Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The project demonstrated for the first time a method for implementing covalent adduct chemical ionization using liquid source mass spectrometry. Covalent Adduct Chemical Ionization (CACI) using a product of acetonitrile self-reaction, (1-methyleneimino)-1-ethenylium (MIE; CH2=C=N+=CH2), has been investigated as a method for localizing double bonds in a series of 16 non-methylene interrupted fatty acid methyl esters (NMI-FAME) of polyenes with 3 and more double bonds. As with polyunsaturated homoallylic (methylene-interrupted) FAME and conjugated dienes, MIE (m/z 54) reacts across double bonds to yield molecular ions 54 mass units above the parent analyte. [M+54]+ ions of several 20 and 22 carbon FAME that include one double bond in the C2-C3 position separated by two to five methylene units from a three, four, or five C homoallylic system dissociate according to rules for the homoallylic system, with an additional fragment corresponding to cleavage between the lone double bond and the carboxyl group and defining the position of the lone double bond. Triene FAME with both methylene and ethylene interruption yielded characteristic fragments distinguishable from homoallylic trienes. Fragmentation of fully conjugated trienes in the MS-1 spectra yield ratios of [M+54]+/[M+54-32]+ (loss of methanol) near unity, which distinguishes them from homoallylic FAME having a ratio of 8 or more; collisionally activated dissociation of [M+54]+ yields a series of ions, including some rearrangement products, indicative of double bond position. Unlike conjugated dienes, fully conjugated triene diagnostic ion signal ratios did not follow any pattern based on double bond geometry. Partially conjugated trienes behave similarly to monoenes and conjugated dienes, yielding [M+54]+/[M+54 32]+ of 2-3 and, permitting them to be assigned as partially conjugated FAME using the MS-1 spectrum. They yield unique MS/MS spectra with weaker but assignable fragment ions, along with a diagnostic fragment that locates the lone double bond and permits 6,10,12-octatrienoate to be distinguished from 6,8,12-octatrienoate. The presence of a triple bond did not affect fragment formation in a methylene-interrupted yne-ene but did change fragments in a conjugated yne-ene. These data extend the principle of double bond localization by acetonitrile CACI-MS/MS to double bond structure in complex FAME found in nature. The results were reported at the Annual Conference of the American Society for Mass Spectrometry conference. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A change in knowledge is found in the association of the many non-trans fatty acid components of partially hydrogenated vegetable oil (PHVO) and the food component's cholesterol raising properties. The methods developed with sponsorship of this grant was required to accurately establish the composition of the PVHO used in the study. A wide variety of trans, conjugated linoleic acids, and higher order unsaturates. These results bear directly on the widespread interest in elimination of trans fatty acids in foods, lead nationally by the New York City health department. A distinct change in knowledge is found in the finding that branched chain fatty acids are normal components of the normal newborn alimentary canal. These results imply that sources of branched chain fatty acids, such as cow's milkfat, may be of value as component of infant nutrition.
Publications
- Branched Chain Fatty Acids Are Constituents of the Normal Healthy Newborn Gastrointestinal Tract. Rinat R. Ran-Ressler, Srisatish Devapatla, P. Lawrence, J. T. Brenna, Pediatric Research 2008, in press.
- Differential Effects of Individual Trans Octadecenoic Acids and Partially Hydrogenated Vegetable Oil on Hepatic Lipid and Lipoprotein Metabolism in the Golden Syrian Hamster. Cynthia Tyburczy, Catherine Major, Adam L. Lock, Frederic Destaillats, Zephirin Mouloungui, Laure Candy, Peter Lawrence, J. Thomas Brenna, Andrew M. Salter, Dale E. Bauman, Journal of Nutrition, 2008, in press.
- Atmospheric Pressure Covalent Adduct Chemical Ionization (APCACI) Tandem Mass Spectrometry for Double Bond Localization in Monoene and Diene-containing Triacylglycerols, Yichuan Xu and J. Thomas Brenna, Analytical Chemistry, 79(6) 2525-2536, 2007.
