ± 0.66.3 ± 0.8
      Monounsaturates
       16:ln-70.02 ± 0.010.03 ± 0.010.03 ± 0.01
       18:ln-70.08 ± 0.020.16 ± 0.040.14 ± 0.03
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       18:ln-90.24 ± 0.052.8 ± 0.43.0 ± 0.3
       20:ln-90.005 ± 0.0020.10 ± 0.010.10 ± 0.01
       22:ln-90.001 ± 0.00020.02 ± 0.000.02 ± 0.003
       24:ln-90.02 ± 0.0050.43 ± 0.040.48 ± 0.07
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total monounsaturates0.37 ± 0.073.6 ± 0.53.8 ± 0.4
      n-6 PUFA
       18:2n-60.19 ± 0.041.4 ± 0.12.1 ± 0.3
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       18:3n-60.001 ± 0.00030.008 ± 0.0010.01 ± 0.001
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:2n-60.009 ± 0.0010.10 ± 0.010.09 ± 0.010
       20:3n-60.04 ± 0.010.19 ± 0.040.25 ± 0.05
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:4n-60.58 ± 0.124.2 ± 0.53.4 ± 0.4
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:2n-60.0006 ± 0.00010.011 ± 0.0030.009 ± 0.002
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:4n-60.04 ± 0.010.12 ± 0.030.06 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-60.01 ± 0.0030.26 ± 0.040.02 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA0.87 ± 0.186.3 ± 0.65.9 ± 0.7
      n-3 PUFA
       d5-18:3n-30.02 ± 0.0050.03 ± 0.00
       18:3n-30.003 ± 0.0010.001 ± 0.0010.001 ± 0.001
       d5-20:5n-30.02 ± 0.0020.05 ± 0.011
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:5n-30.01 ± 0.0050.003 ± 0.0010.27 ± 0.07
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.04 ± 0.010.02 ± 0.003
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-30.03 ± 0.010.01 ± 0.0010.05 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-30.38 ± 0.050.10 ± 0.02
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:6n-30.13 ± 0.030.07 ± 0.011.0 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA0.18 ± 0.050.54 ± 0.061.5 ± 0.3
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids2.4 ± 0.516.3 ± 1.617.5 ± 2.2
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 5Testes fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      μg/both whole testes
      Saturates
       10:00.49 ± 0.1516 ± 271.3 ± 1.3
       12:00.16 ± 0.04129 ± 24018 ± 11
       14:01.9 ± 0.5124 ± 17659 ± 20
       16:048 ± 111,626 ± 5131,414 ± 181
       18:034 ± 6657 ± 231547 ± 93
       20:00.06 ± 0.0314 ± 414 ± 2
       22:00.90 ± 0.1710 ± 211 ± 1
       24:01.1 ± 0.29.5 ± 2.49.3 ± 1.0
      Total saturates86 ± 172,586 ± 11552,073 ± 296
      Monounsaturates
       16:ln-73.1 ± 0.727 ± 2618 ± 5
       18:ln-75.4 ± 1.079 ± 1285 ± 14
       18:ln-932 ± 62,326 ± 2,7631,055 ± 267
       20:ln-90.44 ± 0.0922 ± 1316 ± 3
       22:ln-90.24 ± 0.013.6 ± 2.03.0 ± 1.3
       24:ln-91.5 ± 0.219 ± 721 ± 4
      Total monounsaturates43 ± 82,477 ± 2,8121,198 ± 291
      n-6 PUFA
       18:2n-69.8 ± 2.4469 ± 526280 ± 70
       18:3n-60.12 ± 0.014.5 ± 3.43.0 ± 0.8
       20:2n-60.46 ± 0.1319 ± 718 ± 2
       20:3n-62.0 ± 0.448 ± 1472 ± 10
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:4n-639 ± 61,068 ± 228925 ± 129
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:2n-60.08 ± 0.024.4 ± 0.94.9 ± 2.3
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:4n-68.5 ± 1.8118 ± 26106 ± 13
       22:5n-61.6 ± 0.6613 ± 97350 ± 28
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA62 ± 112,344 ± 7801,757 ± 220
      n-3 PUFA
       d5-18:3n-37 ± 104.5 ± 3.2
       18:3n-30.05 ± 0.020.67 ± 0.370.88 ± 0.22
       d5-20:5n-33.1 ± 1.12.7 ± 0.5
       20:5n-30.95 ± 0.210.83 ± 0.397.0 ± 1.5
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-36.9 ± 4.52.4 ± 1.0
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:5n-32.2 ± 0.41.2 ± 1.16.2 ± 1.9
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-3115 ± 2434 ± 5
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:6n-39.0 ± 1.624 ± 7287 ± 38
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA12 ± 2171 ± 64345 ± 48
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids203 ± 377,564 ± 4,6985,374 ± 809
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 6Skin fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total skin
      Saturates
       10:06.4 ± 3.97.5 ± 3.88.0 ± 6.7
       12:045 ± 2499 ± 1795 ± 22
       14:037 ± 1864 ± 962 ± 12
       16:066 ± 26110 ± 18115 ± 26
       18:016 ± 545 ± 445 ± 3.7
       20:00.33 ± 0.081.2 ± 0.21.1 ± 0.1
       22:00.43 ± 0.071.3 ± 0.31.2 ± 0.2
       24:01.4 ± 0.33.3 ± 0.63.5 ± 0.3
      Total saturates173 ± 77330 ± 49331 ± 70
      Monounsaturates
       16:ln-78.3 ± 4.59.4 ± 2.910 ± 4
       18:ln-76.6 ± 2.16.6 ± 1.15.8 ± 0.9
       18:ln-952 ± 17774 ± 102761 ± 135
       20:ln-90.86 ± 0.203.8 ± 0.53.6 ± 0.4
       22:ln-90.13 ± 0.020.68 ± 0.070.67 ± 0.06
       24:ln-90.21 ± 0.041.6 ± 0.11.6 ± 0.2
      Total monounsaturates68 ± 23796 ± 104783 ± 138
      n-6 PUFA
       18:2n-630 ± 8147 ± 18156 ± 26
       18:3n-60.52 ± 0.170.76 ± 0.140.63 ± 0.13
       20:2n-61.6 ± 0.41.6 ± 0.31.7 ± 0.2
       20:3n-61.7 ± 0.41.7 ± 0.52.1 ± 0.5
       20:4n-66.7 ± 1.417 ± 213 ± 1
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:2n-60.12 ± 0.040.41 ± 0.140.43 ± 0.15
       22:4n-61.4 ± 0.21.9 ± 0.41.0 ± 0.1
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-60.45 ± 0.073.3 ± 0.50.97 ± 0.23
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA42 ± 11174 ± 21176 ± 26
      n-3 PUFA
       d5-18:3n-38.8 ± 1.59.5 ± 2.1
       18:3n-32.6 ± 1.20.72 ± 0.290.68 ± 0.14
       d5-20:5n-30.15 ± 0.030.23 ± 0.05
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:5n-30.48 ± 0.200.05 ± 0.010.36 ± 0.08
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.49 ± 0.130.28 ± 0.06
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-31.2 ± 0.30.14 ± 0.040.49 ± 0.09
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-31.6 ± 0.20.94 ± 0.15
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:6n-32.4 ± 0.50.50 ± 0.1012 ± 3
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA6.8 ± 2.212 ± 224 ± 5
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids291 ± 1121,313 ± 1711,314 ± 234
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 7Skeletal muscle fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total skeletal muscle
      Saturates
       10:00.3 ± 0.31.3 ± 0.41.6 ± 0.3
       12:02.5 ± 1.710 ± 211 ± 2
       14:02.8 ± 1.49.1 ± 1.49.0 ± 1.5
       16:07.9 ± 2.649 ± 746 ± 6
       18:04.6 ± 1.046 ± 539 ± 6.4
      Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:00.07 ± 0.010.36 ± 0.060.32 ± 0.06
       22:00.06 ± 0.010.24 ± 0.030.28 ± 0.09
       24:00.09 ± 0.010.68 ± 0.100.52 ± 0.23
      Total saturates18 ± 7116 ± 13107 ± 14
      Monounsaturates
       16:1n-70.7 ± 0.31.4 ± 0.51.7 ± 0.4
       18:1n-71.0 ± 0.33.6 ± 1.02.4 ± 0.4
      Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
       18:1n-96.1 ± 3.3137 ± 20125 ± 24
       20:1n-90.12 ± 0.041.7 ± 0.21.4 ± 0.2
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:1n-90.03 ± 0.010.25 ± 0.020.24 ± 0.05
