Impact of Early Dietary Intake and Blood Lipid Composition of Long-ChainPolyunsaturated Fatty Acids on Later
Visual Development
*?Dennis R. Hoffman, *?Eileen E. Birch, *?David G.Birch, *§Ricardo Uauy, *Yolanda S. Castan?eda, *Maia G. Lapus, and *Dianna H.Wheaton
*Retina Foundation ofthe Southwest; Departments of ?Pediatrics and ?Ophthalmology, University ofTexas Southwestern Medical Center, Dallas, Texas, U.S.A.; §Institute ofNutrition and Food Technology (INTA), University of Chile, Santiago, Chile
ABSTRACT
Background:In contrast to human milk, current infant formu-lasin the United States do not contain 3 and 6 long-chain polyunsaturated fattyacids. This may lead to suboptimal blood lipid fatty acid profiles and to ameasurable diminution of vi-sual function in developing term infants. The needfor docosa-hexaenoic acid and arachidonic acid supplementation in the infantdiet was evaluated in a double-blind, randomized clinical trial.
Methods:Healthy term infants were randomized to diets of(1)commercial formula, (2)docosahexaenoic acid–enriched for-mula (0.35% of total fatty acids), or (3)docosahexaenoic acid– (0.36%) and arachidonic acid– (0.72%) enriched formula.Eighty-seven infants completed the 17-week nutritional trial, and 58 wereobserved until 52 weeks of life. A reference group was exclusively breast fedfor at least 17 weeks (n 29). Outcomemeasures included electroretinographic responses, vi-sual evoked potentials,and blood fatty acid analysis in infants at birth and at 6, 17, and 52 weeks ofage.
Results:Commercial formula-fed infants had 30% to 50%lower content of docosahexaenoic acidin total red blood cell lipids during the 17-week feeding trial compared withbreast-
fed infants. Significant differencespersisted at the 1-year fol-low-up. Arachidonic acid content was consistentlyreduced in the commercial formula group by 15% to 20%. Infants fed long-chainpolyunsaturated fatty acid–enriched formulas had docosahexaenoic acid andarachidonic acid blood lipid profiles resembling those of human milk-fedinfants. Infants receiving this enriched formula had more matureelectroretinographic responses than commercial formula-fed infants at 6 weeksof age. Human milk-fed and docosahexaenoic acid-enriched for-mula-fed infantshad better visual acuity than commercial for-mula-fed infants at both 17 and 52weeks of age. Early (17-week) fatty acid profiles in blood lipids werecorrelated with later (52-week) visual function development in study infants. Conclusions: Results from this clinicaltrial demonstrate thatlong-chainpolyunsaturated fatty acid supplementation of for-mula in term infants producesblood lipid fatty acid profiles that are similar to those observed inbreast-fed infants. This supple-mentation leads to better visual function laterin life (i.e., 1 year of age) than that shown by infants fed commercialformula.
JPGN 31:540–553, 2000. Key Words:Arachidonicacid—Docosahexaenoic acid—Infant nutrition— 3 Fatty acids— Visual function.© 2000 Lippincott Williams & Wilkins, Inc.
Although a dietarypresence of -linolenic acid ( - LNA; 18:3 3) and linoleic acid (LA; 18:2 6) isconsid-ered essential for proper growth and development of pre-term and terminfants, the need to provide the long-chain derivatives of these essentialacids, namely docosahex-aenoic acid (DHA; 22:6 3) and arachidonic acid (AA;20:4 6) remains undefined. Breast-fed infants receive the essential fattyacids, -LNA and LA, as well as small
Received April 5,2000; revised September 7, 2000; accepted Sep-tember 8, 2000.
Addresscorrespondence and reprint requests to Dr. Dennis R. Hoffman, Retina Foundationof the Southwest, 9900 North Central Expressway, Suite 400, Dallas, TX 75231,U.S.A. (e-mail dhoffman@ retinafoundation.org). Telephone: (214) 363-3911 x125;Fax: (214) 363-4538
amounts of DHA and AA, but infants fedformula in the United States do not receive these long-chain polyun-saturatedfatty acids (LCPUFAs). At issue is whether sufficient amounts of the LCPUFAsare produced de novo to support the demands of a rapidly developing centralnervous system and its associated neural network, including the visual system,or if supplementation of “preformed” LCPUFAs in formula is necessary toopti-mize retinal and cortical visual development in infancy.
The last trimesterand early postnatal months represent a period of rapid increase in the numberof synapses and dendritic growth in the brain (1,2). During this period, rapidmaturation of photoreceptors also occurs in the neural retina (3). Because thecentral nervous system including the retina is derived embryologically fromneu-
540
roectoderm, assessment of visual developmentin early childhood directly reflects developmental progression in the brain. Inrecent years, this evaluation method has been used to investigate the effect ofdiet modification on neurodevelopment. Studies in term infants exemplify thisevaluation method. Dietary LCPUFA supplementation not only results in elevatedblood lipid DHA levels but also in more mature retinal responses, determined byelectroretinography (ERG) (4). Furthermore, infants ran-domized toLCPUFA-supplemented formula had more mature visual cortical processing,determined by visual evoked potentials (VEPs), compared with infants receiv-ingcommercial formula without LCPUFAs (5). Subse-quently, these same infantsreceiving LCPUFAs attained higher scores at 18 months of age inneurodevelopmental testing (6). In addition, visual acuities of these infantsat 52 weeks of age were significantly correlated with the mental andpsychomotor development indices and be-havioral ratings scales of infants at 18months (6).
These results supportthe use of blood lipid fatty acids as a surrogate index of the infant’s tissuefatty acid sta-tus, particularly because direct assessment of neural tis-suesis not practical. Validation of this procedure is de-rived from significantcorrelations between fatty acid profiles in red blood cell (RBC) membranelipids com-pared with neural tissue fatty acid profiles in rats (7), monkeys(8), and humans (9).