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Progress 10/01/06 to 09/30/07
Outputs
The Specific Aims of this research are: Development of atmospheric pressure chemical ionization (APCI) with CH3CN as a solvent for adduct formation and ionization in liquid based separations of lipid classes associated with polyunsaturated fatty acids (PUFA). These are FAME, phospholipids (PL), triglycerides (TG), cholesterol esters (CE), as well as lower abundance lipid classes as time and resources permit. Completion of development of GC/MS/MS for determination of double bond position in arbitrarily complex non-methylene-interrupted (NMI-)FAME. Specific Aim 1. As reported last year, we have now achieved CACI with an APCI source, terming this APCACI. A major publication has now appeared (Xu & Brenna, Anal Chem 2007) that documents triacylglycerols (TAG) analysis. Using acetonitrile as a solvent, we have found conditions that create produce [M+54], [M+40], [M+81] and [M+95]. The [M+54] and [M+40] ions dissociate according to the rules that apply to FAME demonstrated
previously. The series of TAG with fatty acyl structure ABB and BAB from monoenes and dienes, reported last year, form the basis of the data in the paper, and show that we can distinguish double bond position and stereochemistry in all these compounds. This work is very exciting and partially fulfills the promise of our proposal. A challenge has been phospholipids. We have not yet been successful at generating adduct ions with phospholipids. We speculate that this may be due to their desorption into the gas phase as ions, so that reagent ions will not react. We are pursuing two approaches to get around this problem. Our first, unsuccessful approach was to team with Prof Scott McLuckey at Purdue and Dr Tom Covey at Sciex to try out electron transfer dissociation of these compounds. We were unable to generate double charged species for the important phospholipids and have suspended that work. A second approach has yielded promising data in only the last three weeks. In collaboration
with Phytronix of Quebec City, we have conducted the first phospholipid analyses on their novel ion source, called "Laser Diode Thermal Desorption". The source couples sustained fast heating with APCI, and we have now observed the first intact phospholipids, in the form of phosphatidyl choline (PC) with PUFA attached, as intact molecular ions. We are planning experiments to determine if the PC are desorbed as neutrals or as ions - if they are neutrals, it is likely that they would react with reagent ions in the CACI plasma to yield adducts for analysis. We are actively pursuing this collaboration at this writing. Specific Aim 2. Our progress with SA 1 has clarified the path to success with this technique. It shows that adduct formation with the m/z 40 or 54 ions of acetonitrile produces well-behaved adduct ions. Experiments with other reagents, though of some potential utility, would either yield better sensitivity or provide a more convenient alternative. At this writing, we have not
pursued this line of work, in favor of efforts to make the reaction proceed with phospholipids.
Impacts
Research Accomplishments Development of a comprehensive method for rapid determination of double bond location in FAME. Technique is now maturing and being applied to previously difficult biomedical problems. Successful demonstration of CACIMS for monoene and diene TAG promises to enable rapid analysis of double bond structure in these important molecules. Application to transgenic mice show that they make unusual PUFA, providing important clues to the regulation of PUFA biosynthesis.
Publications
- Xu, Y. and J.T. Brenna. 2007. Atmospheric Pressure Covalent Adduct Chemical Ionization (APCACI) Tandem Mass Spectrometry for Double Bond Localization in Monoene and Diene-containing Triacylglycerols, Analytical Chemistry 79(6):2525-2536.
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Progress 01/01/06 to 12/31/06
Outputs
We have now achieved Covalent Adduct Chemical Ionization (CACI) with an APCI source, terming this APCACI. The procedure applies to triacylglycerols (TAG) specifically. Using acetonitrile as a solvent, we have found conditions that create produce [M+54], [M+40], and [M+81]. The [M+54] ion dissociates according to the rules that apply to FAME demonstrated previously. The latter two ions are significant and novel because in many cases they are of greater intensity than the [M+54] ion, thereby giving improved sensitivity. The [M+81] ion dissociates to produce the [M+54] ion and other related fragments. This is reasonable because it has been shown by others that an ion of m/z 81 is the precursor of the m/z 54 reagent ion. The dissociation is familiar and welcome. The [M+40] ion is also a potentially diagnostic ion and study on it is ongoing at this writing. We have synthesized a series of TAG with fatty acyl structure ABB and BAB from monoenes, dienes, and trienes, and are
presently collecting data to demonstrate that they we can distinguish double bond position and stereochemistry in all these compounds. This work is very exciting and partially fulfills the promise of our proposal. We reported that a series of more than 20 standard FAME of various double bond configurations was obtained for studies of NMI-FAME. Fully conjugated trienes and higher order polyenes react with MIE with good intensity. Collisionally-activated dissociation (CAD) of the M+54 adducts results in diagnostic fragments according to rules similar to those established for other FAME, and permit complete assignment of structure. Partially conjugated trienes similarly react with MIE to yield M+54. Fragmentation is more complex for these FAME but produce diagnostic ions that permit double bond assignment. Homoallylic polyenes with a lone double bond distant from the polyene system yields diagnostic ions expected for the polyene system and an additional single diagnostic ion for the lone
double bond. Thus, these polyenes can be fully characterized as well. Fragment intensities are not sufficiently specific to permit assignment of double bond geometry in polyenes. Finally, acetylenic FAME with either a conjugated double bond or a methylene interrupted double bond also produce diagnostic ions that permit position assignment. We discovered that the conjugated triple bond-double bond system fragments like a methylene-interrupted diene, and not like a conjugated diene. Part of this project is explicitly to work with collaborators on lipidomics problems as they arise. We have now published a paper with our Cornell collaborator Dale Bauman, mentioned in the proposal and the -01 report. Dr Bauman, a conjugated fatty acid researcher, had infused a conjugated linolenic acid mixture to cows and required an analysis of the relative mix of isomers. We were able to develop the method and deliver an answer that resulted in an important publication on bovine conjugated fatty acid
metabolism. We have also been contacted by another researcher at another institution doing similar work.