       24:ln-90.12 ± 0.011.5 ± 0.11.3 ± 0.4
      Total monounsaturates8.0 ± 3.9145 ± 21132 ± 25
      n-6 PUFA
       18:2n-63.2 ± 1.062 ± 750 ± 8
      Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
       18:3n-60.06 ± 0.030.35 ± 0.080.25 ± 0.06
      Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:2n-60.26 ± 0.051.6 ± 0.11.3 ± 0.2
      Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:3n-60.42 ± 0.103.0 ± 0.62.6 ± 0.5
       20:4n-63.8 ± 0.637 ± 522 ± 3
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:2n-60.02 ± 0.0040.16 ± 0.050.14 ± 0.02
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:4n-60.77 ± 0.123.9 ± 0.61.2 ± 0.2
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:5n-60.24 ± 0.0512 ± 11.2 ± 0.2
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA8.8 ± 1.9121 ± 1478 ± 11
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      n-3 PUFA
       d5-18:3n-31.2 ± 0.21.3 ± 0.3
       18:3n-30.20 ± 0.090.08 ± 0.020.08 ± 0.02
       d5-20:5n-30.28 ± 0.050.28 ± 0.06
       20:5n-30.13 ± 0.050.07 ± 0.010.55 ± 0.09
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-31.77 ± 0.310.67 ± 0.13
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:5n-30.64 ± 0.120.64 ± 0.111.3 ± 0.3
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-311 ± 14.3 ± 0.8
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:6n-31.1 ± 0.24.3 ± 0.646 ± 9
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA2.1 ± 0.419 ± 254 ± 10
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids37 ± 13401 ± 45371 ± 51
      Total body skeletal muscle values were determined from fatty acid concentration data (mg/g tissue) derived from thigh muscle samples. Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      TABLE 8Bone fatty acid composition of the two experimental diet and 8 day-old dam-related reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total bones
      Saturates
       10:00.03 ± 0.030.01 ± 0.020.01 ± 0.01
       12:00.44 ± 0.21.0 ± 0.71.0 ± 0.5
       14:00.65 ± 0.31.6 ± 0.61.6 ± 0.7
       16:03.2 ± 1.011 ± 212 ± 4
       18:01.9 ± 0.49.1 ± 1.39.6 ± 3.4
       20:00.03 ± 0.010.11 ± 0.020.12 ± 0.04
       22:00.05 ± 0.010.13 ± 0.010.14 ± 0.05
       24:00.09 ± 0.020.26 ± 0.030.28 ± 0.09
      Total saturates6.4 ± 1.823 ± 424 ± 9
      Monounsaturates
       16:1n-70.30 ± 0.100.68 ± 0.140.78 ± 0.25
       18:1n-70.47 ± 0.111.1 ± 0.21.1 ± 0.4
       18:1n-92.0 ± 0.526 ± 927 ± 12
       20:1n-90.04 ± 0.010.61 ± 0.120.57 ± 0.25
       22:1n-90.01 ± 0.0020.14 ± 0.020.14 ± 0.05
       24:1n-90.09 ± 0.021.1 ± 0.11.2 ± 0.4
      Total monounsaturates2.9 ± 0.730 ± 930 ± 13
      n-6 PUFA
       18:2n-61.0 ± 0.36.2 ± 1.77.5 ± 2.8
       18:3n-60.02 ± 0.010.07 ± 0.030.04 ± 0.01
       20:2n-60.14 ± 0.030.43 ± 0.050.51 ± 0.18
       20:3n-60.14 ± 0.030.49 ± 0.030.72 ± 0.20
      Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:4n-61.9 ± 0.49.4 ± 1.48.5 ± 3.0
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:2n-60.01 ± 0.0020.05 ± 0.0050.05 ± 0.01
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:4n-60.32 ± 0.051.6 ± 0.21.1 ± 0.5
      Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:5n-60.08 ± 0.021.3 ± 0.20.18 ± 0.05
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA3.6 ± 0.820 ± 318 ± 7
      n-3 PUFA
       d5-18:3n-30.22 ± 0.110.24 ± 0.11
       18:3n-30.04 ± 0.020.02 ± 0.010.01 ± 0.01
       5-20:5n-30.04 ± 0.010.07 ± 0.02
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       20:5n-30.05 ± 0.010.02 ± 0.000.13 ± 0.03
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.26 ± 0.030.19 ± 0.06
      Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:5n-30.20 ± 0.040.04 ± 0.010.28 ± 0.10
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-30.89 ± 0.140.35 ± 0.14
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
       22:6n-30.40 ± 0.080.21 ± 0.043.8 ± 1.1
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA0.69 ± 0.141.7 ± 0.35.1 ± 1.5
      Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids78 ± 3074 ± 1678 ± 30
      Total body bone values were determined from fatty acid concentration data (mg/g tissue) derived from femur bone samples. Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA + DHA diet groups of P < 0.010.
      TABLE 9Brown adipose fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total brown adipose
      Saturates
       10:00.1 ± 0.11.6 ± 0.51.4 ± 0.3
       12:01.4 ± 0.923 ± 421 ± 2
       14:01.6 ± 1.026 ± 525 ± 4
       16:04.1 ± 1.864 ± 1470 ± 15
       18:03.5 ± 0.927 ± 530 ± 4
       20:00.07 ± 0.030.35 ± 0.060.39 ± 0.06
       22:00.04 ± 0.010.09 ± 0.010.09 ± 0.01
       24:00.08 ± 0.020.11 ± 0.010.10 ± 0.05
      Total saturates11 ± 5142 ± 26148 ± 23
      Monounsaturates
       16:1n-70.25 ± 0.162.6 ± 0.72.9 ± 1.4
       18:1n-70.57 ± 0.202.3 ± 0.52.3 ± 0.6
       18:1n-94.4 ± 1.9207 ± 25208 ± 16
       20:1n-90.16 ± 0.062.4 ± 0.42.6 ± 0.3
       22:1n-90.03 ± 0.010.16 ± 0.030.18 ± 0.03
       24:1n-90.07 ± 0.020.29 ± 0.040.31 ± 0.06
      Total monounsaturates5.5 ± 2.3215 ± 26216 ± 18
      n-6 PUFA
       18:2n-63.4 ± 0.345 ± 549 ± 5
       18:3n-60.03 ± 0.010.3 ± 0.070.22 ± 0.03
       20:2n-60.2 ± 0.11.1 ± 0.201.3 ± 0.1
       20:3n-60.5 ± 0.21.3 ± 0.291.5 ± 0.2
       20:4n-62.7 ± 0.64.9 ± 1.25.2 ± 0.9
       22:2n-60.03 ± 0.010.14 ± 0.050.13 ± 0.02
       22:4n-60.5 ± 0.11.7 ± 0.41.7 ± 0.2
       22:5n-60.21 ± 0.052.2 ± 0.60.8 ± 0.1
      Statistically different values between d5-LNA + DHA diet group of P < 0.010.
      Total n-6 PUFA7.6 ± 2.057 ± 760 ± 5
      n-3 PUFA
       d5-18:3n-32.7 ± 0.42.9 ± 0.2
       18:3n-30.11 ± 0.060.41 ± 0.180.40 ± 0.15
       d5-20:5n-30.08 ± 0.030.11 ± 0.01
      Statistically different values between d5-LNA + DHA diet group of P = 0.010–0.050.
       20:5n-30.07 ± 0.020.06 ± 0.030.20 ± 0.02
      Statistically different values between d5-LNA + DHA diet group of P < 0.010.
       d5-22:5n-30.23 ± 0.110.26 ± 0.02
       22:5n-30.43 ± 0.090.59 ± 0.150.94 ± 0.17
      Statistically different values between d5-LNA + DHA diet group of P < 0.010.
       d5-22:6n-31.3 ± 0.31.2 ± 0.1
       22:6n-30.89 ± 0.191.8 ± 0.515 ± 1
      Statistically different values between d5-LNA + DHA diet group of P < 0.010.
      Total n-3 PUFA1.5 ± 0.37.1 ± 1.521 ± 1
      Statistically different values between d5-LNA + DHA diet group of P < 0.010.
      Total fatty acids25 ± 9421 ± 57445 ± 45
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA + DHA diet group of P = 0.010–0.050.
      b Statistically different values between d5-LNA + DHA diet group of P < 0.010.
      TABLE 10White adipose fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total white adipose
      Saturates
       10:09.8 ± 8.159 ± 962 ± 8
       12:052 ± 33279 ± 51302 ± 32
       14:044 ± 25166 ± 33182 ± 22
       16:089 ± 38280 ± 62347 ± 54
       18:018 ± 681 ± 1490 ± 7
       20:00.21 ± 0.061.1 ± 0.21.2 ± 0.4
       22:00.08 ± 0.020.41 ± 0.140.49 ± 0.47
       24:00.02 ± 0.010.29 ± 0.112.0 ± 1.4
