J. Thomas Brenna

α-Linolenic acid supplementation and conversion to n-3 long-chain polyunsaturated fatty acids in humans.

Blood levels of polyunsaturated fatty acids (PUFA) are considered biomarkers of status. Alpha-linolenic acid, ALA, the plant omega-3, is the dietary precursor for the long-chain omega-3 PUFA eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA). Studies in normal healthy adults consuming western diets, which are rich in linoleic acid (LA), show that supplemental ALA raises EPA and DPA status in the blood and in breast milk. However, ALA or EPA dietary supplements have little effect on blood or breast milk DHA levels, whereas consumption of preformed DHA is effective in raising blood DHA levels. Addition of ALA to the diets of formula-fed infants does raise DHA, but no level of ALA tested raises DHA to levels achievable with preformed DHA at intakes similar to typical human milk DHA supply. The DHA status of infants and adults consuming preformed DHA in their diets is, on average, greater than that of people who do not consume DHA. With no other changes in diet, improvement of blood DHA status can be achieved with dietary supplements of preformed DHA, but not with supplementation of ALA, EPA, or other precursors.

Dietary fat intakes for pregnant and lactating women

Dietary fat intake in pregnancy and lactation affects pregnancy outcomes and child growth, development and health. The European Commission charged the research project PERILIP, jointly with the Early Nutrition Programming Project, to develop recommendations on dietary fat intake in pregnancy and lactation. Literature reviews were performed and a consensus conference held with international experts in the field, including representatives of international scientific associations. The adopted conclusions include: dietary fat intake in pregnancy and lactation (energy%) should be as recommended for the general population; pregnant and lactating women should aim to achieve an average dietary intake of at least 200 mg DHA/d; intakes of up to 1 g/d DHA or 2.7 g/d n-3 long-chain PUFA have been used in randomized clinical trials without significant adverse effects; women of childbearing age should aim to consume one to two portions of sea fish per week, including oily fish; intake of the DHA precursor, alpha-linolenic acid, is far less effective with regard to DHA deposition in fetal brain than preformed DHA; intake of fish or other sources of long-chain n-3 fatty acids results in a slightly longer pregnancy duration; dietary inadequacies should be screened for during pregnancy and individual counselling be offered if needed.

High-precision continuous-flow isotope ratio mass spectrometry

Although high-precision isotope determinations are routine in many areas of natural science, the instrument principles for their measurements have remained remarkably unchanged for four decades. The introduction of continuous-flow techniques to isotope ratio mass spectrometry (IRMS) instrumentation has precipitated a rapid expansion in capabilities for high-precision measurement of C, N, O, S, and H isotopes in the 1990s. Elemental analyzers, based on the flash combustion of solid organic samples, are interfaced to IRMS to facilitate routine C and N isotopic analysis of unprocessed samples. Gas/liquid equilibrators have automated O and H isotopic analysis of water in untreated aqueous fluids as complex as urine. Automated cryogenic concentrators permit analysis at part-per-million concentrations in environmental samples. Capillary gas chromatography interfaced to IRMS via on-line microchemistry facilitates compound-specific isotope analysis (CSIA) for purified organic analytes of 1 nmol of C, N, or O. GC-based CSIA for hydrogen and liquid chromatography-based interfaces to IRMS have both been demonstrated, and continuing progress promises to bring these advances to routine use. Automated position-specific isotope analysis (PSIA) using noncatalytic pyrolysis has been shown to produce fragments without appreciable carbon scrambling or major isotopic fractionation, and shows great promise for intramolecular isotope ratio analysis. Finally, IRMS notation and useful elementary isotopic relationships derived from the fundamental mass balance equation are presented.

Breast-fed infants achieve a higher rate of brain and whole body docosahexaenoate accumulation than formula-fed infants not consuming dietary docosahexaenoate.