Impacts
Our success with monoene, diene, and triene-containing TAGs is very exciting, and promises to be a comprehensive method for TAG analysis. Our acquisition of a linear ion trap has proven pivotal because we need MS-MS-MS to sort out all fragments. The FAME work has generated considerable interests by collaborators and potential collaborators.
Publications
- Lawrence, P. and Brenna, J.T. 2006. Acetonitrile Covalent Adduct Chemical Ionization (CACI) Mass Spectrometry for Double Bond Localization in Non-Methylene-Interrupted Polyene Fatty Acid Methyl Esters. Analytical Chemistry 78(4): 1312-7.
- Michaud, A.L. and Brenna, J.T. 2006. Structural Characterization of CLA Methyl Esters with Acetonitrile Chemical Ionization Tandem Mass Spectrometry. Advances in Conjugated Linoleic Acid Research, Vol. 3, 2006, AOCS Press.
- Brenna, J.T. 2006. Acetonitrile-CI-GC-MS of fatty acids and their isomers. Book chapter in: New Techniques and Applications in Lipid Analysis and Lipidomics, M.M. Mossoba, J.K.G. Kramer, J.T. Brenna, R.E. McDonald, Eds., AOCS Press.
- Wolyniak, C.J., Brenna, J.T., Murphy, K.J. and Sinclair, A.J. 2005. Gas Chromatography-Chemical Ionization-Mass Spectrometric Fatty Acid Analysis of a Commercial Supercritical Carbon Dioxide Lipid Extract from New Zealand Green Lipped Mussel (Perna canaliculus). Lipids, 40(4)355-360.
- Saebo, A., Perfield II, J.W., Delmonte, P., Yurawecz, M.P., Lawrence, P., Brenna, J.T. and Bauman, D.E. 2005. Milk Fat Synthesis is Unaffected by Abomasal Infusion of the Conjugated Diene 18:3 Isomers cis-6, trans-8, cis-12 and cis-6, trans-10, cis-12. Lipids, 40(1):89-95.
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Progress 01/01/05 to 12/31/05
Outputs
We have completed the animal phase of the project and are in the midst of an extended data analysis phase. The impact of higher levels DHA than currently used in formulas are showing physiological effects. In particular, higher levels influence gene expression in the liver and brain for a wide variety of processes. Work continuing into 2006 will further define these effects
Impacts
Results of this project confirm and extend previous results indicating advantages to higher amounts of DHA in formula
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
The live animal phase is now complete. Samples have been collected and are being analyzed. Data should be ready for first reporting in mid-March 2005.
Impacts
Published results bolster the case for supplementary DHA in formula of premature infants. The new project investigates optimal levels.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
This year saw publication of several results from this project. We showed that supplementary docosahexaenoic acid and arachidonic acid improves visual response in premature baboons but does not normalize endogenous synthesis of these PUFA from precursors. We showed also that they improve surfactant production in premature animals. A project was initiated with 12 baboons at the Southwest Foundation for Primate Research in San Antonio, Texas. The animals will be fed three different levels of docosahexaenoic acid. Electrophysiological and biochemical outcomes will be recorded.
Impacts
Published results bolster the case for supplementary DHA in formula of premature infants. The new project investigates optimal levels.