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      Total saturates213 ± 109867 ± 163986 ± 117
      Monounsaturates
       16:1n-713 ± 729 ± 1138 ± 10
       18:1n-772 ± 2414 ± 414 ± 4
       18:1n-98.1 ± 2.22096 ± 3412228 ± 161
       20:1n-91.2 ± 0.310 ± 210 ± 1
       22:1n-90.10 ± 0.020.46 ± 0.090.44 ± 0.05
       24:1n-90.12 ± 0.050.65 ± 0.290.71 ± 0.16
      Total monounsaturates94 ± 322,150 ± 3542,291 ± 172
      n-6 PUFA
       18:2n-636 ± 10384 ± 59438 ± 30
       18:3n-60.90 ± 0.242.5 ± 0.482.2 ± 0.4
       20:2n-62.0 ± 0.64.6 ± 1.305.1 ± 0.7
       20:3n-62.1 ± 0.53.3 ± 1.124.2 ± 0.9
       20:4n-65.8 ± 1.715 ± 412 ± 1
       22:2n-60.13 ± 0.030.75 ± 0.200.70 ± 0.52
       22:4n-62.0 ± 0.52.5 ± 0.81.6 ± 0.4
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:5n-60.75 ± 0.235.6 ± 1.42.1 ± 0.5
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA50 ± 14418 ± 67466 ± 33
      n-3 PUFA
       d5-18:3n-328 ± 433 ± 3
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       18:3n-33.2 ± 1.51.7 ± 0.71.7 ± 0.7
       d5-20:5n-30.47 ± 0.180.53 ± 0.11
       20:5n-30.71 ± 0.370.16 ± 0.080.79 ± 0.20
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.92 ± 0.420.78 ± 0.16
       22:5n-31.8 ± 0.60.34 ± 0.171.2 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-32.2 ± 0.52.9 ± 0.4
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       22: 6n-33.4 ± 1.30.86 ± 0.2834 ± 7
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA9.1 ± 3.734 ± 674 ± 10
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids492 ± 3613,469 ± 5873,818 ± 315
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 11Visceral adipose fatty acid composition of the two experimental diet and 8-day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total visceral adipose
      Saturates
       10:00.22 ± 0.166.8 ± 1.14.7 ± 1.4
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       12:01.9 ± 1.034 ± 633 ± 4
       14:01.9 ± 0.922 ± 423 ± 3
       16:04.2 ± 1.344 ± 952 ± 8
       18:00.94 ± 0.1913 ± 213 ± 1
       20:00.01 ± 0.0020.19 ± 0.070.16 ± 0.03
       22:00.006 ± 0.0020.05 ± 0.020.09 ± 0.03
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       24:00.006 ± 0.0010.05 ± 0.010.04 ± 0.03
      Total saturates9.2 ± 3.5121 ± 19126 ± 16
      Monounsaturates
       16:ln-70.54 ± 0.243.8 ± 1.34.6 ± 1.0
       18:ln-70.43 ± 0.060.19 ± 0.070.18 ± 0.03
       18:ln-93.4 ± 0.6332 ± 53315 ± 30
       20:ln-90.07 ± 0.011.1 ± 0.71.5 ± 0.2
       22:ln-90.007 ± 0.0010.08 ± 0.020.13 ± 0.04
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       24:ln-90.01 ± 0.0040.08 ± 0.020.14 ± 0.06
      Total monounsaturates4.5 ± 0.9337 ± 54322 ± 31
      n-6 PUFA
       18:2n-61.8 ± 0.358 ± 960 ± 6
       18:3n-60.04 ± 0.010.37 ± 0.080.27 ± 0.05
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       20:2n-60.11 ± 0.020.60 ± 0.140.63 ± 0.09
       20:3n-60.02 ± 0.0030.37 ± 0.290.59 ± 0.11
       20:4n-60.31 ± 0.052.0 ± 0.41.5 ± 0.2
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:2n-60.01 ± 0.0010.06 ± 0.010.07 ± 0.01
       22:4n-60.11 ± 0.010.42 ± 0.120.23 ± 0.05
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-60.04 ± 0.010.86 ± 0.170.26 ± 0.07
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA2.4 ± 0.363 ± 1063 ± 6
      n-3 PUFA
       d5-18:3n-34.4 ± 0.74.2 ± 0.5
       18:3n-30.16 ± 0.050.18 ± 0.050.15 ± 0.03
       d5-20:5n-30.07 ± 0.040.09 ± 0.04
       20:5n-30.04 ± 0.010.02 ± 0.010.12 ± 0.03
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.11 ± 0.040.12 ± 0.05
       22:5n-30.10 ± 0.020.03 ± 0.010.16 ± 0.04
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-30.35 ± 0.070.42 ± 0.08
       22:6n-30.19 ± 0.030.11 ± 0.024.5 ± 1.1
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA0.49 ± 0.105.3 ± 0.89.8 ± 1.8
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids17 ± 5526 ± 84520 ± 52
      Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet).
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      TABLE 12Brain fatty acid composition of the two experimental diet and 8 day-old dam-reared reference groups
      28 Day-old
      Fatty Acid8 Day Dam-rearedd5-LNA Dietd5-LNA + DHA Diet
      mg/total brain
      Saturates
       10:00.02 ± 0.010.05 ± 0.030.01 ± 0.003
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       12:00.01 ± 0.0030.01 ± 0.010.008 ± 0.005
       14:00.30 ± 0.030.13 ± 0.020.21 ± 0.10
       16:04.2 ± 0.311 ± 110 ± 0
       18:02.0 ± 0.19.3 ± 0.89.5 ± 0.5
       20:00.01 ± 0.0010.20 ± 0.040.22 ± 0.03
       22:00.007 ± 0.0010.20 ± 0.040.24 ± 0.03
       24:00.007 ± 0.0020.39 ± 0.090.45 ± 0.04
      Total saturates7.1 ± 0.623 ± 223 ± 1
      Monounsaturates
       16:ln-70.21 ± 0.010.13 ± 0.010.16 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       18:ln-70.35 ± 0.031.4 ± 0.11.4 ± 0.1
       18:ln-91.5 ± 0.17.6 ± 0.77.7 ± 0.5
       20:ln-90.03 ± 0.0030.25 ± 0.260.50 ± 0.07
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
       22:ln-90.002 ± 0.00040.06 ± 0.010.06 ± 0.01
       24:ln-90.01 ± 0.020.66 ± 0.300.65 ± 0.09
      Total monounsaturates2.2 ± 0.211 ± 111 ± 1
      n-6 PUFA
       18:2n-60.13 ± 0.020.35 ± 0.040.45 ± 0.04
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       18:3n-60.01 ± 0.0010.004 ± 0.0010.004 ± 0.001
       20:2n-6
       20:3n-60.06 ± 0.010.21 ± 0.020.31 ± 0.05
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:4n-61.8 ± 0.15.0 ± 0.34.7 ± 0.2
       22:2n-6
       22:4n-60.39 ± 0.031.5 ± 0.11.3 ± 0.1
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-60.21 ± 0.021.2 ± 0.10.25 ± 0.02
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-6 PUFA2.6 ± 0.28.3 ± 0.57.0 ± 0.3
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      n-3 PUFA
       d5-18:3n-30.002 ± 0.00020.002 ± 0.0002
       18:3n-30.0008 ± 0.0003NDND
       d5-20:5n-30.002 ± 0.0010.004 ± 0.001
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       20:5n-30.007 ± 0.0010.001 ± 0.00010.01 ± 0.004
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:5n-30.03 ± 0.0020.02 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:5n-30.05 ± 0.0040.02 ± 0.0030.06 ± 0.01
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       d5-22:6n-32.4 ± 0.30.63 ± 0.03
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
       22:6n-31.6 ± 0.13.1 ± 0.46.3 ± 0.3
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total n-3 PUFA1.6 ± 0.15.6 ± 0.47.1 ± 0.3
      Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.
      Total fatty acids13 ± 146 ± 348 ± 2
      ND, not detected(i.e., < 0.0001 mg/total brain). Data represent means ± SD (n = 7 for 8 day dam-reared and d5-LNA diet, n = 6 for d5-LNA + DHA diet). 20:2n-6 and 22:2n-6 were not reported; and this table was originally found in Ref.
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      .
      a Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P = 0.010–0.050.
      b Statistically different values between d5-LNA and d5-LNA + DHA diet groups of P < 0.010.