Docosahexaenoate (DHA) has been increasingly recognized as an important fatty acid for neural and visual development during the first 6 mon of life. One important point of controversy that remains is the degree to which adequate levels of DHA can be acquired from endogenous synthesis in infants vs. what should be provided as dietary DHA. We have approached this problem by a retrospective analysis of published body composition data to estimate the actual accumulation of DHA in the human infant brain, liver, adipose tissue, remaining lean tissue, and whole body. Estimating whether infants can synthesize sufficient DHA required comparison to and extrapolation from animal data. Over the first 6 mon of life, DHA accumulates at about 10 mg/d in the whole body of breast-fed infants, with 48% of that amount appearing in the brain. To achieve that rate of accumulation, breast-fed infants need to consume a minimum of 20 mg DHA/d. Virtually all breast milk provides a DHA intake of at least 60 mg/d. Despite a store of about 1,050 mg of DHA in body fat at term birth and an intake of about 390 mg/d α-linolenate (α-LnA), the brain of formula-fed infants not consuming DHA accumulates half the DHA of the brain of breast-fed infants while the rest of the body actually loses DHA over the first 6 mon of life. No experimental data indicate that formula-fed infants not consuming DHA are able to convert the necessary 5.2% of α-LnA intake to DHA to match the DHA accumulation of breast-fed infants. We conclude that dietary DHA should likely be provided during at least the first 6 mon of life.

The influence of long chain polyunsaturate supplementation on docosahexaenoic acid and arachidonic acid in baboon neonate central nervous system.

BackgroundDocosahexaenoic acid (DHA) and arachidonic acid (ARA) are major components of the cerebral cortex and visual system, where they play a critical role in neural development. We quantitatively mapped fatty acids in 26 regions of the four-week-old breastfed baboon CNS, and studied the influence of dietary DHA and ARA supplementation and prematurity on CNS DHA and ARA concentrations.MethodsBaboons were randomized into a breastfed (B) and four formula-fed groups: term, no DHA/ARA (T-); term, DHA/ARA supplemented (T+); preterm, no DHA/ARA (P-); preterm and DHA/ARA supplemented (P+). At four weeks adjusted age, brains were dissected and total fatty acids analyzed by gas chromatography and mass spectrometry.ResultsDHA and ARA are rich in many more structures than previously reported. They are most concentrated in structures local to the brain stem and diencephalon, particularly the basal ganglia, limbic regions, thalamus and midbrain, and comparatively lower in white matter. Dietary supplementation increased DHA in all structures but had little influence on ARA concentrations. Supplementation restored DHA concentrations to levels of breastfed neonates in all regions except the cerebral cortex and cerebellum. Prematurity per se did not exert a strong influence on DHA or ARA concentrations.Conclusion1) DHA and ARA are found in high concentration throughout the primate CNS, particularly in gray matter such as basal ganglia; 2) DHA concentrations drop across most CNS structures in neonates consuming formulas with no DHA, but ARA levels are relatively immune to ARA in the diet; 3) supplementation of infant formula is effective at restoring DHA concentration in structures other than the cerebral cortex. These results will be useful as a guide to future investigations of CNS function in the absence of dietary DHA and ARA.

Efficacy of Dietary Arachidonic Acid Provided as Triglyceride or Phospholipid as Substrates for Brain Arachidonic Acid Accretion in Baboon Neonates

Arachidonic acid (AA) is a long-chain polyunsaturate (LCP) present in human breast milk as both triglyceride (TG) and as phospholipid (PL). There has been little attention to the metabolic consequences of lipid form of AA in infant formulas. Our objective was to investigate the efficacy of dietary TG and PL as carriers of AA for accretion in the brain and associated organs of term baboon neonates consuming a formula with LCP composition typical of human milk. TG and phosphatidylcholine (PC) with [U-13C]-AA in the sn-2 position and with unlabeled 16:0 in the remaining positions (TG-AA* or PL-AA*, respectively) were used as tracers to study the tissue AA* incorporation. Baboon neonates received a single oral dose of either TG-AA* (n = 3) or PL-AA* (n = 4) at 18–19 d of life. Tissues were obtained 10 d later (28–29 d of life) and isotopic enrichment was measured. In the brain, 4.5% of the PL-AA* dose and 2.1% of the TG-AA* dose were recovered as brain AA*, respectively, indicating that PL was about 2.1-fold more effective than TG as a substrate for brain AA accretion. Preferential incorporation of PL-derived AA* over TG source of AA* was also observed in the liver, lung, plasma, and erythrocytes. Because of the quantitative predominance of TG-AA in formula, total brain AA accretion, expressed as absolute weight, was 5.0-fold greater from TG-AA than from PL-AA. We estimate that about half of postnatal brain AA accretion is derived from dietary preformed AA in term baboon neonates consuming a formula with lipid composition similar to that of human milk.