Publications
- Sarkadi-Nagy, Eszter, Vasuki Wijendran, Guan Yeu Diau, Angela Chueh Chao, Andrea T. Hsieh, Anu Turpeinen, Peter W. Nathanielsz, J. Thomas Brenna. 2003. The Influence of Prematurity and Long Chain Polyunsaturate Supplementation in Four-Week Adjusted Age Baboon Neonate Brain and Related Tissues. Pediatric Research 54: 244-252, 2003.
- Guan-Yeu Diau, Ellis R. Loew, Vasuki Wijendran, Eszter Sarkadi-Nagy, Peter W. Nathanielsz, J. Thomas Brenna. 2003. Docosahexaenoic and Arachidonic Acid Influence on Preterm Baboon Retinal Composition and Function. Investigative Ophthalmology Vision Research, 2003, 44(10) 4559-4566.
- Angela Chueh Chao, Guan-Yeu Diau, Vasuki Wijendran, Eszter Sarkadi-Nagy, Andrea T. Hsieh, Peter W. Nathanielsz, J. Thomas Brenna. 2003. Influence of Dietary Long Chain Polyunsaturated Fatty Acids on Premature Baboon Lung Fatty Acid Compo-sition. , Lipids, 38(4): 425-429, 2003.
- Eszter Sarkadi-Nagy, Vasuki Wijendran, Guan-Yeu Diau, Angela Chao, Andrea T. Hseih, Anu Turpeinen, Peter Lawrence, Peter W. Nathanielsz, J. Thomas Brenna. 2003. Formula Feeding Potentiates Docosahexaenoic and Arachidonic Acid Biosynthesis in Preterm and Term Four-week Adjusted Age Baboons. J Lipid Res, 2003, in press.
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Progress 01/01/02 to 12/31/02
Outputs
Five treatment groups have been run through the protocol: B, breastfed; T-, term formula-fed; T+, term formula-fed with 22:6 and 20:4 supplement; P-, preterm, formula fed; P+, preterm, formula fed with supplements. Samples of retina and 26 regions of the CNS have been analyzed. ERGs, VEPs and heart rate varfiability data have been analyzed as well. Sample analysis for brain fatty acid mapping is complete for 26 regions of the primate brain, which we believe to be the most extensive map of its kind. The project is well ahead of schedule. Major outcomes: Breastfeeding supported the greatest accretion of 22:6 in the CNS, prematurity and feeding of unsupplemented formula supported the lowest accretion of all long chain polyunsaturates. 20:4 was much less affected by diet and prematurity than 22:6. Tracer data showed that supplementation of formula with 22:6 at similar levels as breastmilk did not produce similar accretion of 22:6. ERG parameters were best in breastfeeding
and poorer in other groups. VEPs and heart rate variability were much more noisy than anticipated and did not yield useful correlations. Supplementary outcomes: 36 fatty acids positively identified by chemical ionization tandem mass spectrometry were quantitatively analyzed in 26 regions of the CNS in all five treatment groups. This constitutes by far the most comprehensive mapping of fatty acids in the primate CNS. Trends apparent are: 22:6n-3 and 22:5n-6 are positively correlated with one another, but negatively correlated with 22:5n-3. 20:4n-6 is the less sensitive to treatments than most n-3 fatty acids. Saturates and monounsaturates are essentially immune to treatments. 22:6 was highly correlated with energy use as reported in age matched sedated vervet monkeys using PET, as was dopamine, dopa, and gaba. For the first time, we show that 22:6 was particularly rich in the deep brain structures in and surrouding the brainstem. The globus pallidus was highest with greater than 14
percent of fatty acids as 22:6. Surrounding white matter was very low in 22:6 (around 5 percent).