Membranephospholipids of the retina have been known for many years to be enriched inDHA (10), with particularly high concentrations localized at the neuro-sensorycellular junctions (11). In rod photoreceptors, DHA is tightly associated withand known to have a regulatory influence on rhodopsin, the chromophorere-sponsible for capture of photons and initiation of the visual response (12).The specific mechanism(s) by which DHA may be associated with visualdevelopment remains unknown but may involve biochemical and bio-physical functionalmodifications (12), effects on apop-totic death of photoreceptors (13), and/ormodulation of activity of ionic channel proteins in neuroexcitatory cells (14).
During infantdevelopment, considerable accretion of LCPUFAs, DHA in particular, occurs inneural and reti-nal membranes during the last trimester of gestation andcontinues throughout the first year of life (15,16). A maternal-to-fetal “biomagnification”of LCPUFAs oc-curs as a result of unidirectional DHA transport from maternal tofetal circulation driven by high-affinity DHA-specific transport proteins inthe placenta (17). Al-though this LCPUFA enrichment mechanism may sup-ply ampleDHA for proper neural development in utero, there remain concerns about theLCPUFA supply in the neonate. Immature endogenous synthesis of LCPUFAs has longbeen thought to limit accretion of important fatty acids in neural tissuesduring infant development (16,18). However, recent studies using stableisotopes of -LNA and LA have demonstrated that infants have the
ability to metabolizethese fatty acids through chain elon-gation, desaturation, and partialperoxisomal -oxida-tion, to DHA and AA, respectively (19,20). Yet,signifi-cantly lower concentrations of DHA have consistently been reported inbrain tissue of formula-fed infants than in breast-fed infants (9,21,22).Furthermore, differences in blood lipid composition between infants fed dietswith and without LCPUFAs (23,24) suggest that infants are unable to synthesizesufficient amounts of DHA to meet developmental needs. Thus, the need forpreformed DHA in the infant diet (such as in human milk or en-riched formula)remains a viable alternative to the sole reliance on endogenous conversion ofthe parent essen-tial fatty acid -LNA, to provide sufficient DHA for optimalvisual and neural development.
In this study, weexamined which blood lipid fractions provide the most information on infantfatty acid status and dietary compliance and best correlate with visualdevelopment. Results from this clinical trial demonstrate that provision ofLCPUFAs early in life favorably modify the infant’s blood lipid fatty acidprofile and are correlated with development of visual function later in life.
MATERIALS AND METHODS
Subjects
Biochemical andvisual function results for two study co-horts of healthy term infants (37–40weeks postmenstrual age) are reported herein. Both groups of infants receivedstudy diets within the first 4 days of life (range, 0–4 days; mean ± standarddeviation [SD], 2.1 ± 1.0 days). Separate cohorts for ERG and VEP testing wereused to minimize the number of blood samples and functional measures obtainedper infant, limit test-ing demands on the infants and parents, and reducesubject withdrawal. Thirty-three infants were enrolled in the ERG arm of thestudy and 79 in the VEP arm.
Before the first ERGmeasurement at 6 weeks, nine infants were withdrawn from the study, five ofthese primarily because of lactose intolerance. No infants were lost betweenthe 6- and 17-week measurements in the ERG study cohort. Recruitment andenrollment of study subjects as well as exclusion criteria, demographicprofiles, subject compliance, and withdrawal data have been reported previouslyfor the VEP cohort (5). In brief, 16 infants were lost to follow-up by 17 weeksin the VEP arm, 8 of whom had symptoms associated with lactose or cow’s milkprotein intolerance.
Each cohort consistedof three randomized infant groups receiving either standard diet or one of twoexperimental diets. At 17 weeks of age, retinal maturation was assessed by ERGin one cohort (n 24), and in thesecond cohort (n 63), development ofvisual cortical function was followed by sweep VEP. The VEP acuity measureswere obtained in 58 infants in the second cohort at 52 weeks. An additional groupof term infants (n 29) wereexclusively breast fed for at least the first 17 weeks of life and served as anonrandomized control group for blood fatty acid and VEP data.
ETHICALCONSIDERATIONS
Written informedconsent was obtained from one or both parents 0 to 4 days after birth andbefore the infant’s enrollment. The research protocol adhered to the tenets ofthe Declaration of Helsinki and was approved by the Institutional Review Boardof the University of Texas Southwestern Medical Center, Presbyterian MedicalCenter, and Medical City Columbia Hospital (all located in Dallas, TX, U.S.A.).A blocked randomization sched-ule was developed by the Mead–Johnson ResearchCen-ter (Evansville, IN, U.S.A.) and provided in sealed en-velopes to the studysite.
Diets
Study diets were (1)commercial formula (CF; Enfa-mil with iron), (2) formula supplemented with DHA(DHA formula; Enfamil supplemented with iron and 0.35% DHA), or (3) formulasupplemented with DHA and AA (DHA+AA formula; Enfamil supplemented with iron,0.36% DHA, and 0.72% AA). All formulas were provided by Mead–JohnsonNutritionals (Evansville, IN, U.S.A.) in 32-oz ready-to-feed cans and contained2.2 g protein, 5.6 g fat and 10.3 g carbohydrate per 100 kcal. Formulas metexisting nutrient standards recommended by the Committee on Nutrition, AmericanAcademy of Pediatrics (25). Vitamin E content was maintained at near 2 mg-tocopherol per gram of unsaturated fatty acid in formulas to limit lipidperoxidation. The fatty acid profiles of commercial formula and the experimen-
tal formulas are presented in Table 1.Supplementation with DHA and AA was accomplished by addition of single-celloils (SCOs), specifically DHASCO and ARASCO (Martek Biosciences, Columbia, MD,U.S.A.).