      Saturated fatty acid composition of rat pup organs

      The most abundant and physiologically relevant saturated fatty acids typically found in tissues are 16:0 and 18:0 (palmitic and stearic acids), which also represent the main products of the saturated fatty acid biosynthetic pathway (
      • Wakil S.J.
      • Stoops J.K.
      • Joshi V.C.
      Fatty acid synthesis and its regulation.
      ). Only the retina showed significant differences in these saturated fatty acids, where there was a 1.3- and 1.2-fold increase in 16:0 and 18:0 content, respectively, for the d5-LNA + DHA compared with the d5-LNA diet group. For the retina, total saturated fatty acid content was also significantly increased by 1.2-fold in the d5-LNA + DHA diet group.

      Monounsaturated fatty acid composition of rat pup organs

      The most abundant and physiologically relevant monounsaturated fatty acid typically found in tissues is 18:1n-9 (oleic acid), which can be biosynthesized by the Δ-9 desaturation of 18:0 (
      • Sampath H.
      • Ntambi J.M.
      The fate and intermediary metabolism of stearic acid.
      ). Only retina was found to significantly differ between the two dietary groups in oleic acid content, with a decrease of 1.3-fold in the d5-LNA + DHA compared with d5-LNA diet group. Correspondingly, retina was also decreased by 1.3-fold in total monounsaturated fatty acid content in the d5-LNA + DHA diet group.

      n-6 PUFA composition of rat pup organs

      The most abundant and physiologically relevant n-6 PUFAs typically found in tissues are 18:2n-6 (LA), a dietary essential PUFA, and its biosynthetic end product, 20:4n-6 (arachidonic acid, AA) (
      • Sprecher H.
      Metabolism of highly unsaturated n-3 and n-6 fatty acids.
      ). During times of n-3 PUFA deprivation, it is apparent that the accretion of AA from LA is increased and AA is also further metabolized to produce 22:5n-6 (DPAn-6), which accumulates in tissues so as to reciprocally replace lost DHA (
      • Greiner R.S.
      • Catalan J.N.
      • Moriguchi T.
      • Salem Jr., N.
      Docosapentaenoic acid does not completely replace DHA in n-3 FA-deficient rats during early development.
      ,
      • Lim S.Y.
      • Hoshiba J.
      • Salem Jr., N.
      An extraordinary degree of structural specificity is required in neural phospholipids for optimal brain function: n-6 docosapentaenoic acid substitution for docosahexaenoic acid leads to a loss in spatial task performance.
      ,
      • Moriguchi T.
      • Loewke J.
      • Garrison M.
      • Catalan J.N.
      • Salem Jr., N.
      Reversal of docosahexaenoic acid deficiency in the rat brain, retina, liver, and serum.
      ,
      • Moriguchi T.
      • Lim S.Y.
      • Greiner R.
      • Lefkowitz W.
      • Loewke J.
      • Hoshiba J.
      • Salem Jr., N.
      Effects of an n-3-deficient diet on brain, retina, and liver fatty acyl composition in artificially reared rats.
      ). Of all tissues examined, only kidney and brain were found to be changed in LA content, with an increase of 1.5- and 1.3-fold, respectively, in the d5-LNA + DHA compared with d5-LNA diet group. Despite an overall lack in effect of the diets on LA in the tissues, the AA content of the tissues was significantly decreased in the d5-LNA + DHA compared with d5-LNA diet group, except in plasma, brown adipose, white adipose, visceral adipose (P = 0.025), brain (P = 0.042), and liver (P = 0.019). The largest decrease in AA content for the internal organs and other various tissues was found in heart and skeletal muscle, at 1.5- and 1.7-fold, respectively, of the d5-LNA diet group. As anticipated, all tissues examined in this study also had significantly lower amounts of DPAn-6, a marker of DHA deficiency, in the d5-LNA + DHA compared with the d5-LNA diet group, ranging from a 2- to a 36-fold decrease. For the internal organs and various other tissues, the liver and skeletal muscle displayed the largest decreases in DPAn-6 content at 36- and 10-fold, respectively, of the d5-LNA diet group. Plasma also had a large decrease in DPAn-6 at 19-fold of the d5-LNA diet group. Despite decreased accumulation of LA, AA, and/or DPAn-6 in the d5-LNA + DHA diet group for all tissues, only the heart, lungs, retina, skeletal muscle, brain, and liver showed a significantly decreased content of total n-6 PUFA (1.6-, 1.3-, 1.7-, 1.5-, 1.2-, and 1.5-fold, respectively, of the d5-LNA diet group).

      Unlabeled LNA and d5-LNA composition of rat pup organs

      LNA is the initial precursor n-3 PUFA for the biosynthesis of DHA; thus, under conditions of a diet replete in DHA (d5-LNA + DHA diet), tissue pools of unlabeled LNA and d5-LNA would be anticipated to be less actively utilized. In this experiment, all dietary LNA fed to the animals from p8 to p28 consisted of d5-labeled LNA, which leads to the formation of biosynthesized d5-DHA (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ). Any unlabeled LNA present in the tissues at p28 is endogenous LNA that accumulated in the p8 dam-reared animals before the start of the feeding experiment. There were no tissues that displayed a significant increase in d5-LNA and/or unlabeled LNA content in the d5-LNA + DHA compared with d5-LNA diet group; but the white adipose content approached a significant increase in d5-LNA content (1.2-fold; P = 0.025).