Impacts
Supplementation of formula with 22:6, approved by the FDA in Dec 01, improves brain 22:6 accretion in preterm neonates but does not equalize it with breastfed neonates, even for term-born neonates. Breastfed neonates have improved visual function compared to the P+, P-, and T- groups. For the first time, we show that 22:6 was correlated with energy consumption in the CNS, a novel clue to its function, which remains elusive. The presence of high concentrations of 22:6 in deep brain structures suggests an important and previously unsuspected role for 22:6 in fundamental functions.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The current year is the third year of the grant period. Five treatment groups have been run through the protocol: B, breastfed; T-, term formula-fed; T+, term formula-fed with 22:6 and 20:4 supplement; P-, preterm, formula fed; P+, preterm, formula fed with supplements. Samples of retina and 26 regions of the CNS have been analyzed. ERGs, VEPs and heart rate varfiability data have been analyzed as well. Sample analysis for brain fatty acid mapping is complete for 26 regions of the primate brain, which we believe to be the most extensive map of its kind. The project is well ahead of schedule. Major outcomes: Breastfeeding supported the greatest accretion of 22:6 in the CNS, prematurity and feeding of unsupplemented formula supported the lowest accretion of all long chain polyunsaturates. 20:4 was much less affected by diet and prematurity than 22:6. Tracer data showed that supplementation of formula with 22:6 at similar levels as breastmilk did not produce similar
accretion of 22:6. ERG parameters were best in breastfeeding and poorer in other groups. VEPs and heart rate variability were much more noisy than anticipated and did not yield useful correlations. Supplementary outcomes: 36 fatty acids positively identified by chemical ionization tandem mass spectrometry were quantitatively analyzed in 26 regions of the CNS in all five treatment groups. This constitutes by far the most comprehensive mapping of fatty acids in the primate CNS. Trends apparent are: 22:6n-3 and 22:5n-6 are positively correlated with one another, but negatively correlated with 22:5n-3. 20:4n-6 is the less sensitive to treatments than most n-3 fatty acids. Saturates and monounsaturates are essentially immune to treatments. 22:6 was highly correlated with energy use as reported in age matched sedated vervet monkeys using PET, as was dopamine, dopa, and gaba. For the first time, we show that 22:6 was particularly rich in the deep brain structures in and surrouding the
brainstem. The globus pallidus was highest with >14%of fatty acids as 22:6. Surrounding white matter was very low in 22:6 (around 5%).
Impacts
The significance of results in hand are as follows: 1) Supplementation of formula with 22:6, approved by the FDA in Dec 01, improves brain 22:6 accretion in preterm neonates but does not equalize it with breastfed neonates, even for term-born neonates. 2) Breastfed neonates have improved visual function compared to the P+, P-, and T- groups. 3) For the first time, we show that 22:6 was correlated with energy consumption in the CNS, a novel clue to its function, which remains elusive. 4) The presence of high concentrations of 22:6 in deep brain structures suggests an important and previously unsuspected role for 22:6 in fundamental functions.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
a. Specific Aims The Specific Aims for this grant period (3/00-4/01) are defined in the proposal and can be summarized as follows. A study using 5 groups of 9 neonatal baboons in each group will be conducted to determine the effects of fatty acid nutritional treatments on stable isotopically labeled polyunsaturated fatty acid (PUFA) metabolism and retinal and vision-related brain function. The animals are drawn from pregnant animals transported to Ithaca, NY and maintained in a nursery at Cornell. The treatments were modified for formula-feed neonates by including the administration of prenatal corticosteroid to the pregnant animals to more accurately model human clinical practice. Also, two more outcome measurements this year: a) determination of heart rate variability using complexity-based software, and b) mapping of the fatty acid concentration in 26 regions of the breastfed and long-chain-PUFA-free-formula-fed neonates. b. Progress in Year 06 Year 06 is the
second year of the grant period. Tissues of the animals dosed in the first year of the new period, year 05, are almost completely analyzed, an abstract has been presented on the data and a manuscript is nearing completion. There was a very strong supply of baboons this year. The nursery was open in 3 periods this year. In summer 00, 12 neonates were studied, in fall 00, 15 animals were studied, and in the spring 01, at this writing, 12 more animals will be studied. This total of 39 animals in the second period constitutes the majority of the animal phase of the research. ERGs, VEPs and heart rate body temperature data have been logged for these animals. Sample analysis for brain fatty acid mapping is complete for 26 regions of the primate brain, which we believe to be the most extensive map of its kind. The project is well ahead of schedule.
Impacts
c. Significance Data from year 05 animals has been presented at a conference. That trial has shown that arachidonic acid consumed orally as a component of phospholipid (PL) is twice as effective as a substrate for brain arachidonic accretion compared to arachidonic acid consumed as triglyceride. This is the first tracer-based evidence that PL is a more effective carrier for PUFA and implies that sources of PUFA for formula that include arachidonic acid as PL are at least as efficacious as more common TG carriers, and in fact may well be preferred.
Publications
- Breast-fed Infants Achieve A Higher Rate Of Brain And Whole Body Docosahexaenoate Accumulation Than Formula-Fed Infants Not Consuming Dietary Docosahexaenoate, Stephen C. Cunnane, Valerie Francescutti, J. Thomas Brenna, Michael A. Crawford. Lipids 2000, 35:105-111.