General Protocol
All infants wereexclusively fed study diets or human milk through 17 weeks of age.Subsequently, study for-mula was no longer provided, and only commercialfor-mula, devoid of LCPUFAs, was available. Approxi-mately half of the humanmilk–fed infants (11 of 20) were weaned to formula at or shortly after 17weeks, whereas the remainder continued to breast-feed for up to 52 weeks ofage. Blood samples (2.0 mL) were collected in Vacutainer tubes (BectonDickinson, Franklin Lakes, NJ) containing the anticoagulantethylenediaminetetra-acetic acid (EDTA) at birth from cord blood and inMi-crotainer tubes (Becton Dickinson) at 6, 17, and 52 weeks of age by heelstick aided by infant heel-warming packs. Blood samples were obtained from theERG co-hort at enrollment (placental cord blood) and at 6 and 17 weeks of age.In the ERG cohort, retinal responses were obtained at 6 and 17 weeks of age.Blood samples in the VEP cohort were obtained at enrollment and at 17 and 52weeks of age. Visual acuity, as determined by VEP, was obtained at the 6-, 17-,26- and 52-week time points (study results reported in Reference 5) but bloodlipid-VEP sample pairs were available only for the 17- and 52-week time points.Data sets for ERG and VEP matched to blood lipids were combined for 17-weekre-sults. Investigators conducting the blood lipid analysis
and visual functiontesting were masked to type of for-mula provided to infants.
Blood Lipid FattyAcid Analysis
Plasma and RBCs wereseparated by centrifugation (3000gfor 10 minutes). The RBCs were washed with saline and lysed with water duringthe extraction process. A modified Bligh and Dyer (26) procedure was used toextract lipids from plasma and RBC lipids. To limit oxi-dative damage, sampleswere extracted immediately (2–4 hours) with methanol-chloroform solventcontaining 0.02% butylated hydroxytoluene (BHT), and oxygen was flushed fromtubes at each step with N2.
Lipid extracts in BHT-containing solvent werestored briefly (<7 days) under N2 at ?20°C untiltransmethyl-ation or further fractionation by thin-layer chromatogra-phy. Analiquot of plasma total lipid was separated into a total phospholipid fraction,and a pool of neutral lipids using hexane-diethyl ether-acetic acid (80:20:1,by vol-ume) as the chromatographic solvent system. Lipid stan-dards werephosphatidylcholine (PC), oleoylglycerol, dioleoylglycerol, trioleoylglycerol,cholesteroyl oleate, and oleic acid. The phospholipid fractions, PC,phospha-tidylethanolamine (PE) and phosphatidylinositol-plus-phosphatidylserine(PI+PS) were isolated from an ali-quot of RBC total lipid using the solventsystem of chlo-roform-petroleum ether-methanol-acetic acid–boric acid(40:30:25:10:1.8; vol:vol:vol:vol:wt) described by Gilfil-lan et al. (27).Fatty acid methyl esters from total plasma, RBCs, and isolated lipid fractionswere prepared by transesterification under N2 with 14% borontrifluoride in methanol at 100°C for 20 minutes (28). Fatty acid methyl estersin methylene chloride were separated and quantified using capillary column gaschromatography and flame ionization detection on a gas chromatograph (model5890; Hewlett–Packard, Palo Alto, CA, U.S.A.) equipped with a 0.25-mm innerdiameter, 30-m capillary column containing Omegawax stationary phase (Su-pelco,Bellefonte, PA, U.S.A.). Chromatographic param-eters included helium as carriergas flowing at 1.0 mL/ min, a split ratio of 10-to-1, injector temperature of240°C, detector temperature of 250°C, and an oven tem-perature program of 195°Cfor 2 minutes, decrease at 0.5°C/min to 180°C, 180°C for 1 minute, increase at5°C/min to 210°C, and 210°C for 25 minutes. At this point, biologiccontaminates were removed from the col-umn by increasing the oven temperatureat 20°C/min to 245°C and running at 245°C for 40 minutes. This tem-peratureprogram includes a unique cooling ramp to re-solve the fatty acids (e.g., 20:39) that were important in this clinical nutrition trial.
Figure 1 showsrepresentative chromatographic sepa-rations of fatty acid methyl esters fromRBC lipids of a term study infant and the standard (GLC68+11). GLC68+11 was rundaily along with samples as a stan-dard laboratory practice and consisted of 19fatty acid
FIG. 1. Gas chromatographic separation of fattyacid methyl es-ters from (a)GLC68+11 standard and (b) total redblood cell lipid fraction from a 6-week-old term infant. Truncated peaks werefully integrated and reflect strip-chart recorder limitations.
methyl esters(GLC68A; NuChek Prep, Elysian, MN, U.S.A.) enriched with 11 individualstandards (Sigma Chemical Co., St. Louis, MO, U.S.A. and Cayman Chemical Co.,Ann Arbor, MI, U.S.A.). Retention times from the GLC68+11 standard chromatogramwere rou-tinely used for peak identification. The 22:5 6 (docosa-pentaenoicacid; DPA 6) peak was identified by com-parison to an ovine retinal preparationknown to be en-riched in this fatty acid.
Fatty acid data,expressed as both relative weight per-centage and mass, were obtained fromseven blood lipid fractions (total RBC, RBC-PC, RBC-PE, RBC-PI+PS, totalplasma, plasma phospholipids, and plasma neutral lipids) at birth (cord blood)and at 6, 17, and 52 weeks of age in the two infant cohorts. Although data alsowere calculated as a relative percentage, we focused on mass units reported asmicrograms of fatty acid per milliliter plasma or per milliliter packed RBCs,computed by com-parison to an internal standard. The fatty acid 23:0 (10 g) wasadded as an internal standard to the methanol-chloroform before extraction oftotal lipids or extraction of lipid fractions from silica gel after thin-layerchroma-tography. In the literature, fatty acid values are generally reported aspercentage of relative weight; however, mass analysis provides an absoluteconcentration that is not influenced by the abundance of other fatty acids inthe sample.