      Unlabeled EPA and d5-EPA composition of rat pup organs

      EPA is one of the principal stable intermediates and follows LNA in the biosynthetic pathway toward DHA; thus, d5-LNA would be converted to d5-EPA. In the experimental diet replete in DHA (d5-LNA + DHA diet), the formation of unlabeled EPA and biosynthesized d5-EPA would be anticipated to be decreased. In none of the tissues, however, was there a significant or near-significant decrease in the amount of biosynthesized d5-EPA content in the d5-LNA + DHA compared with the d5-LNA diet group. In fact, plasma, lung, kidney, brain, and liver all exhibited significant increases in biosynthesized d5-EPA on the d5-LNA + DHA diet; whereas heart, skin, bone, brown adipose, and carcass approached a significant increase in d5-EPA content (P = 0.020, 0.015, 0.014, 0.013, and 0.011, respectively). For the internal organs and various other tissues, liver and bone exhibited the greatest increases in biosynthesized d5-EPA, at 3.2- and 1.7-fold, respectively, of the d5-LNA diet group. Plasma, however, was found to have a much larger increase in biosynthesized d5-EPA, at 4.8-fold of the d5-LNA diet group. Likewise, in all of the whole organs/tissues, unlabeled EPA was found to be significantly increased in the d5-LNA + DHA compared with d5-LNA diet group. Compared with the other internal organs and body tissues, kidney and skeletal muscle showed the greatest increases in unlabeled EPA content at 98- and 8-fold, respectively. Conversely, plasma showed a more marked response than these tissues in that unlabeled EPA was undetectable in the d5-LNA diet group, but was present in the d5-LNA + DHA diet group at 22% of the levels found in the liver.

      Unlabeled DPA and d5-DPA composition of rat pup organs

      DPA is the elongation product of EPA in the biosynthetic pathway toward DHA, and thus biosynthesized d5-EPA would be directly converted to d5-DPA. In the experimental diet replete in DHA (d5-LNA + DHA diet), the formation of unlabeled DPA and biosynthesized d5-DPA would be anticipated to be decreased. Consistent with this, in all tissues except brown, white, and visceral adipose, there was a significant or near-significant (bone, spleen, and testes; P = 0.022, 0.012, and 0.037, respectively) decrease in the amount of biosynthesized d5-DPA in the d5-LNA + DHA compared with the d5-LNA diet group. Brown, white, and visceral adipose showed no significant differences in biosynthesized d5-DPA content between the two diet groups. For the internal organs and various other tissues, heart and skeletal muscle showed the greatest decreases in biosynthesized d5-DPA for the d5-LNA + DHA diet group, at 3.8- and 2.6-fold, respectively, of the d5-LNA diet group. Plasma also exhibited a comparatively large decrease in d5-DPA at 2.5-fold of the d5-LNA diet group. In contrast to the findings for biosynthesized d5-DPA, in all of the whole organs/tissues, unlabeled DPA was found to be significantly increased in the d5-LNA + DHA compared with the d5-LNA diet group. For the internal organs, liver showed the greatest increase in unlabeled DPA, at 12-fold of the d5-LNA diet group. With respect to the various other tissues, bone and visceral adipose exhibited the largest increase in unlabeled DPA, both at ∼6.3-fold of the d5-LNA diet group. Plasma had a comparatively larger increase in unlabeled DPA at 13-fold of the d5-LNA diet group.

      Unlabeled DHA and d5-DHA composition of rat pup organs

      DHA is the terminal product in the biosynthetic pathway that begins with dietary LNA. For the experimental diet replete in preformed unlabeled DHA (d5-LNA + DHA diet), the formation of biosynthesized d5-DHA would be anticipated to be decreased, but the tissue accumulation of unlabeled DHA, from the constant input of dietary DHA, would be large. Consistent with this, in all tissues except brown, white, and visceral adipose, there was a significant decrease in the amount of biosynthesized d5-DHA content in the d5-LNA + DHA compared with the d5-LNA diet group. Whereas brown and visceral adipose showed no significant or near-significant differences between the two diet groups, the effect on biosynthesized d5-DHA in white adipose, although only of near significance (P = 0.022), was in the opposite direction to that observed for the other tissues, inasmuch as it displayed increased d5-DHA in the d5-LNA + DHA diet group. The retina showed the most marked and significant decrease in biosynthesized d5-DHA, at 5.1-fold of the d5-LNA diet group. With respect to the major internal organs, however, brain, liver, and kidneys showed the greatest decrease in the amount of biosynthesized d5-DHA present, with each at ∼3.9-fold of the d5-LNA diet group. For the other tissues, bone and skeletal muscle exhibited the greatest decrease, with each at ∼2.5-fold of the d5-LNA diet group. Plasma exhibited a decrease in unlabeled DPA of 2.9-fold that of the d5-LNA diet group.
      As anticipated, all tissues showed a dramatic increase in unlabeled DHA content in the d5-LNA + DHA compared with the d5-LNA diet group. Of the internal organs, digestive tract and spleen showed the largest increases, both at ∼21-fold of the d5-LNA diet group. Of the other tissues, visceral fat exhibited the greatest increase, at 43-fold of the d5-LNA diet group. Plasma exhibited an increase in unlabeled DHA of 20-fold that of the d5-LNA diet group. As a direct result of the marked accumulation of unlabeled DHA in the d5-LNA + DHA diet group, there was a corresponding significant increase in the total n-3 PUFA content of all organs/tissues within this diet group.

      Percent distribution of d5-DHA in rat pup organs

      Table 14 shows the biosynthesized d5-DHA in the tissues of the d5-LNA + DHA versus d5-LNA diet groups (n = 6 and 7, respectively) expressed as a percentage of the total-body d5-DHA that was accumulated. The total-body amount of d5-DHA found in the d5-LNA + DHA diet group was 2.3-fold lower than that of the d5-LNA diet group; whereas the corresponding amount of total-body unlabeled DHA was 12-fold higher than the d5-LNA diet group. Of the internal organs and other tissues, the greatest percent of whole-body distribution of d5-DHA was found in the liver and skeletal muscle, respectively, for both diet groups. Although skeletal muscle did not differ between the diet groups in the percent distribution of d5-DHA, there were differences detected for many of the other tissues examined. The differing internal organs as represented by brain, retina, digestive tract, liver, kidneys, and testes all had a significantly lower percentage (1.7-, 2.2-, 1.3-, 1.8-, 1.7-, and 1.4-fold, respectively) of the total d5-DHA in the d5-LNA + DHA compared with the d5-LNA diet group. In contrast, the other tissues, as represented by skin and brown, white, and visceral adipose, all had a significantly higher percentage (1.3-, 2.1-, 3.0-, and 2.7-fold, respectively) of the total d5-DHA in the d5-LNA + DHA diet group. This may indicate that a redistribution of biosynthesized DHA out of these latter tissues, which are key sites of fat storage, and into the major internal organs occurs when DHA is absent from the diet, as in the d5-LNA diet group.
      Table 15 shows the unlabeled DHA content, in milligrams, of the whole organs and tissues of the 8 day-old dam-reared reference group compared with 28 days in the d5-LNA and d5-LNA + DHA dietary groups (n = 7, 7, and 6, respectively). These data indicate how much DHA has been accumulated in each organ over the 3 week period of the experiment. Table 15 indicates that most organs had a substantial accumulation of DHA when animals were fed the d5-LNA + DHA diet. In contrast, most organs had a net loss of DHA when animals were fed the d5-LNA diet.