- Maternal intravenous administration of long chain n-3 polyunsaturates to the pregnant ewe in late gestation results in specific inhibition of prostaglandin H synthase (PGHS) 2, but not PGHS1 and oxytocin receptor mRNA in myometrium during betamethasone-induced labor. Xiao Hong Ma, Wen Xuan Wu, J. Thomas Brenna, P. W. Nathanielsz, Journal of the Society for Gynecological Investigation, 2000,7(4):233-237.
- On The Relative Efficacy of a-Linolenic acid and Preformed Docosahexaenoic Acid as Substrates for Tissue Docosahexaenoate During Perinatal Development, Meng-Chuan Huang and J. Thomas Brenna, Chapter in Fatty Acids: From Neuronal Membrane to Physiological and Behavioral Function, ed by D. I. Mostofsky, S. Yehuda, N. Salem, Jr., in press, 2000.
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Progress 01/01/99 to 12/31/99
Outputs
The goals of this continuation project are to couple biochemical tracer studies of essential fatty acid metabolism with functional outcomes related to retinal and brain development in preterm baboons, and including a randomized breastfed term reference group, to generate highly applicable information to aid in nutritional management of premature human infants. The proposed work focuses on preterm neonates (153 days gestation) with term neonate (185 days gestation) reference groups, and invesigates the effects of diet on conversion and organ accumulation of linoleic acid (LA), a-linolenic acid (LNA), and docosahexaenoic acid (DHA), and on functional outcomes of importance to vision. Three diets similar to those tested in experimental clinical settings will be studied in premature neonates: 1) LA/LNA ratio of 9:1, long chain polyunsaturate (LCP)?free; 2) LA/LNA=9, supplemented with 0.3%en DHA and 0.6%en arachidonic acid (AA); and 3) LA/LNA=4, LCP-free. These diets are
applied in a completely randomised design and studied at equivalent post-conceptual ages in two groups of term neonates: a) term studied at 6 weeks consuming diet 1; b) randomized breastfed term baboons, also studied at 6 weeks, serving as a gold standard reference group, which is never available in human studies. The primary outcomes to be measured are: i) The bioequivalence of dietary LNA and DHA as precursors for brain DHA accretion using stable isotope tracers, ii) Fractional conversion of LNA and LA to LCP in brain, retina, and liver, iii) Electroretinogram (ERG) implicit times and amplitudes, with calculation of the amplification factor (A) associated with initial photoreceptor transduction, iv) flash and pattern-reversal visual evoked potentials, v) development of circadian rhythm-dependent sleep/wake states, vi) eye focusing properties. The combination of biochemical and functional outcomes in a well controlled intervention study with a randomized breatfed group will permit
correlations to be drawn between physiology and function not possible in humans. Because of the similarity of primate brain requirements, the dietary bioequivalence of LNA and DHA for brain DHA accretion will apply to preterm humans and facilitate calculation of adequate dietary n-3 fatty acid levels. Deficiency states will be directly measured via functional tests to assess the impact of low omega-3 fatty acid status on development of vision and related health parameters.
Impacts
Because of the similarity of primate brain requirements, the dietary bioequivalence of LNA and DHA for brain DHA accretion will apply to preterm humans and facilitate calculation of adequate dietary n-3 fatty acid levels. Deficiency states will be directly measured via functional tests to assess the impact of low omega-3 fatty acid status on development of vision and related health parameters.
Publications
- An Octaene Fatty Acid, 4,7,10,13,16,19,22,25-Octacosaoctaenoic Acid (C28:8n-3), Found in Marine Oils, C. Van Pelt, M.-C. Huang, C. Tschanz, J. T. Brenna, Journal of Lipid Research, 1999, 40(8)1501-1505.
- Bioequivalence of dietary a-linolenic and docosahexaenoic acids as sources of docosahexaenoate ac-cretion in brain and associated organs of neonatal baboons, by Hui-Min Su, Luca Bernardo, Majid Mirmiran, X. H. Ma, T. N. Corso, P. W. Nathanielsz and J. T. Brenna, Pediatric Research, 1999, 45:87-93.
- Dietary 18:3n-3 And 22:6n-3 As Sources Of 22:6n-3 Accretion In Neonatal Baboon Brain And As-sociated Organs H.-M.Su, L. Bernardo, M. Mirmiran, X.-H. Ma, P. W. Nathanielsz & J. T. Brenna, Lipids, 1999, 34:S347-50.