For fatty acidanalysis of study formulas, an extraction procedure was used to limit loss ofshort-chain (6–12 carbon) fatty acids and methyl esters during samplepro-cessing. The procedure is based on that described by the Association ofOfficial Analytical Chemists (29) and,
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544 D. R. HOFFMAN ET AL.
briefly, involvesextraction of lipids using 3% NH4OH-100% ethanol-diethyl ether-petroleumhydrocarbons (2:2:5:5, by volume). Methyl esters were prepared as describedearlier under N2 and extracted twice with 0.5 mL methylene chloride, essentiallyaccording to MacGee and Allen (30).
Electroretinography
The maturity ofretinal function in infants was as-sessed by measurement of ERG responses tolight stimuli using methodology previously described (31,32). Briefly,full-field ERG responses to short- (450-nm) and long- (650-nm) wavelengthstimuli over an extensive range of retinal illuminances were evaluated indark-adapted infants at 6 and 17 weeks of age. The ERG responses to short-wavestimuli were used to help isolate rod photoreceptor function at the two testages (33). Na-ka–Rushton (34) plots of responses as a function of il-luminancewere used to determine maximum response amplitude (Vmax), rod thresholds (i.e.,light required to generate a 2- V response) and the semisaturation con-stant(log k). Retinal maturation corresponded to higher values of Vmax and lower rodthreshold and log k val-ues.
Sweep Visual EvokedPotential Acuity
The VEP measure of visual acuity determinesthe neu-ral integrity of the pathway from the retina to the primary visualcortex. The test procedure uses variable spatial frequency square-wave gratingsphase reversing at 6.6 Hz and has been described in detail elsewhere (5).Briefly, two bipolar placements of Oz versus O1 and O2 were used to record(gain 10,000–20,000, ?3-dB cut-off at 1 and 100 Hz) the electroencephalogram,which was adaptively filtered in real time to isolate the VEP (397-Hz samplingrate). Amplitude and phase of the re-sponse at the second harmonic of thestimulation fre-quency was calculated for each channel. Noise was mea-sured bydetermining the amplitude and phase of the two adjacent nonharmonicfrequencies. Grating acuity was estimated with an automated algorithm thatexamines signal-to-noise ratio and phase coherence and performs a linearregression for the final descending limb of the vector-averaged function(minimum of three trials; typi-cally five trials) relating VEP second harmonic ampli-tude(amplitude at the reversal frequency of 13.2 Hz) to spatial frequency. SweepVEP acuities were expressed in log minimum angle of resolution (logMAR; e.g.,20/20 corresponds to a minimum angle of resolution of 1 minute of arc [min arc]and logMAR of 0.0, whereas 20/200 corresponds to a minimum angle of resolutionof 10 min arc and logMAR of 1.0).
Statistical Analysis
Blood lipid analysisand vision tests were conducted in a masked fashion. The randomization code wasrevealed
to both testers and parents afterneurodevelopmental test-ing of study infants at 18 months of age. Comparisonsamong the three infant groups randomized to study for-mula for fatty acidcomposition at each age were con-ducted by one-way analysis of variance(ANOVA); be-cause of the number of comparisons (n 51 in Tables 3–5), the stringency of statistical significance wasset at P < 0.001, with theBonferroni adjustment. Multiple-comparison analysis of randomized diet groupresults were conducted with the Newman–Keuls procedure at an of 0.05.Correlations between blood lipid fatty acids and visual function acuities inrandomized, formula-fed infants were calculated by Pearson correlationanalysis. P < 0.05 was consideredsignificant.
RESULTS
Diets
The fatty acidcomposition of the three study formulas is shown in Table 1. The 6-to- 3 fattyacid ratios ranged between 8 to 1 and 10 to 1 and were comparable with valuesreported for human milk (23,35,36). The target concentrations for DHA (0.30–0.36%of total fatty acids) and AA (0.60–0.72%) in the current experimental formulaswere based on our previous analysis of human milk (23) and literature reportsof human milk fatty acid profiles in various populations (35,36).
Red Blood Cell LipidFatty Acids
Full data sets fromthe RBC total lipid fraction at birth (cord blood) and at 6, 17, and 52 weeksare presented in Tables 2 through 5. Data from other lipid fractions showedexcellent agreement with data from the RBC total lipid fraction. Percentagevalues for the four essen-tial and provisionally essential 3 and 6 fatty acids(LA, AA, -LNA, and DHA) in 17-week-old study in-fants are given in Table 6.
Cord Blood
At birth, there wereno significant differences in fatty acid content of RBCs obtained from cordblood of term infants before randomization to study diets (Table 2). Meanconcentrations of Mead acid (20:3 9) were el-evated at birth and ranged from 5to 8 g/mL RBCs among the various diet groups compared with 4.4, 0.8, and 0.9g/mL at 6, 17, and 52 weeks, respectively. Mead acid is the end product ofoleic acid (18:1 9) metabolism and generally accumulates when there is adeficiency of essential fatty acids (37). A universal index of essential fattyacid status is the ratio of Mead acid to AA (20:3 9 to 20:4 6). Ratios of Meadacid to AA exceeding 0.025 are considered indicative of essential fatty acidinsufficiency (38). At birth, infants in all diet
Includes 14:0, 16:0, 17:0, 18:0, 20:0, 22:0 and 24:0.
cIncludes 16:1, 18:1, 20:1, 22:1 and 24:1. LCPUFA are long-chainpolyunsaturated fatty acids of >18 carbon chain length. Mead acid/AA ratio20:3 9/20:4 6. Unsaturation Index is the sum of (# of double bonds × mass ofeach fatty acid). No significant differences were found among randomizedgroups; all analysis of variance F statistics <1.8, P > 0.2.
