Eszter Sarkadi-Nagy1, Vasuki Wijendran2, Guan Yeu Diau3, Angela Chueh Chao, Andrea T ...

37 Pages·2003·0.44 MB·Indonesian

JLR Papers In Press. Published on October 1, 2003 as Manuscript M300106-JLR200
FORMULA-FEEDING POTENTIATES DOCOSAHEXAENOIC AND ARACHIDONIC ACID BIOSYNTHESIS IN
TERM AND PRETERM BABOON NEONATES

Eszter Sarkadi-Nagy1, Vasuki Wijendran2, Guan Yeu Diau3, Angela Chueh Chao,
Andrea T. Hsieh, Anu Turpeinen4, Peter Lawrence, Peter W. Nathanielsz,5
and J. Thomas Brenna*
Division of Nutritional Sciences
Savage Hall
Cornell University
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Ithaca, NY 14853 n
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USA d
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Revised according to reviewer comments and returned, August, 2003. .jlr.o
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Submitted to: Journal of Lipid Research, March, 2003 y g
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Running footline: Preterm Polyunsaturate Biosynthesis ril 4
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*Correspondence: Phone: [phone]; Fax: [phone]; <[email protected]>.

Present addresses: 1Dept of Nutritional Sciences and Toxicology, University of California,
Berkeley, California, USA,; 2Foster Biomedical Laboratory, MS029 Brandeis University, P.O.
Box 549110, Waltham, MA, 02454 USA; 3325 Chen-Kon Road, Section 2, Division of Pediatric
Surgery, Department of Surgery Tri-Service General Hospital Nai-Whu, Taipei 10070, Taiwan
ROC; 4Dept of Nutritional Sciences, University of Helsinki, Finland; 5New York University
Medical Center, NY, NY.
Copyright 2003 by Lipid Research, Inc.
Sarkadi-Nagy et al. Page 2

Formula-Feeding Potentiates Docosahexaenoic and Arachidonic Acid Biosynthesis in
Term and Preterm Baboon Neonates

Abstract. Infant formulas supplemented with docosahexaenoic acid (DHA) and
arachidonic acid (ARA) are now available in the U.S., however little is known about the factors
that affect biosynthesis. Baboon neonates were assigned to one of four treatments: term,
breastfed; term, formula-fed; preterm (155 of 182 days gestation), formula-fed; preterm,
formula+DHA/ARA-fed. Standard formula had no DHA/ARA; supplemented formula had
0.61%wt DHA (0.3%calories) and 1.21%wt ARA (0.6%calories), and baboon breastmilk
contained 0.68±0.22%wt DHA and 0.62±0.12%wt ARA. At 14 days adjusted age, neonates
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received a combined oral dose of [U-13C]-linolenic acid (LNA) and [U-13C]-linoleic acid (LA), and n
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tissues were analyzed 14 days post-dose. Brain accretion of LNA-derived-DHA was about 3-fold d
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greater for the formula groups than the breastfed group, and dietary DHA partially attenuated w
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excess DHA synthesis among preterms. A similar, significant pattern was found in other organs. .jlr.o
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Brain LA-derived-ARA accretion was significantly greater in the unsupplemented term group g
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but not in the preterm groups compared to breastfed. jlr These data show that formula t, o
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potentiates biosynthesis/accretion of DHA/ARA in term and preterm neonates compared to ril 4
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breastfed neonates, and that inclusion of DHA/ARA in preterm formula partially restores 19
DHA/ARA biosynthesis to lower, breastfed levels. Current formula DHA concentrations are
inadequate to normalize LCP synthesis to that of breastfed levels.

Keywords: stable isotope tracers, isotope ratio mass spectrometry, n-3 long chain
polyunsaturated fatty acids, n-6 long chain polyunsaturated fatty acids
Sarkadi-Nagy et al. Page 3

Abbreviations: ARA, arachidonic acid; CNS, central nervous system; B, breastfed; T-, term,
formula-fed; P-, preterm, formula-fed; P+, preterm, formula-fed and LCP-supplemented; DHA
or 22:6n-3, docosahexaenoic acid; EDTA, Ethylenediaminetetraacetic acid; FA, fatty acid;
FAME, fatty acid methyl ester(s); LA, linoleic acid; LA*, [U-13C]-LA; LCP, long chain
polyunsaturated fatty acids; LNA, α- linolenic acid; LNA*, [U-13C]-LNA; RBC, red blood cells;
RPE, retinal pigment epithelium
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Sarkadi-Nagy et al. Page 4