- Carbon Recycling into de novo Lipogenesis is a Major Pathway in Neonatal Metabolism of Polyun-saturates, S. C. Cunnane, C. R. Menard, S. S. Likhodii, J. T. Brenna, M. A. Crawford, Prostagland-ins, Leukotrienes, and Essential Fatty Acids, 1999, 60: (5-6) 387-392.
- Linoleic Acid Kinetics and Conversion to Arachidonic Acid in the Pregnant and Fetal Baboon, H.-M. Su, T. N. Corso, P. W. Nathanielsz, J. T. Brenna, Journal of Lipid Research, 1999, 40(7):1304-12.
- Docosahexaenoate Requirement and Infant Development. Stephen C. Cunnane, Valerie Frances-cutti, J. Thomas Brenna, Nutrition 1999, 15: 801-802. (Letter to the editor).
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Progress 01/01/98 to 12/31/98
Outputs
Studies on term neonate baboons were completed and published during the last period. Term baboons were taken by CS and placed in a neonatal nursery. The nursery is controlled for temperature and humidity, access is through an intermediate room where all caretakers were gowned and wear mouth/nose masks, hair nets, and shoe protectors. Diets consist of commercial formula, Enfamil, and corresponded to the basal diet planned in this pro-posal. The animals were maintained initially in incubators designed for human infants and were transferred at about 1 week of age to steel cages equipped with telemetry antennas. Growth and neurological indices were measured regularly. Regardless of initial weights, after one week of life all neonates grew at similar rates. The breadth of the distribu-tion of initial weights reflects biological heterogeneity in baboons, more comparable to that of humans than that of small inbred laboratory species. Weight data (g, mean SD(%CV)) for all
neonates (male and female) is summarized as follows: Birth: 822 86 (10%); 6-weeks: 1214 128 (11%); weight gain=48%. These data document that animals thrived on the basal diet. At 1-2 weeks of age, telemetry systems that transmitted heart rate, respiration rate, body temperature, and electromyograms were implanted surgically into the abdomens. Animals were monitored continuously for one week by means of a computerized data collection system. Data have not yet been fully analyzed. At 4 weeks of age, neonates were given orally 13C-fatty acid added to freshly prepared formula. Four neonates received 13C-LNA (7 mg) and 4 others received 13C-DHA (4 mg). At 14 days post-dose, (i.e., at 6 weeks of age), neonates were euthanized and selected organs were harvested. Isotope enrichments and quan-titative fatty acid profiles were determined in plasma, red blood cells, brain, retina, liver, and other organs. The percent of the preformed dose that appeared as DHA in the brain was 1.7%, nearly the
figure (1.6%) we found for fetal accretion of the preformed DHA dose presented in Figure 4 above. The ratio of preformed DHA to LNA-derived DHA in brain DHA was approximately 7:1. This value is about 3 times greater than found in DHA accumulated during gestation in fetuses of mothers dosed with the same tagged substrates during pregnancy. The implication of this finding is that LNA is more efficiently converted and/or transported by the neonate during the period of rapid brain growth than the mother/fetus pair. From these data we are able to estimate the turnover of brain 22:6 as less than 5% per week during this period.
Impacts
(N/A)
Publications
- "Bioequivalence of dietary a-linolenic and docosahexaenoic acids as sources of docosahexaenoate accre-tion in brain and associated organs of neonatal baboons", by Hui-Min Su, Luca Bernardo, Majid Mirmi-ran, X. H. Ma, T. N. Corso, P. W. Nathanielsz and J. T. Brenna, Pediatric Research, 1999, 45:1-7.
- "Simultaneous Measurement of Desaturase Activities Using Stable Isotope Tracers or a Non-Tracer Method", H.-M. Su and J. T. Brenna, Analytical Biochemistry 1998, 261(1): 43-50.
- "Brain Docosahexaenoate Accretion In Fetal Baboons: Bioequivalence Of Dietary a-Linolenic And Do-cosahexaenoic Acids", R. S. Greiner, P. W. Nathanielsz, J. T. Brenna, 1997, Pediatric Research, 42: 826-834.