By 6 weeks of age,marked diet-induced differences in RBC fatty acids were evident among formulagroups (Table 3), indicating that dietary compliance was excel-lent. Infantswho received 3LCPUFA in their diets (DHA and DHA+AA groups; Table 1) hadcorrespond-ingly higher concentrations of total 3LCPUFAs in their blood lipids.A similar trend was found for infants fed the 6LCPUFA-deficient commercialformula, so that total blood lipid 6LCPUFAs were significantly reduced comparedwith the other diet groups. This pattern was observed in data expressed aseither mass or relative percentage and was also found in the other blood lipidfractions (Table 6). For infants receiving either experi-mental diet (DHA orDHA+AA groups), total 3 and 6 LCPUFAs closely matched that of the human milk(HM) group.
At 6 weeks (Table 3),dietary supplementation of the major 6LCPUFA, namely AA, resulted in anelevation of RBC AA in the DHA+AA group compared with the CF group. Theelevation of LA in the DHA group was
unexpected; 3/ 6competition (39) should decrease up-take and incorporation of LA into RBCphospholipids. Although there were differences in individual 3 fatty acidsamong diet groups at 6 weeks, only the DHA con-tent could be identified asstatistically significant based on stringent multiple-comparison criteria.
Two fatty acidindices of 3 sufficiency include the ratio of 3-to- 6 end products (i.e.,DHA-to-DPA 6) and the 6-to- 3LCPUFA ratio. The significantly lower DHA-to-DPA 6and higher 6-to- 3LCPUFA ratios in RBC total lipids of the CF fed infant groupwere indica-tive of a dietary 3-deficient status at 6 weeks of age (Table 3).However, these indices in the two experimen-tal diet groups (DHA and DHA+AA)compared with the HM group were consistent with an improved 3 fatty acidprofile. Because all infants receive diets containing ample amounts of both 3and 6 essential fatty acid, a true deficiency is not likely to occur. At 6weeks of age, the Mead acid-to-AA ratio was reduced from cord blood levels inRBCs of the three formula groups, although only the DHA and DHA+AA groupsreached levels equivalent to that of HM-fed infants. The unsaturation indexrepresents the sum of the number of double bonds multiplied by the mass of eachfatty acid in the sample. In both experimental formula-fed groups at 6 weeks ofage (DHA and DHA+AA), this index was significantly
higher,indicating an improved unsaturation status com-pared with the CF-fed infantgroup.
Seventeen Weeks
After 17 weeks of the study, marked diet-induced ef-fects were evidentamong the infants randomized to the
CF groups (Table 4).A significant reduction in 6LCPUFA in the DHA group probably reflected thecompetitive nature of 3 and 6 fatty acids provided exogenously in the diet. Aswas found at 6 weeks, total 3LCPUFA content of the CF group was significantlyreduced compared with the DHA and DHA+AA groups
at 17 weeks. TheDHA-formula group had 3LCPUFA levels elevated above that of the HM group;however, the DHA+AA group had 3LCPUFA levels that were indis-tinguishable fromthose in the HM-fed group. At 17 weeks, RBC LA concentrations in the CF andDHA-enriched formula groups were significantly elevated over the DHA+AA group,suggesting that dietary provision of preformed AA in the DHA+AA group (and theHM group) may have inhibited metabolic activity of LA (39). Similar resultswere found at the 6- and 17-week time points for RBC AA content. By comparisonto the CF group, the DHA+AA group had a high level of AA (as did the HM group);however, in the DHA-study group, RBC AA levels were reduced, probably becauseof 3 fatty acid competition.
Supplementation ofDHA elevated RBC DHA levels at 17 weeks of age in the DHA- and DHA+AA formulagroups as expected, but dietary inclusion of the LCPUFAs from the SCOs did notelevate the levels of the DHA precursors, eicosapentaenoic acid (EPA; 20:5 3)and docosapentaenoic acid (DPA 3; 22:5 3) compared with the CF group or theHM-fed infants. In fact, in the group receiving AA supplementation, are-duction in the level of these fatty acids may have re-flected 3– 6competition.
The low DHA-to-DPA 6ratio and high 6-to-3LCPUFA ratio in the CF group at 17 weeks also in-dicated acompromised status of both DHA and 3LCPUFAs of CF-fed infants. Neuringer et al.(40) noted the importance of a DPA 6 accumulation in 3-deficient monkeys andproposed that this fatty acid re-
places 3 fatty acids,particularly DHA, in retinal mem-branes. This replacement phenomenon wassupported by the presence of a constant level of 22-carbon unsaturated fattyacids in the tissue, regardless of the shifting 3-to-6 ratio. In contrast, at17 weeks, we found that the sum of the 3 and 6 22-carbon fatty acids in totalRBC lipids was significantly lower in the CF infant group (108 g/mL RBCs; P < 0.0005) and nearly identical inthe HM, DHA, and DHA+AA groups (139, 138, and 139 g/mL, respectively). Similarresults were found if the data were expressed as relative weight percentage.
Fifty-Two Weeks
After 17 weeks,infants no longer received study for-mulas and subsequently, by 52 weeks, manydifferences in RBC fatty acids had disappeared (Table 5); however, 3LCPUFAswere the exception. Despite a change in dietary regimen at 17 weeks and aturnover of RBCs (half life 120 days) (41), there were persistent differ-encesamong the diet groups in DHA and total 3LCPUFA at 52 weeks of age. The balanceof 3-to-6 fatty acids also continued to be skewed at 52 weeks as determined bythe DHA-to-DPA 3 and 6-to-3LCPUFA ratios.