An alternate pathway to long-chain polyunsaturates: the FADS2 gene product Δ8-desaturates 20:2n-6 and 20:3n-3

The mammalian Delta6-desaturase coded by fatty acid desaturase 2 (FADS2; HSA11q12-q13.1) catalyzes the first and rate-limiting step for the biosynthesis of long-chain polyunsaturated fatty acids. FADS2 is known to act on at least five substrates, and we hypothesized that the FADS2 gene product would have Delta8-desaturase activity. Saccharomyces cerevisiae transformed with a FADS2 construct from baboon neonate liver cDNA gained the function to desaturate 11,14-eicosadienoic acid (20:2n-6) and 11,14,17-eicosatrienoic acid (20:3n-3) to yield 20:3n-6 and 20:4n-3, respectively. Competition experiments indicate that Delta8-desaturation favors activity toward 20:3n-3 over 20:2n-6 by 3-fold. Similar experiments show that Delta6-desaturase activity is favored over Delta8-desaturase activity by 7-fold and 23-fold for n-6 (18:2n-6 vs 20:2n-6) and n-3 (18:3n-3 vs 20:3n-3), respectively. In mammals, 20:3n-6 is the immediate precursor of prostaglandin E1 and thromboxane B1. 20:3n-6 and 20:4n-3 are also immediate precursors of long-chain polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid, respectively. These findings provide unequivocal molecular evidence for a novel alternative biosynthetic route to long-chain polyunsaturated fatty acids in mammals from substrates previously considered to be dead-end products.

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (−/−), which enabled us to study HUFA deficiency without depleting their precursors. In −/−, no in vivo AA synthesis was detected after administration of [U-13C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the −/− developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The −/− also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male −/− lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in −/− declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in −/−. DPAn-6 was absent in −/− brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (−/−) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.—Stroud, C. K., T. Y. Nara, M. Roqueta-Rivera, E. C. Radlowski, P. Lawrence, Y. Zhang, B. H. Cho, M. Segre, R. A. Hess, J. T. Brenna, W. M. Haschek, and M. T. Nakamura. Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.

Docosahexaenoic Acid Modulates the Interactions of the Interphotoreceptor Retinoid-binding Protein with 11-cis-Retinal

Rapid transport of retinoids across the interphotoreceptor matrix is a critical part of the visual cycle, since it serves to replenish bleached rhodopsin with its chromophore 11-cis-retinal. The transport of retinoids in the interphotoreceptor matrix is believed to be mediated by the interphotoreceptor retinoid-binding protein (IRBP), a protein that, in addition to possessing two retinoid-binding sites, associates in vivo with long chain fatty acids. Here, the interrelationships between binding of the two types of ligands to IRBP were studied. The composition of fatty acids associated with IRBP in bovine retina was determined, and it was found that polyunsaturated fatty acids constitute a significant fraction of those. It was further found that docosahexaenoic acid, but not palmitic acid, induced a rapid and specific release of 11-cis-retinal from one of the proteins retinoid-binding sites. Based on these results and on the additional observation that a steep concentration gradient of docosahexaenoic acid exists between photoreceptor and pigment epithelium cells, a model for the mechanism by which IRBP may target 11-cis-retinal to photoreceptor cells is proposed.

Background Paper on Fat and Fatty Acid Requirements during Pregnancy and Lactation

been recognized as being of possible importance to longterm infant health. With respect to fatty acid nutrition, consumption of various oils during the last week of pregnancy was found to permanently program avoidance behavior in rats [Messeri et al., 1975] despite the postnatal brain growth spurt in this species, which is consistent with several other studies demonstrating perinatal effects of PUFA nutrition on behavior [Borgman et al., 1975; Lamptey and Walker, 1978]. Similarly, cholesterol intake in infancy was hypothesized to program cholesterol levels in later life, first supported by data in rats [Reiser and Sidelman, 1972; Jensen et al., 1978]. More recently, baboon [Mott et al., 1990] and human studies [Owen et al., 2002] indicate that breast feeding has positive effects on the infant’s later cholesterol physiology, and in addition is consistently associated with later cognitive benefits [SchackNielsen and Michaelsen, 2006]. Notably, experimental studies of the influence of perinatal nutrition on chronic disease development in humans are impossible due to ethical and practical considerations. However, evidence from randomized controlled trials (RCT) that evaluate acute outcomes, reviewed below, are fully consistent with the hypothesis that long-chain PUFA (LCPUFA) nutrition is a key factor linking breast feeding to the infant’s CNS development and the mother’s mental health. Specific health conditions in pregnancy influence either the mother or the infant, or both. The most unambiguous evidence tends to be associated with health conIntroduction