      Fatty acid composition of rear leg muscle subtypes

      Supplementary Tables VII–IX show the fatty acid compositional results obtained for red gastrocnemius (fast-oxidative), white gastrocnemius (fast-glycolytic), and soleus (slow-oxidative) leg muscle fiber types, respectively. Because these muscle fiber types are not a distinct whole-organ compartment, and for purposes of comparison, the data are expressed as milligrams fatty acid/gram tissue. Of the three muscle types, red gastrocnemius displayed the most significant differences between the d5-LNA + DHA and d5-LNA diet groups. Red gastrocnemius in the d5-LNA + DHA diet group had significantly decreased amounts of 18:1n-9, 20:1n-9, total monounsaturates, LA, 20:2n-6, AA, 22:4n-6, 22:5n-6, total n-6 PUFA, d5-LNA, unlabeled LNA, d5-DPA, and d5-DHA, with d5-LNA and unlabeled LNA approaching significance (P = 0.041 and 0.018, respectively); but there were significantly increased amounts of unlabeled EPA, unlabeled DPA, unlabeled DHA, and total n-3 PUFA. White gastrocnemius and soleus leg muscles were nearly identical in their significant differences, both displaying in the d5-LNA + DHA diet group significantly decreased amounts of 22:4n-6, 22:5n-6, d5-DPA, and d5-DHA, but there were significantly increased amounts of unlabeled DPA, unlabeled DHA, and total n-3 PUFA. White gastrocnemius additionally showed significantly decreased amounts of AA in the d5-LNA + DHA diet group, whereas soleus had significantly decreased amounts of total n-6 PUFA, as well as increased amounts of d5-EPA approaching significance (P = 0.044). The significant differences between the diet groups as noted above for red gastrocnemius were the most similar of the three muscle types to that reported for total-body skeletal muscle (Table 7), whose data were derived by extrapolation from analysis of mixed thigh muscle tissue.