Docosahexaenoic andArachidonic Acids
Concentrationprofiles of DHA and AA in RBC lipids of study infants are shown as a functionof time in Fig-ures 2 and 3. The 50% reduction in the RBC DHA level
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548 D. R. HOFFMAN ET AL.
b HM, human milk-fed group; CF, commercial formula-fed group; DHA,DHA-enriched formula-fed group; DHA+AA, DHA- and AA-enriched formula-fed group;ANOVA, analysis of variance; F, ANOVA F statistic.
cLA, 18:2 6; -LNA, 18:3 3; AA, 20:4 6; DHA, 22:6 3.
d PC, RBC-phosphatidylcholine; PE, RBC-phosphatidylethanolamine; PI+PS,RBC-phosphatidylinositol+phosphatidylserine; NL, neutral lipds; PL,phospholipids.
e,f,g Differentsuperscripts indicate significant difference (P < 0.05) by Newman-Keuls multiple comparison analysis betweenrandomized formula-
fed infant groups.
in CF-fed infants compared with HM-fedinfants was readily apparent at the 17-week time point. The blood lipid DHAcontent of the CF group at 17 weeks was also 30% lower than birth levels ofDHA. In contrast, dietary supplementation of DHA (with or without AA) resultedin RBC DHA levels that surpassed the level achieved by breast-feeding at both 6and 17 weeks. By the time the infants reached 52 weeks of age, feeding of studyfor-mulas had been discontinued for 35 weeks, and differ-ences among the threeformula-fed groups were reduced, yet statistically significant. Nine of 20infants in the HM group continued to breast-feed until 1 year of age, yet meanRBC DHA levels in the HM group decreased by 28% at 52 weeks compared with thoseat 17 weeks.
The AA content of RBClipids from infants in the CF group was markedly reduced during the dietaryinterven-
tion period but,because solid foods were provided ad libitum after 17 weeks, RBC AA levelsapproached that of the HM-fed infants by 52 weeks (Fig. 3). Competition betweenDHA and AA for incorporation into membrane lipids was evident by the reductionof RBC AA in the DHA infant group until formula supplementation ceased at 17weeks. Provision of AA in the diet (i.e., DHA+AA group) resulted in RBC AAlevels that were comparable to those of the HM-fed infant group during the17-week supplementation period, but levels decreased markedly by 52 weeks oflife.
Lipid Fractions
The distributionamong the various blood lipid frac-tions of the two essential (LA, -LNA) andtwo provi-
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FIG. 2. Docosahexaenoic acid (DHA) concentrationin red bloodcell total lipids ofterm infants as a function of time in study. Infants were fed human milk (HM),commercial formula (CF), formula enriched with DHA (DHA), or formula enrichedwith DHA and arachidonic acid (DHA+AA). Values are mean ± SE; values for infantgroups denoted by different letters are significantly dif-ferent (P < 0.05) within an age group ofrandomized formula-fed infants.
FIG. 4. Association between early fatty acidcomposition (redblood celldocosahexaenoic acid [DHA] concentration at 17 weeks) and later visual function(sweep visual evoked potential acuity at 52 weeks) in randomized formula-fedstudy infants. Data sets were significantly correlated (r = ?0.54; P = 0.00001; n = 58) so that infants with higher DHAlevels had lower log minimum angle of resolution (logMAR) values (i.e., bettervisual acuity).
sionally essential (AA, DHA) fatty acids atthe end of the dietary supplementation period (i.e., 17 weeks) is shown inTable 6. Marked diet-induced differences among ran-domized formula-fed groupswere found for LA, AA, and DHA for all blood lipid fractions. LA and -LNA weremost enriched in the plasma fractions, whereas RBC lipids had higher contentsof the LCPUFAs, AA and DHA. Based on the ANOVA F factor, the PC frac-tion inRBCs was most sensitive to dietary differences in 6 fatty acids, whereas totalRBC lipids and PE were most responsive to dietary variations in DHA. The sma
FIG. 3. Arachidonic acid (AA) concentration inred blood cell totallipids of terminfants as a function of time in study. Infants were fed human milk (HM),commercial formula (CF), formula enriched with docosahexaenoic acid DHA (DHA),or formula enriched with DHA and AA (DHA+AA). Values are mean ± SE; values forinfant groups denoted by different letters are significantly different (P < 0.05) within an age group ofrandomized formula-fed infants. n.s., not significant at P < 0.05.
amounts of LNAincorporated into either RBC or plasma lipids made statistical comparisonsbetween diet groups less reliable.
Docosahexaenoic Acidand
ElectroretinographicFunction
At 6 weeks, the log k(semisaturation constant) of the rod ERG response was significantly differentin the three formula groups (Table 7). The lower log k value of ?0.15 logscotopic troland-seconds reflected a more mature ERG response in the DHA+AAformula group compared with the other study groups. This diet-induceddifference was no longer present at 17 weeks of age. The Vmax and rodthresholds were not statistically significant among diet groups at either 6 or17 weeks of age.