It is well established that the long chain polyunsaturated fatty acids (LCP) docosahexaenoic
acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are required for proper neural
development. Mammals can synthesize DHA from linolenic acid (LNA) and ARA from linoleic
acid (LA) by a series of elongation and desaturation reactions (1). LNA and LA are considered to
be dietary essential fatty acids because mammals are incapable of synthesizing these fatty acids.
The n-3 and n-6 fatty acids are not interconvertible and it has long been assumed that they
compete for the same elongation and desaturation enzymes; it has only recently been shown
that the ∆6-desaturase, the first step in DHA and ARA biosynthesis from LNA and LA,
respectively, operates on both LNA and LA (2) . Tracer studies show that preterm and term
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human infants (3-5), as well as term and fetal baboons (6-8) convert LNA to DHA and LA to lo
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ARA. fro
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The most intense period of human brain growth is from about 28 weeks gestation to 18 w
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months after birth, during which time brain DHA accretion peaks (9). Premature infants born rg
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at 28 weeks now routinely survive but have very little adipose tissue stores compared to infants u
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born at term and must obtain all fatty acids from their diets, rather than via placental transfer. n
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Those consuming unsupplemented formula must synthesize all LCP from precursors, and ample ril 4
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evidence demonstrates that unsupplemented formula-fed term and preterm human infants have
lower DHA in the central nervous system (CNS), plasma, and red blood cells (RBC) (10-14).
Autopsy data suggest that term formula-fed infants deplete adipose tissue DHA stores by 6
month of age compared to breastfed infants who generally maintain DHA levels (15, 16), thus
factors concerning LCP biosynthesis are relevant for all human infants. Results are mixed for
ARA, as some studies report lower ARA values in plasma and RBC lipids in formula-fed infants
than in breastfed infants (17, 18), while others do not (19).
The functional consequences of LCP supplementation are controversial. Consistent
functional deficits are found for premature infants in visual function (20, 21) and in other CNS
Sarkadi-Nagy et al. Page 5

functions (22). Improvement in function is not uniformly found in studies of term infants, for
whom the effects of a deficiency may resolve with later development (23, 24).
Human breastmilks around the world all contain LCP (25), and commercial infant formulas
containing DHA and ARA, introduced in the USA only in early 2002, have been in use in many
countries for several years. Dietary LCP support greater LCP concentrations in tissues of
experimental animals and in plasma and RBC of human infants, although there is evidence that
dietary LCP and LA suppress ∆6-desaturase activity and mRNA in rats (2, 26). There are no
data to indicate whether dietary LCP decrease biosynthesis of DHA and ARA in human infants
or primates.
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Quantification of total LCP biosynthesis in target organs is not possible in human infants o
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because tissue sampling is required. Accessible tissues, blood or buccal mucosal cells (27, 28), a
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must be sampled and used as proxies for organ status (29). Data on the relationship between from
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tracers detected in plasma/RBC and organs is sparse. Animal studies are required for direct w
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sampling of target organs; because of the unique size and structure of the human CNS, it is best b
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modeled by non-human primates. Much important early data on the relationship of dietary n-3 es
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fatty acids to visual function was generated in term neonatal rhesus monkeys (30). The baboon A
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is a common model for prematurity (31) and is omnivorous in the wild (32), making it an , 2
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excellent model for preterm human LCP nutrition.
In a recent paper, we reported on the influence of prematurity and LCP supplementation on
total fatty acids of brain and related tissues of preterm baboon neonates at four weeks adjusted
age (33). Here we report the results of isotopic tracers administered to these same animals to
determine the influence of LCP supplementation and prematurity on ARA and DHA
biosynthesis. We also assess relative quantitative LCP biosynthesis and relate brain and retina
precursor-derived ARA and DHA accretion to blood-borne precursor-derived ARA and DHA
accretion to assist in interpretation of human studies.
Sarkadi-Nagy et al. Page 6