      DISCUSSION

      This study focused upon the issue of whether inclusion of preformed DHA in the diet decreases the net accretion of DHA derived from dietary LNA in growing rat pups. We have expanded upon our previous work (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ) that examined only in the brains and livers of rat pups during development (p8–p28) whether inclusion of preformed DHA in the diet decreases the net accretion of DHA derived from dietary LNA. We have now obtained similar data for all the other major tissues of this same set of animals. Our study is novel in that it is able to examine the total accumulation of biosynthesized d5-DHA in various tissues over an extended time period. We achieved this result due to the complete and continuous replacement of LNA in the animals' diet with the stable isotope-labeled LNA (d5-LNA). If the animals are not provided any unlabeled n-3 PUFA during the feeding experiment, any unlabeled n-3 PUFA present in the tissues at the end of the study must be that remaining from prior accumulated stores of these fatty acids. Our study is also unique in that the experimental feeding and development time period examined, p8–p28, is one in which the rat pups are undergoing an intense spurt in brain growth (
      • Dobbing J.
      Vulnerable periods in developing brain.
      ). Accelerated growth of all other tissues throughout the body is also clearly evident in the data presented in supplementary Table I, which show an increase in organ/tissue masses of 2- to 24-fold between 8 and 28 days of age. Overall, the p8–p28 rat pup model is somewhat analogous to the growth period of brain and other organs that occurs in humans from ∼28 week gestation to 2–3 years of age (
      • Dobbing J.
      Vulnerable periods in developing brain.
      ); however, it must be taken into account that the basal metabolic rate of infant rats and humans differs greatly, which may produce major differences in lipid metabolism within some tissues.
      One central question in human infant nutrition is whether the DHA demands of the developing brain, retina, and other organs can be supported by providing sufficient dietary LNA alone or if the inclusion of dietary DHA is necessary. It is known that young mammals provided with diets sufficient in LNA (>1% of energy intake) do not attain the brain or retinal levels of DHA associated with animals that consume preformed dietary DHA (>0.25% of energy intake) (
      • Ward G.
      • Woods J.
      • Reyzer M.
      • Salem Jr., N.
      Artificial rearing of infant rats on milk formula deficient in n-3 essential fatty acids: a rapid method for the production of experimental n-3 deficiency.
      ,
      • Abedin L.
      • Lien E.L.
      • Vingrys A.J.
      • Sinclair A.J.
      The effects of dietary alpha-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig.
      ,
      • Bowen R.A.
      • Clandinin M.T.
      High dietary 18:3n-3 increases the 18:3n-3 but not the 22:6n-3 content in the whole body, brain, skin, epididymal fat pads, and muscles of suckling rat pups.
      ,
      • Woods J.
      • Ward G.
      • Salem Jr., N.
      Is docosahexaenoic acid necessary in infant formula? Evaluation of high linolenate diets in the neonatal rat.
      ). Several stable-isotope studies have supported this contention by demonstrating that dietary LNA is not bioequivalent to preformed dietary DHA in supplying DHA to various tissues (
      • Greiner R.C.
      • Winter J.
      • Nathanielsz P.W.
      • Brenna J.T.
      Brain docosahexaenoate accretion in fetal baboons: bioequivalence of dietary alpha-linolenic and docosahexaenoic acids.
      ,
      • Su 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.
      ,
      • Su H.M.
      • Bernardo L.
      • Mirmiran M.
      • Ma X.H.
      • Nathanielsz P.W.
      • Brenna J.T.
      Dietary 18:3n-3 and 22:6n-3 as sources of 22:6n-3 accretion in neonatal baboon brain and associated organs.
      ). The conversion efficiency of LNA to DHA appears to be rather low as measured within the plasma, with the highest human values reportedly found in pregnant women and term infants at 9% and 4%, respectively, and the lowest in adult males at ≤0.1% (
      • Su 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.
      ,
      • Su H.M.
      • Bernardo L.
      • Mirmiran M.
      • Ma X.H.
      • Nathanielsz P.W.
      • Brenna J.T.
      Dietary 18:3n-3 and 22:6n-3 as sources of 22:6n-3 accretion in neonatal baboon brain and associated organs.
      ,
      • Burdge G.C.
      • Wootton S.A.
      Conversion of alpha-linolenic acid to eicosapentaenoic, docosapentaenoic and docosahexaenoic acids in young women.
      ,
      • Burdge G.C.
      • Jones A.E.
      • Wootton S.A.
      Eicosapentaenoic and docosapentaenoic acids are the principal products of alpha-linolenic acid metabolism in young men*.
      ,
      • Pawlosky R.J.
      • Hibbeln J.R.
      • Novotny J.A.
      • Salem Jr., N.
      Physiological compartmental analysis of alpha-linolenic acid metabolism in adult humans.
      ,
      • Pawlosky R.J.
      • Hibbeln J.R.
      • Lin Y.
      • Goodson S.
      • Riggs P.
      • Sebring N.
      • Brown G.L.
      • Salem Jr., N.
      Effects of beef- and fish-based diets on the kinetics of n-3 fatty acid metabolism in human subjects.
      ,
      • Pawlosky R.J.
      • Hibbeln J.R.
      • Salem Jr., N.
      Compartmental analyses of plasma n-3 essential fatty acids among male and female smokers and nonsmokers.
      ,
      • Sauerwald T.U.
      • Hachey D.L.
      • Jensen C.L.
      • Chen H.
      • Anderson R.E.
      • Heird W.C.
      Effect of dietary alpha-linolenic acid intake on incorporation of docosahexaenoic and arachidonic acids into plasma phospholipids of term infants.
      ); however, this is a combined reflection of the liver synthesis, plasma secretion, catabolic rate, and uptake of DHA by the peripheral tissues such as the brain (
      • Igarashi M.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      • DeMar Jr., J.C.
      Low liver conversion rate of alpha-linolenic to docosahexaenoic acid in awake rats on a high-docosahexaenoate-containing diet.
      ,
      • Igarashi M.
      • DeMar Jr., J.C.
      • Ma K.
      • Chang L.
      • Bell J.M.
      • Rapoport S.I.
      Upregulated liver conversion of alpha-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet.
      ). In our previous study (
      • Lefkowitz W.
      • Lim S.Y.
      • Lin Y.
      • Salem Jr., N.
      Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat.
      ), we found that accretion of unlabeled DHA in the liver and brain of the d5-LNA + DHA diet group was 2- and 3-fold, respectively, higher than the d5-DHA accreted in the d5-LNA diet group, which is basically consistent with the amount of dietary unlabeled DHA being twice that of the d5-LNA included in both diets. In our present study, however, we have now found that the total-body unlabeled DHA accumulation in the d5-LNA + DHA diet group was 5-fold higher than the total d5-DHA found in the LNA diet group (Tables 14 and 15); and this strongly suggests that preformed DHA is a much more efficient source for tissue accretion of DHA than is dietary LNA in developing rat pups, as has been reported for infant primates (
      • Greiner R.C.
      • Winter J.
      • Nathanielsz P.W.
      • Brenna J.T.
      Brain docosahexaenoate accretion in fetal baboons: bioequivalence of dietary alpha-linolenic and docosahexaenoic acids.
      ,
      • Su 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.
      ). A very high accumulation of the total d5-DHA in both diet groups was seen for the connective tissues (skin, adipose, skeletal muscle, and bones combined), at 50–70% of the total body distribution, with skeletal muscle containing the most at ∼30% (Table 14). The brain and liver in both diets, however, each contained 4–7% and 17–30%, respectively, of the total biosynthesized d5-DHA. Preferential accumulation of dietary DHA has been noted before in the whole skin and carcass (adipose, skeletal muscle, and bones) of rats (
      • Poumes-Ballihaut C.
      • Langelier B.
      • Houlier F.
      • Alessandri J.M.
      • Durand G.
      • Latge C.
      • Guesnet P.
      Comparative bioavailability of dietary alpha-linolenic and docosahexaenoic acids in the growing rat.
      ). Overall, our results suggest that the principal source of DHA for rat pup tissues is preformed dietary DHA and not DHA synthesized from dietary LNA. Although in our study, on a whole-body basis, dietary preformed DHA is more efficient than dietary LNA for supplying DHA to tissues in the developing rat pup, this does not imply that a much higher dietary level of LNA than that used in our study would provide the same tissue content of DHA, inasmuch as it has been shown that even extremely high intakes of LNA cannot support the nervous system levels observed for much smaller DHA dietary content (
      • Abedin L.
      • Lien E.L.
      • Vingrys A.J.
      • Sinclair A.J.
      The effects of dietary alpha-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig.
      ,
      • Bowen R.A.
      • Clandinin M.T.
      High dietary 18:3n-3 increases the 18:3n-3 but not the 22:6n-3 content in the whole body, brain, skin, epididymal fat pads, and muscles of suckling rat pups.
      ,
      • Woods J.
      • Ward G.
      • Salem Jr., N.
      Is docosahexaenoic acid necessary in infant formula? Evaluation of high linolenate diets in the neonatal rat.
      ).
      In the present study, we found that the presence of dietary preformed DHA decreases the net biosynthesis/accretion of d5-DHA from d5-LNA in the tissues of growing rat pups by 2- to 5-fold, in comparison to those receiving a diet containing LNA alone. As anticipated, the nervous system, brain, and retina were among the tissues most sensitive to the inclusion of DHA in the diets (5.1- and 4.0-fold decrease, respectively), but we also found that the kidneys, liver, and testes ranked rather high in their responses (3.9-, 3.9-, and 3.3-fold decrease, respectively). These findings are consistent with liver being the major site of DHA biosynthesis in the body (
      • Scott B.L.
      • Bazan N.G.
      Membrane docosahexaenoate is supplied to the developing brain and retina by the liver.
      ), with the kidneys and testes also having a high importance for DHA content for renal function and spermatogenesis (
      • Dusing R.
      • Struck A.
      • Gobel B.O.
      • Weisser B.
      • Vetter H.
      Effects of n-3 fatty acids on renal function and renal prostaglandin E metabolism.
      ,
      • Hagve T.A.
      • Woldseth B.
      • Brox J.
      • Narce M.
      • Poisson J.P.
      Membrane fluidity and fatty acid metabolism in kidney cells from rats fed purified eicosapentaenoic acid or purified docosahexaenoic acid.
      ,
      • Lin D.S.
      • Neuringer M.
      • Connor W.E.
      Selective changes of docosahexaenoic acid-containing phospholipid molecular species in monkey testis during puberty.
      ,
      • Rodemer C.
      • Thai T.P.
      • Brugger B.
      • Kaercher T.
      • Werner H.
      • Nave K.A.
      • Wieland F.
      • Gorgas K.
      • Just W.W.
      Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice.