Therole of DHA as a factor associated with develop-ment of retinal function ininfancy was evaluated by correlation analysis. In formula-fed infants of theERG cohort, log k at 6 weeks of age was significantly corre-lated with massconcentration of DHA in the PC fraction of RBC (r ?0.48; P 0.02). Noother Naka–Rushton parameters at either 6 or 17 weeks were significantlycorrelated with DHA in total lipids from plasma, RBCs, or subfractions (i.e.,plasma phospholipids, plasma neu-tral lipids, RBC PE, or RBC PI+PS). Thehighest corre-lations found between ERG parameters and fatty acids in RBCs werebetween log k and Mead acid (20:3 9) at 6 weeks (r 0.55; P 0.005).Similarly, the ratio of Mead acid to AA (20:3 9 to 20:4 6) was correlated withlog k (r 0.56; P 0.004). Thus, higher concen-trations of Mead acid in infants at 6weeks were associ-ated with higher log k values (i.e., more immaturere-sponses).
b HM, human milk-fed group; CF, commercial formula-fed group; DHA,DHA-enriched formula-fed group; DHA+AA, DHA- and AA-enriched formula-fed group;ANOVA, analysis of variance; F, ANOVA F statistic.
c Electroretinographic (ERG) parameters fitted to Naka-Rushton equations:Log Vmax V maximum ERG response; Log rod threshold (scotopic troland-seconds);Log k (scotopic troland-seconds).
d Sweep visual-evoked potential (VEP) acuity given as logMAR (minimumangle of resolution).
e, f Different superscripts indicate significant difference (P < 0.05) by Newman-Keuls multiplecomparison analysis of randomized formula-fed infant groups.
g Data from reference 5.
More mature responses are represented by higherVmax values, lower rod threshold, log k and logMAR values. n.a. not availabledue to insufficient numbers of subjects recruited.
Docosahexaenoic Acidand Visual Evoked
Potential Acuity
At both 17 and 52 weeks,VEP acuity was signifi-cantly (P <0.05) poorer in the CF infant group than in the DHA and DHA+AA groups (Table 7;note that better visual function is defined by lower logMAR values). At both 17and 52 weeks, VEP acuity in the DHA and DHA+AA infant groups wasindistinguishable from that in the HM group, whereas the CF group hadapproxi-mately 0.1 logMAR (one line on an eye chart) poorer vision.
In the 17-week data,the highest correlation was found between sweep VEP acuity and theconcentration (in micrograms per milliliter) of DHA in the total RBC lipidfraction (Table 8; r ?0.35; P 0.004). VEP acuity was alsosignificantly correlated with DHA in RBC PE, RBC PI+PS, plasma total lipid, andplasma neutral lipid fractions. In 17-week-old infants, the DHA levels in RBCPC and plasma phospholipid fractions were not correlated with VEPs measured at17 weeks. Based on blood samples and visual function testing at 52 weeks,significant correlations were found between VEP acuity and DHA in the RBCfractions, PE, and PI+PS (P 0.049 andP 0.008); however, no other lipidfraction demonstrated this relation. Significant correlations be-tween VEPacuity and DHA in RBC PE and RBC PI+PS at 52 weeks may reflect a preservationof DHA in these vital membrane phospholipids. We also found a signifi-
cant correlationbetween VEP and the ratio of 3-to- 6 end products, DHA to DPA 6, in total RBClipids (r ?0.46; P 0.003) and in RBC phospholipid fractions at 52 weeks.
The highest overallcorrelation was found between sweep VEP acuity measured at 52 weeks and DHA inthe total RBC lipid fraction at 17 weeks (r?0.54; P 0.00001; see Fig. 4).Statistical significance for compari-son of late (52-week) VEP acuity and early(17-week) DHA values was found in all lipid fractions measured (Table 8). Thus,accretion and composition of LCPUFAs in blood and membrane lipids early in life(i.e., <4 months of age) influenced functional performance later in life (1year).
TheVEP acuity did not correlate with the sum of saturated fatty acids, monounsaturatedfatty acids or 6LCPUFAs in total RBC lipids at either 17 or 52 weeks. In totalRBC lipids of 52-week-old infants, VEP acuities were significantly correlatedwith early (17-week) sums of 3LCPUFAs (r?0.54; P < 0.0005; n 58) as well as the ratios of6-to-3LCPUFAs (r 0.48; P < 0.0005) and the ratio of DHA toDPA 6 (r ?0.49; P < 0.0005). The unsat-uration index (UI) reporting the totalnumber of double bonds in total RBC lipids at 17 weeks was significantlycorrelated with VEP acuity at 52 weeks (r?0.34; P 0.008) but not at 17 weeks,further supporting the im-portance of the early dietary LCPUFA supply in latervisual function.
nalysis;
PC, phosphatidylcholine; PE,phosphatidylethanolamine; PI+PS, phosphatidylinositol+phosphatidylserine; PL,phospholipid; RBC, red blood cell; VEP, visual-evoked potentials.
DISCUSSION
Healthy term infantsreceiving commercial formula lost from 30% to 50% of their DHA body stores (asindexed by blood lipid fatty acid profiles) in the first 4 months of life,whereas breast-fed infants retained com-parable DHA levels from birth.Provision of experimen-tal formula enriched with 0.36% DHA and 0.72% AA to terminfants for the first 17 weeks of life resulted in blood lipid fatty acidprofiles that were balanced in 3-to- 6 LCPUFAs and closely matched that ofbreast-fed infants at 6, 17, and 52 weeks of age. Infants receiving dietaryLCPUFA supplementation had more mature reti-nal function, as determined by ERG,at 6 weeks and improved visual function, as determined by VEP, at 17 and 52weeks compared with CF-fed infants. Significant correlations were found betweenDHA levels and ERG function measured at 6 weeks and between DHA levels and VEPacuity measured at 17 weeks of age. Overall, total RBC lipid and RBC PEprovided the most consis-tent diet-related information and correlation resultsamong the RBC and plasma blood lipid fractions. The highest correlation betweenDHA and visual function was found with RBC total lipid DHA in 17-week-oldinfants and sweep VEP acuity in 52-week-old infants.
These data supportthe concept that early nutrition influences proper development of visualfunction later in life. In addition, it appears that the RBC total lipidfrac-tion from these infants provided the most meaningful information relatingDHA accretion in the body with neural and visual functional development.