MATERIALS AND METHODS
The design of this study was described in detail in a previous publication (33). Important
details will be outlined here.
Animals and Diets. The animal protocol was approved by the Cornell Institutional
Animal Care and Use Committee (IACUC) and the facility is approved by the Association for
Assessment and Accreditation of Laboratory Animal Care (AAALAC). Nineteen pregnant
baboons (Papio cynocephalus) were transported in the first half of gestation from the Southwest
Foundation for Biomedical Research in San Antonio, Texas to Cornell University, Ithaca, NY.
Animals were housed in separate cages within sight of at least one other animal and were fed a
commercial primate diet containing fishmeal, which served as an abundant source of n-3 LCP. D
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Pregnant females/neonate pairs were randomly assigned to one of four categories: term, a
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breastfed (B); term, formula-fed (T-); preterm, formula-fed (P-); preterm, fed formula fro
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supplemented with LCP (P+). Nine animals delivered spontaneously at approximately 182 days w
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of gestational age. From these nine, four neonates were assigned to the B group and nursed for rg
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four weeks. Samples of breastmilk were obtained on the day of neonate necropsy. The e
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remaining five animals, the T- group, were transferred to the primate nursery within 12 hours of A
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birth and fed a standard formula (commercially available preterm infant formula without LCP , 2
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Enfacare, Mead-Johnson Nutritionals, Evansville, IN) for 4 weeks. Cesarean sections were
performed on ten animals at 155±5 days of gestational age, as estimated by ultrasound
measurement of fetal head circumference and biparietal diameter (34). The P- group consisted
of five premature animals fed the standard formula, Enfacare, identical to the T- group. The
remaining five preterm neonates, the P+ group, were fed a formula of powdered Enfacare,
blended with an encapsulated oil providing 151 mg DHA and 302 mg ARA per 100 g Enfacare
formula powder (all formulas and encapsulated oils kindly provided by Mead-Johnson
Nutritionals). Neonates were fed ad libitum.
Sarkadi-Nagy et al. Page 7