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Progress 01/01/97 to 12/31/97
Outputs
Pregnant baboons on a diet with 6%en as fat, a 10:1 w6/w3, and no long chain polyunsaturates is an excellent model of vegan pregnancy in humans. Our calculations show that pregnant vegans should be able to meet the fatty acids needs of their growing fetuses with a dietary level of about 0.8%en as 18:3, as long as the w6/w3 ratio remains 10:1 or less. Further, dietary supplements of about 0.025%en as 22:6 would also meet requirements. This level is considerably lower than previous reports in rats and chicks would suggest, and shows that most vegans on balanced diets should have no problem meeting the needs of their growing fetuses. Our findings with respect to fatty acid recycling show that essential fatty acids are important sources of carbon for the developing brain, and are consistent with very recent measurements of others. We are beginning to apply these results to other human conditions in which essential fatty acid status is compromised in order to discover
clues as to why a broad range of disease conditions result in low essential fatty acids.
Impacts
(N/A)
Publications
- "Brain Docosahexaenoate Accretion In Fetal Baboons: Bioequivalence Of Dietary a-Linolenic And Do-cosahexaenoic Acids", R. S. Greiner, P. W. Nathanielsz, J. T. Brenna, 1997, Pediatric Research, 42: 826-834.
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Progress 01/01/96 to 12/30/96
Outputs
1. Specific Aims The specific aims outlined in the original proposal, and with minor modification as outlined in previous progress reports, are on schedule. We can now report definitive conclusions completing the maternal dosings of Specific Aims #1, and 4, for detection of maternal transfer and metabolism of w3 fatty acids in pregnant primates. As of this writing, a manuscript on this principle aim is in its final revision and is expected to be mailed in December/early January, and the another mentioned in the previous progress report is now published. Details can be found in those references. We summarize below. 2. Studies and Results Protocol Modification The high retention of 22:6 in brain has resulted in a slight modification of the original protocol, making the experiments simpler and the data more easily interpreted. Rather than look at two different time points in gestation we analyzed preliminary data and found that no detectable changes in desaturation or
accretion occurred with time. This observation allows us to administer doses and, while monitoring myometrial activity by implanted electrodes, let the animals metabolize the dosed fatty acids as long as the surgical preparations support pregnancy.
Impacts
(N/A)
Publications
- Sheaff, R, Zhang, Q., Goodman, K.J., Guissani, D.A., Nathanielsz, P.W., and Brenna, J.T. 1996. Linoleate, a-Linolenate, and Docosahexaenoate Recycling into Saturated and Monounsaturated Fatty Acids is a Major Pathway in Pregnant of La 2.
- Sheaff, R. C., Winter, J.A., Nathanielsz, P.W., Brenna. J.T. 1997. The Bioequivalence of Docosahexaenoic and a-Linolenic Acids: Accretion in the Fetal Brain and Retina After A Stable Isotope Dose to Pregnant Baboon. Pediatric Research.
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Progress 01/01/95 to 12/30/95
Outputs
Year 1, Specific Aim 2:Baboon fetuses were removed by C#sarian section at 48 h, and retinas, brains, livers, and plasma analyzed for labeled fatty acids. We have found that animals fed a diet low in long chain polyunsaturates retain about 30% of the labeled fatty acids as fatty acids in fetal tissues compared to about 1% in chow-fed animals. However, labeled fatty acids are efficiently converted to saturated and monounsaturated fatty acids in the developing brain. Recent data in rats (S. Cunnane, Toronto Medical School) indicates that surfeit carbon levels are used as general carbon sources for cholesterol synthesis in rats, and we expect our primates do as well. These observations led us to dose pregnant rhesus monkeys with labeled linoleate (18:2n-6) to determine similarity to 18:3n-3 metabolism. We find that linoleate is used as a carbon source ("recycled") to a slighter greater extent than linolenate. These findings suggest that unsaturated fatty acids may be an
important, and perhaps critical, source of carbon for the developing brain. Year 2, Specific Aim 3:We have investigated linoleate and linolenate elongation and recycling occurs in all 20+ animals investigated, showing that the enzymes required for both processes are operative at the beginning of the brain growth spurt at levels comparable to those found at later time points. We plan to make a detailed quantitative analysis of the time course of this process to determine whether conversion increases with gestational age. We must also.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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Progress 01/01/94 to 12/30/94
Outputs
Five animals have been dosed with 13-C-labeled linolenate and fetal plasma has been collected. The preliminary results indicate that linolenate is transported to the fetus and actively metabolized within 4 h of maternal dosing. Labeled linolenate is not confined to a single lipid class but is distributed among triglycerides, phospholipids, cholesterol ester, and non-esterified fatty acids in the plasma. Samples of retina, cornea, lens, retinal pigment epithelium, brain occipital lobe, and liver are being processed for label analysis at this writing.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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