      ). Interestingly, the heart, lungs, and bones were found to have somewhat lower responses in this respect (2.6-, 2.4-, and 2.5-fold, respectively). This is somewhat unexpected, because DHA has been implicated in maintaining cardiac rhythm, respiratory surfactant formation, and mineralization by osteoclasts (
      • Chao A.C.
      • Ziadeh B.I.
      • Diau G.Y.
      • Wijendran V.
      • Sarkadi-Nagy E.
      • Hsieh A.T.
      • Nathanielsz P.W.
      • Brenna J.T.
      Influence of dietary long-chain PUFA on premature baboon lung FA and dipalmitoyl PC composition.
      ,
      • Leaf A.
      • Xiao Y.F.
      • Kang J.X.
      Interactions of n-3 fatty acids with ion channels in excitable tissues.
      ,
      • Mallampalli R.K.
      • Salome R.G.
      • Spector A.A.
      Regulation of CTP:choline-phosphate cytidylyltransferase by polyunsaturated n-3 fatty acids.
      ,
      • Pound E.M.
      • Kang J.X.
      • Leaf A.
      Partitioning of polyunsaturated fatty acids, which prevent cardiac arrhythmias, into phospholipid cell membranes.
      ,
      • Reinwald S.
      • Li Y.
      • Moriguchi T.
      • Salem Jr., N.
      • Watkins B.A.
      Repletion with (n-3) fatty acids reverses bone structural deficits in (n-3)-deficient rats.
      ,
      • Sun D.
      • Krishnan A.
      • Zaman K.
      • Lawrence R.
      • Bhattacharya A.
      • Fernandes G.
      Dietary n-3 fatty acids decrease osteoclastogenesis and loss of bone mass in ovariectomized mice.
      ), but it should be noted that these tissues still exhibit a marked effect (more than 2-fold) of dietary DHA upon d5-DHA accretion.
      The decreases in d5-DHA accumulation that we noted for the tissues are probably due primarily to changes in DHA biosynthesis in liver hepatocytes (
      • Scott B.L.
      • Bazan N.G.
      Membrane docosahexaenoate is supplied to the developing brain and retina by the liver.
      ), with the other tissues more reflective of changes in DHA uptake from the plasma thereafter. It is known, however, that skin fibroblasts, lung/bronchial epithelium, retinal pigmented epithelium, and brain astrocytes show some capability to synthesize DHA from LNA (
      • Kang J.X.
      • Man S.F.
      • Brown N.E.
      • Labrecque P.A.
      • Garg M.L.
      • Clandinin M.T.
      Essential fatty acid metabolism in cultured human airway epithelial cells.
      ,
      • Moore S.A.
      • Yoder E.
      • Murphy S.
      • Dutton G.R.
      • Spector A.A.
      Astrocytes, not neurons, produce docosahexaenoic acid (22:6 omega-3) and arachidonic acid (20:4 omega-6).
      ,
      • Moore S.A.
      • Hurt E.
      • Yoder E.
      • Sprecher H.
      • Spector A.A.
      Docosahexaenoic acid synthesis in human skin fibroblasts involves peroxisomal retroconversion of tetracosahexaenoic acid.
      ,
      • Wang N.
      • Anderson R.E.
      Synthesis of docosahexaenoic acid by retina and retinal pigment epithelium.
      ). Thus a portion of the biosynthesized d5-DHA may possibly have arisen from tissues outside of the liver.
      Inside the liver hepatocytes, expression of the Δ-5 and Δ-6 desaturases, utilized in the biosynthesis of DHA from LNA, is positively controlled by the transcription factors SREBP-1 and NF-Y (sterol regulatory element binding protein-1 and nuclear factor-Y), which are both turned off when dietary DHA is abundant (
      • Matsuzaka T.
      • Shimano H.
      • Yahagi N.
      • Memiya-Kudo M.
      • Yoshikawa T.
      • Hasty A.H.
      • Tamura Y.
      • Osuga J.
      • Okazaki H.
      • Iizuka Y.
      • et al.
      Dual regulation of mouse Delta(5)- and Delta(6)-desaturase gene expression by SREBP-1 and PPARalpha.
      ,
      • Nara T.Y.
      • He W.S.
      • Tang C.
      • Clarke S.D.
      • Nakamura M.T.
      The E-box like sterol regulatory element mediates the suppression of human delta-6 desaturase gene by highly unsaturated fatty acids.
      ). Anticipated decreases did occur in d5-DPA and d5-DHA content of most tissues in the d5-LNA + DHA diet group, providing support for the existence of this product feedback inhibition mechanism in the DHA biosynthetic pathway of the developing rat pup. In contrast, there were substantial increases in tissue d5-EPA, unlabeled EPA, and unlabeled DPA content, but this may be due in part to retro-conversion processes acting on these fatty acids and DHA (
      • Brossard N.
      • Croset M.
      • Pachiaudi C.
      • Riou J.P.
      • Tayot J.L.
      • Lagarde M.
      Retroconversion and metabolism of [13C]22:6n-3 in humans and rats after intake of a single dose of [13C]22:6n-3-triacylglycerols.
      ,
      • Gronn M.
      • Christensen E.
      • Hagve T.A.
      • Christophersen B.O.
      Peroxisomal retroconversion of docosahexaenoic acid (22:6(n-3)) to eicosapentaenoic acid (20:5(n-3)) studied in isolated rat liver cells.
      ). Unexpectedly, tissue levels of d5-LNA were not increased in the d5-LNA + DHA diet group, an anticipated end result of decreased utilization of LNA toward DHA synthesis; however, some excess d5-LNA could have been lost through increased gene expression of enzymes involved in fatty acid β-oxidation by PPAR (peroxisomal proliferation-activated receptor) transcription factors that use DHA as an activating ligand (
      • Clarke S.D.
      Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome.
      ,
      • Cunnane S.C.
      • Menard C.R.
      • Likhodii S.S.
      • Brenna J.T.
      • Crawford M.A.
      Carbon recycling into de novo lipogenesis is a major pathway in neonatal metabolism of linoleate and alpha-linolenate.
      ,
      • Emken E.A.
      • Adlof R.O.
      • Duval S.M.
      • Nelson G.J.
      Effect of dietary docosahexaenoic acid on desaturation and uptake in vivo of isotope-labeled oleic, linoleic, and linolenic acids by male subjects.
      ).
      Surprisingly, in brown, white, and visceral adipose tissues, we found that the accumulation of d5-DHA as well as d5-DPA was insensitive to the inclusion of preformed DHA in the diet. This unresponsiveness was not observed for any of the other tissues examined, in that they all showed significant decreases in d5-DPA and d5-DHA content for the d5-LNA + DHA diet group relative to the d5-LNA diet group. Although we cannot rule out possible tissue-specific differences in fatty acid uptake and transport, the insensitivity of d5-DHA incorporation into adipose to the presence of dietary DHA is likely to be due to adipose tissue storing fatty acids primarily in triglycerides, whereas other tissues store this fatty acid mainly in the phospholipid form. The activities of enzymes controlling the acylation of DHA into phospholipids are sensitive to tissue levels of DHA (
      • DeMar 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.
      ,
      • Contreras M.A.
      • Greiner R.S.
      • Chang M.C.
      • Myers C.S.
      • Salem Jr., N.
      • Rapoport S.I.
      Nutritional deprivation of alpha-linolenic acid decreases but does not abolish turnover and availability of unacylated docosahexaenoic acid and docosahexaenoyl-CoA in rat brain.
      ,
      • Rao J.S.
      • Ertley R.N.
      • DeMar Jr., J.C.
      • Rapoport S.I.
      • Bazinet R.P.
      • Lee H.J.
      Dietary n-3 PUFA deprivation alters expression of enzymes of the arachidonic and docosahexaenoic acid cascades in rat frontal cortex.
      ). In contrast, it has been shown that dietary DHA does not influence the activity of triglyceride acylation; thus, plasma d5-DHA would be more equally incorporated into adipose triglycerides in both diet groups (
      • Berge R.K.
      • Madsen L.
      • Vaagenes H.
      • Tronstad K.J.
      • Gottlicher M.
      • Rustan A.C.
      In contrast with docosahexaenoic acid, eicosapentaenoic acid and hypolipidaemic derivatives decrease hepatic synthesis and secretion of triacylglycerol by decreased diacylglycerol acyltransferase activity and stimulation of fatty acid oxidation.
      ,
      • Willumsen N.
      • Hexeberg S.
      • Skorve J.
      • Lundquist M.
      • Berge R.K.
      Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats.
      ).
      In our study, we examined the tissue fatty acid composition of 8 day-old dam-reared rat pups (Tables 113 and supplementary Tables III–IX) prior to the 20 day (p8–p28) administration of the d5-LNA and d5-LNA + DHA diets, as a baseline reference group. This allowed us to quantify the changes in unlabeled endogenous fatty acids at 20 days over baseline (p28 versus p8), especially that of unlabeled DHA. As anticipated, providing dietary preformed DHA dramatically increased unlabeled DHA in all tissues by 3- to 41-fold above the p8 baseline group level (Table 15). Coinciding with its large mass, skeletal muscle had the greatest increase in unlabeled DHA, at 41-fold above the p8 baseline group. Of all the internal organs, the testes had the largest elevation in unlabeled DHA, at 32-fold above the p8 baseline group, and this agrees with rapid sexual maturation of the p8–p28 male animals (
      • Lin D.S.
      • Neuringer M.
      • Connor W.E.
      Selective changes of docosahexaenoic acid-containing phospholipid molecular species in monkey testis during puberty.
      ,
      • Rodemer C.
      • Thai T.P.
      • Brugger B.
      • Kaercher T.
      • Werner H.
      • Nave K.A.
      • Wieland F.
      • Gorgas K.
      • Just W.W.
      Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice.
      ). On a total-body basis, the d5-LNA + DHA diet group had a 10-fold increase in unlabeled DHA over the p8 baseline group. In the d5-LNA diet group, an absence of preformed dietary DHA apparently led to the redistribution among tissues of body stores of unlabeled DHA (Table 15). Although on a total-body basis there was no difference detected in unlabeled DHA content between the d5-LNA diet and the p8 baseline groups, all organs/tissues except plasma showed significant alterations in unlabeled DHA content over baseline. The lack of a difference in total-body unlabeled DHA content between the two groups, however, suggests that a balanced reshuffling of unlabeled DHA occurred between the various organs/tissues. Brain, retina, testes, skeletal muscle, and brown adipose all showed an increase of unlabeled DHA of 2- to 3-fold above the p8 baseline group, suggesting movement of unlabeled DHA into these highly important organs and tissues. In contrast, liver, digestive tract, bone, skin, white adipose (near significance; P = 0.021), and visceral adipose incurred net decreases in unlabeled DHA at 2- to 5-fold less than the p8 baseline group, which implies an efflux of DHA from these organs and tissues, which are known to process and/or store fatty acids. Even more troubling, however, were the decreases noted in unlabeled DHA content of the heart (near significance; P = 0.010), lungs, kidneys, and spleen at 1.3-, 7.6-, 1.9-, and 2.0-fold less than the p8 baseline group, respectively. The optimal functioning of these latter organs is no doubt critical for early development in infant mammals, and decreases in DHA content during rapid growth may potentially lead to a compromise in their functioning in order to allow the developing brain, retina, skeletal muscle, and testes to accumulate DHA. Movement of DHA into brown adipose, which is lost as infants mature, may be critical for supporting thermogenesis. Thus, our findings may have unforeseen health implications for human infants being continually fed formula that contains LNA as the sole source of n-3 PUFA.

      Supplementary Material

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