The three animals that were six weeks of age at euthanasia, and were included in our other
report (33) on total fatty acid compositions, were excluded from this analysis. Thus, all animals
reported in this study were 4 weeks adjusted age at time of euthanasia.
Isotopically-labeled Tracer Doses. All animals received a single combined oral
dose of 20.9 ± 1.5 mg [U-13C]-LNA (LNA*) and 21.0 ± 2.3 mg [U-13C]-LA (LA*) (Spectra Stable
Isotopes, Columbia, MD). LNA* and LA* were 98% uniformly 13C labeled, and were in free fatty
acid form. The dose was administered to each of the 19 neonates 14 days prior sacrifice ; this
corresponded to 14 days postnatal age for terms and for preterms 14 days adjusted age (39±5
days postnatal age).
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Dose was prepared as follows. LNA* and LA* were pipetted and weighed into a sterilized 20 o
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mL vial containing four mL of freshly prepared warm formula. The mixture was vortexed a
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thoroughly and 100 µL was withdrawn and saved for isotopic analysis. The remaining vial from
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contents were drawn into a 5 mL syringe. The vial was washed twice with 1 mL warm fresh .jlr.o
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formula which was then drawn into a second syringe. The contents of both syringes were b
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administered orally within an hour of preparation to the neonates, immediately prior to a s
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regular feeding. Ap
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Sampling. Neonates were euthanized by exsanguination under halothane general , 2
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anesthesia. Organs were collected and individually placed in freezer bags. Blood was collected
in EDTA-containing vacutainer tubes, and RBC and plasma were rapidly separated by
centrifugation. The eyes were immediately dissected, and retina and RPE were placed in sterile
saline solution. All samples, including retina and RPE in saline, were flash frozen in liquid
nitrogen and transferred to a -80°C freezer where they were kept until analysis.
Analyses. Total lipids were extracted from tissue homogenates using the Bligh and Dyer
method (35) modified to insure that the solvent mixture for each tissue type was a single phase
(36). FAs were derivatized to fatty acid methyl esters (FAME) with BF in methanol (Sigma, St
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Louis, MO) and the FAME were dissolved in heptane containing butylated hydroxytoluene
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(Sigma, St Louis, MO) as an antioxidant. Freshly prepared diheptadecanoyl-
phosphatidylcholine was added as internal standard to each tissue sample before
homogenization. Freshly prepared triheptadecanoin (Matreya, Inc., Pleasant Gap, PA) was
added to the formula and breastmilk extraction mixtures as internal standard.
Total FA concentrations were determined by gas chromatography (GC; HP 5890 GC with a
flame ionization detector) using a BPX-70 capillary column (60m×0.32mm×0.25µm film
thickness; SGE, Austin, TX) and H carrier gas. Response factors for each fatty acid were
2
obtained using an equal weight FAME mixture, and data were calibrated with methyl
heptadecanoate as an internal standard.
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Tracer analysis for 13C was performed using a high precision gas chromatography- o
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combustion isotope ratio mass spectrometer (GCC-IRMS; Finnigan MAT 252, Bremen, a
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Germany) described in detail previously (37). from
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Isotope Calculations. Calibrated isotopic data are acquired as delta values expressing w
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the relative deviation from an international standard Pee Dee Belemnite (PDB) in part per b
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thousand. (13C/12C = RPDB=0.0112372) est, o
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R − R A
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δ13CPDB = fRPDBPDB ×1000 (1) ril 4, 2
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R is the ratio of the heavy to light isotope for the sample. Extracting R from equation (1), atom
f f
percent (AP) of the heavier isotope in analyte peak can be calculated as:
R
AP = f ×100
(2)
f 1+ R
f
Atom percent excess (APE) is calculated by subtracting the baseline sample atom percent from
the post-dose sample atom percent. Baseline values were derived from analysis of samples of a
single undosed-fetus sacrificed at cesarean section as described previously (6). The total amount
of tracer which accumulates in a particular tissue can be calculated by multiplying the APE by
the tracee concentration (Q) in tissues. If the dose fatty acid is elongated from endogenous non-
f
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labeled sources (i.e. LNA to DHA and LA to ARA), this value must be corrected by the number of
moles of C in the analyte divided by the number of moles of C in the dose FA, to be expressed in
terms of the moles of precursor that has entered the product pool. This value is termed the
molar dose equivalent (D*) (38) and is calculated as:
[ ]
APE C
D* = f ×Q × f (3)
100 f [C]
d
To eliminate artifactual differences resulting from different oral amounts, results are normalized
by the amount of labeled FA consumed by the animal (d):
D*
%Dose =
(4)
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The %Dose value reflects transformation of labeled FA into products and incorporated into a
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organs in terms of molar equivalents of the dosed fatty acids, LNA* and LA* for labeled DHA fro
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(DHA*) and ARA (ARA*), respectively. w
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The labeled fatty acids are tracers for the unlabeled fatty acid tracees consumed in the feed. brg
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The relevant pool sizes that must be taken into account are the differing tracee concentrations, e
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which in this case are LNA and LA in the diets. Normalizing the data to the relative tracee levels A
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permits conclusions to be drawn about the fate of dietary fatty acids in the meal immediately , 2
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subsequent to dosing. Changes in fatty acid pools, such as plasma fatty acids, induced by
differing experimental diets are characteristic of the metabolism under study and therefore need
not be considered separately. In our treatments, the LNA and LA concentrations were
essentially identical for the T-, P-, and P+ groups, but different for the B group. The breastmilk
%Dose values were adjusted by dividing them by the ratio M /M , where M is molarity, FA
FA,F FA,B
is either LA or LNA, B is breastmilk, and F is formula. In the present context, the result can be
interpreted as the %Dose that would have been found if the concentrations of breastmilk LA and
LNA concentrations were equivalent to that of the other treatments. For simplicity and
consistency with previous reports (e.g. (8, 39)), we refer to this as %Dose.
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Correlations among tracer concentrations in plasma, RBC, and tissue pools were calculated
from individual data from each animal without regard to treatment group to determine the
degree to which pools accessible in human infants correlate with tissues. Each animal was
represented by a single data point for DHA* and ARA* for each pool.
Statistics. Data are expressed as mean ± SD. Within each organ, means were
compared with the SAS System for Windows, Version 8.02 (Cary, NC) using the Mixed
Procedure by writing specific contrasts for the main effects of group (B, T-, P+, P-), and of
labeled fatty acids (DHA*, ARA*). Significance was declared at p<0.05. Regression equations
were calculated in Excel 2000 for Windows98 (Microsoft, Seattle, WA).
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