Several biochemicalindices were consistent with de-velopment of a suboptimal fatty acid status ininfants provided standard CF. One primary indicator was a sig-nificantlyreduced level of blood lipid DHA, which may correspond to similar reductions inDHA of neural and retinal tissues. A second indicator was a reduction in themajor 6LCPUFA, AA, and despite low blood levels of LCPUFAs, the balance of3-to- 6 LCPUFAs was markedly skewed in the 3-deficient CF group. Of fur-
ther importance is a small but significantreduction in total fatty acid unsaturation (i.e., unsaturation index [UI]) inthe CF infant group compared with the breast-fed and two experimental dietgroups; UI in the CF group was also lower than in breast-fed infants at alltime points. Changes in fatty acid unsaturation have been known to influencethe function of various membrane-related en-zymes, receptors and nutrienttransport systems (12). More specifically, DHA has repeatedly been shown tomodify both biophysical and biochemical functions in neural and retinalmembranes (12,42), although the spe-cific mechanism impacting visualdevelopment remains unknown. The importance of unsaturation of cell mem-branefatty acids was underscored by the significant cor-relation between early (17week) fatty acid UI and VEP acuity in later development (52 weeks).
An additional markerof aberrant metabolism in the CF group was the marginally elevated level ofMead acid (20:3 9) and elevated ratio of Mead acid to AA in total RBC lipids.These indices are consistent with an excess dietary conversion of oleic acid(18:1 9) to Mead acid and have been reported in neonates by other investigators(24,43). Cord blood samples from all infants exhibited an elevation of the Meadacid-to-AA ratio in both total lip-ids from RBCs and plasma (mean, 0.035 ±0.014 [SD] for RBCs; 0.040 ± 0.023 for plasma). These values were above thelimit (>0.025) suggested by Siguel et al. (38) that identifies a status ofessential fatty acid insuffi-ciency. However, no infants reached the Meadacid-to-AA threshold ratio of 0.2 associated with severe defi-ciency ofessential fatty acids (37). By 6 weeks of age, only infants receiving CFwithout added LCPUFAs re-mained in the insufficiency state as defined by thisindex. At 6 weeks, infants in the CF group also had less mature ERG responses,which correlated with elevated Mead acid and Mead acid-to-AA ratios.
Researchers in the1970s and 1980s consistently re-ported lower blood levels of DHA in formula-fedinfants than in breast-fed infants (24,43,44). This difference was ofbiochemical interest but was not clinically significant,
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552 D. R. HOFFMAN ET AL.
because no physiological functional measureswere associated with the attenuated DHA content. In more recent studies(31,32,45,46), visual function was found to be influenced by the absence orpresence of 3LCPUFAs in preterm infant diets. Our research group reported thatboth retinal and visual maturation were sub-optimal in preterm infantsreceiving commercial formula (31,32,46). These formulas had high amounts of the6 essential fatty acid, LA, but low -LNA and no 3 or 6 LCPUFA. In 1992, more-LNA was included in infant diet formulations by major manufacturers to providead-ditional substrate for subsequent conversion to DHA. Al-though LA wasconsidered the most important essential fatty acid, its content was reduced tomatch that in human milk and to limit 3– 6 substrate competition.
Although it is stillprohibited in the United States (47), enrichment of preterm and term infant formulaswith LCPUFAs has been recommended by numerous world-wide organizations andcommittees setting guidelines for infant diets (48–50). The current resultsreinforce the importance of an optimized biochemical fatty acid pro-file andits association with functional performance in term infants. In addition, thesedata demonstrate that an optimized blood fatty acid profile in early life (4months of age) can have an impact on visual development later in life (1 yearof age).
Acknowledgments: The authors thank thenumerous pedia-tricians associated with Presbyterian and Columbia Medical CityHospitals for their cooperation, the many parents and in-fants for theirparticipation in this study, and Mead–Johnson Nutritional Division (Evansville,IN, U.S.A.) for providing the infant formulas used in the study.
This work wassupported in part by National Institute of Child Health and Human DevelopmentGrant HD22380.
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Clinical Quiz
Joseph F. Fitzgerald,NASPGN Clinical Quiz Editor
RiccardoTroncone, ESPGHAN Clinical Quiz Editor
Janet K. Harnsberger, Contributor
University of Utah, Salt Lake City, UtahU.S.A.
A l9-month-old girl was brought for pediatricgastroenterology consultation with a 2-week history of episodic, intense,colicky abdominal pain. Her appetite decreased progressively during the periodof illness, and she lost weight. It was reported that she had been evaluated inemergency rooms and physicians’ offices numerous times during the preceding 7months when she experienced similar symptoms. She enjoyed good health andweight gain during symptom-free periods. There was no recent history ofvomiting, diarrhea, or fever. Her medical history included treatment forsalmonellosis at 5 months of age.
She had an elevatedsedimentation rate (38 mm/hr) and metabolic acidosis on admission. An abdominalsonographic survey revealed an enlarged, multicystic ovary. Evaluation for anovarian neoplasm was negative, and another study produced normal results.Contrast radiography of the colon with reflux of contrast into the terminalileum revealed no disease. An enteroclysis study was performed, which revealeddelayed transit and dilated, fluid-filled loops of distal ileum. An abdominalcomputed tomographic (CT) scan with intravenous contrast was subsequentlyobtained (Fig. 1). What is the most likely diagnosis?
A. Intussusception
B. Crohn’s disease with fistula formation
C. Gastric duplication
D. Pancreatic cyst
E. Salmonella-associated subphrenicabscess
ANSWER: see page 571 FIG. 1.
J Pediatr Gastroenterol Nutr, Vol. 31, No. 5, November 2000
