A randomized controlled trial of early dietary supply of longchain polyunsaturated fatty acids and mental development in term infants

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1 A rndomized controlled tril of erly dietry supply of longchin polyunsturted ftty cids nd mentl development in term infnts Eileen E Birch* PhD; Shron Grfield MS; Dennis R Hoffmn PhD; Ricrdo Uuy MD PhD; Dvid G Birch PhD, Retin Foundtion of the Southwest, 90 N Centrl Expresswy, Suite 400, Dlls, TX 75231, USA. *Correspondence to first uthor t ddress ove. E-mil: eirch@retinfoundtion.org The effects of dietry docoshexenoic cid (DHA) supply during infncy on lter cognitive development of helthy term infnts were evluted in rndomized clinicl tril of infnt formul milk supplemented with 0.35% DHA or with 0.36% DHA nd 0.72% rchidonic cid (AA), or control formul which provided no DHA or AA. Fifty-six 18-month-old children (26 mle, 30 femle) who were enrolled in the tril within the first 5 dys of life nd fed the ssigned diet to 17 weeks of ge were tested using the Byley Scles of Infnt Development, 2nd edition (BSID-II) (Byley 1993) t the Retin Foundtion of the Southwest, Dlls, TX. These children hd lso een ssessed t 4 months nd 12 months of ge for lood ftty-cid composition, sweep visul evoked potentil (VEP) cuity, nd forced-choice preferentil looking (FPL) cuity (Birch et l. 1998). Supplementtion of infnt formul with DHA+AA ws ssocited with men increse of 7 points on the Mentl Development Index (MDI) of the BSID-II. Both the cognitive nd motor suscles of the MDI showed significnt developmentl ge dvntge for DHA- nd DHA+AA-supplemented groups over the control group. While similr trend ws found for the lnguge suscle, it did not rech sttisticl significnce. Neither the Psychomotor Development Index nor the Behvior Rting Scle of the BSID-II showed significnt differences mong diet groups, consistent with specific dvntge of DHA supplementtion on mentl development. Significnt correltions etween plsm nd RBC-DHA t 4 months of ge ut not t 12 months of ge nd MDI t 18 months of ge suggest tht erly dietry supply of DHA ws mjor dietry determinnt of improved performnce on the MDI. The role of specific nutrients present in humn milk in promoting optiml cognitive development is controversil (Beijers 1992, Crwford et l. 1992, Doing 1992, Doyle et l. 1992, Houghton 1992, Jcoson nd Jcoson 1992, Jmes 1992, Lucs nd Cole 1992, McArthur et l. 1992, Rice 1992, Wright nd Dery 1992). There re mny pulictions deling with the dvntges of restfeeding over formul feeding on lter mentl ptitude nd scholstic chievement scores; however, it is difficult in most studies to discern whether the dvntge of restfeeding resides in the nutrient composition of humn milk or is due to mternl vriles ssocited with the decision to restfeed (e.g. IQ, eduction level, socioeconomic sttus, mternl infnt interction) nd the ct of restfeeding itself (Hoefer nd Hrdy 1929, Rogerson nd Rogerson 1939, Pollock 1989, Bouchrd nd Lykken 19, Doyle et l. 1992, Jcoson nd Jcoson 1992, Lucs et l. 1992). Recently, in rndomized clinicl trils of infnt formul composition, there hs een evidence tht specific nutritionl component of humn milk, docoshexenoic cid (DHA), my confer n dvntge in cognitive development. DHA in the diet of preterm or term infnts hs een ssocited with higher mentl development scores (mesured on the Mentl Development Index [MDI] of the Byley Scles of Infnt Development) (Crlson 19, Dmli et l. 1996), higher psychomotor development scores (Brunet-Lezine Test) (Agostini et l. 1995), shorter-look durtions to novel stimuli on the Fgn test (Crlson nd Werkmn 1996), nd etter prolem-solving skills (Infnt Plnning Test) (Willts et l. 1998). On the other hnd, DHA in the infnt diet hs lso een reported to e ssocited with poorer psychomotor development scores (Byley Scles Psychomotor Development Index [PDI]) (Crlson 1993) nd poorer voculry comprehension nd production (McArthur Communictive Inventory) (Jnowsky et l. 1995). More commonly, rndomized clinicl trils of DHA supplementtion of preterm or term infnt formul milks hve employed mesures of visul cuity s indices of the functionl sttus of the rin (e.g. Birch et l. 1992; Crlson et l. 1996, ; Auestd et l. 1997). The focus on visul-cuity outcomes mkes sense from the point of view tht quntittive, relile, vlid techniques re ville for the infnt ge rnge, including visul evoked potentil cuity nd preferentil looking cuity (Birch 1989). It is importnt to keep in mind tht the motive for visul-cuity ssessment in studies of DHA supplementtion hs not een detection of visul deficits requiring ophthlmologicl cre or erly eductionl intervention ut, rther, the quntifiction of sutle differences in visul cuity s indirect mesures of differences in the mturtion of rin function. However, in the context of clinicl trils of infnt nutrition, it is unknown whether erly mesurement of infnt visul cuity is predictive of lter cognitive function. The primry im of our study ws to evlute the effects of dietry DHA supply during erly infncy on lter cognitive development of helthy term infnts in the context of rndomized doule-lind clinicl tril. A second im ws to determine whether erly mesures of infnt visul cuity re predictive of lter cognitive development. Method PARTICIPANTS Seventy-nine helthy term infnts orn in the Dlls, Texs re were enrolled in the rndomized clinicl tril within 174 Developmentl Medicine & Child Neurology 2000, 42:

2 the first 5 dys of life. All prticipnts were orn t 37 to 40 weeks postmenstrul ge s determined y erly sonogrm, dte of lst menstrul period, nd physicl nd neurodevelopmentl ssessment t irth. Only singleton irths with irthweights pproprite for gesttionl ge were included. Exclusion criteri were fmily history of milk-protein llergy, genetic or fmilil eye disese (e.g. hereditry retinl disese, strismus), vegetrin or vegn mternl dietry ptterns, mternl metolic disese, nemi, or infection, presence of congenitl mlformtion or infection, jundice, perintl sphyxi, meconium spirtion, nd ny perintl event which resulted in plcement of the infnt in the neontl intensive cre unit. Prents of eligile neontes were provided with rief informtion sheet out the dietry study only fter hospitl records noted tht they hd elected to formul feed. Informed consent ws otined from one or oth prents 24 to 96 hours fter irth nd efore the infnt s prticiption. This reserch protocol oserved the tenets of the Declrtion of Helsinki nd ws pproved y the Institutionl Review Bords of the University of Texs Southwestern Medicl Center (Dlls, TX), Presyterin Medicl Center (Dlls, TX), nd Medicl City Columi Hospitl (Dlls, TX). RANDOMIZATION Infnts were rndomized on the dy of enrollment (rnge, 0 to 4 dys; men 2.1; SD 1.0 ) to one of three diets, descried in the prgrph elow. Fmilies were recruited from two seprte hospitls to encourge ethnic nd socioeconomic diversity in the cohort; infnts recruited from oth hospitls were rndomized using single rndomiztion schedule t centrl loction. Demogrphics of the cohort hve een pulished previously nd demonstrte tht the rndomiztion ws effective in equlizing the three diet groups (Birch et l. 1998). A rief summry of demogrphics is provided in Tle I, including sex, rce, irthweight, nd prents eductionl level. Ech of the three diets ws msked y two color/numer codes, for totl of six possile diet ssignments for ech infnt. A locked rndomiztion schedule ws developed y the Med Johnson Reserch Center (Evnsville, IN) nd provided in seled envelopes to the study site. DIETS Study diets were Enfmil with iron, Enfmil with iron supplemented with 0.35% DHA (of totl ftty cids), or Enfmil with iron supplemented with 0.36% DHA nd 0.72% rchidonic cid (AA). A formul which provided oth DHA nd AA ws included in the protocol ecuse there ws some evidence in preterm infnts tht dietry DHA supplementtion might depress AA levels (Crlson et l. 1993, Uuy et l. 1994) nd/or lter growth (Crlson et l. 1993, 1996). All formuls provided pproximtely 15% linoleic cid (LA) nd 1.5% α-linolenic cid (LNA) (of totl ftty cids). DHA-supplemented nd DHA+AAsupplemented formuls contined single-cell oils, specificlly DHASCO nd ARASCO (Mrtek Biosciences, Columi, MD, USA). All formuls were provided in 946 ml redy-to-feed cns nd contined 14.7 g protein, 37.5 g ft, nd 69.0 g crohydrte nd 25 JK/L; ll nutrients met existing stndrds for commercil formul nd estlished Infnt Formul Act requirements. Assigned diets were fed exclusively until 17 weeks of ge. No solid foods were introduced efore 17 weeks. Beyond 17 weeks, the infnts received commercil formul (not provided y the study). As previously reported, loodlipid nlyses confirmed complince with the ssigned diets (Birch et l. 1998). GENERAL PROTOCOL Mentl development ws ssessed using the Byley Scles t 18 months of ge (± 2 weeks) y one of the uthors (SG), child psychologist specilizing in psychoeductionl testing who ws msked to the infnts diets nd other test results. As reported previously, these infnts lso hd een ssessed t 4 months nd 12 months of ge for lood fttycid composition, sweep visul evoked potentil (VEP) cuity, nd forced-choice preferentil looking (FPL) cuity (Birch et l. 1998). SAMPLE SIZE Smple sizes were estimted using the method descried y Rosner (19) for α=0.05 nd 1 β=0. to detect men differences in the MDI of the Byley Scles of 1SD or greter mong diet groups (SD=15). The finl smple size required t 18 months is 16 children. Anticipting 25% loss to followup over 18 months, we plnned recruitment of 25 infnts for ech of the three diet groups nd chieved enrollment of 26 to 27 per group. A flow chrt for enrollment nd follow-up is presented in Figure 1. Most of the infnts lost to follow-up dropped out of the study during the initil 6 weeks fter enrollment, due to the peditricins recommendtions to switch to soy protein-sed formul following symptoms suggestive of lctose or cow-milk protein intolernce (seven infnts). Of the 71 infnts who remined in the protocol to 6 weeks of ge, 58 (82%) completed the protocol until they were 18 months of ge. Two children were excluded from the study t the 18-month visit y the psychoeductionl consultnt; one ws not included ecuse of fmily history of Tle I: Demogrphics of the cohort Formul N Sex % White/ Men Mternl eduction Pternl eduction group (M/F) % minority irthweight (g) (% high school/ (% high school/ (SD) % college nd % college nd postgrdute) postgrdute) CF 20 9/ / (404) 35.0/ /85.0 DHA 17 7/ / (236) 35.3/ /76.5 DHA+AA 19 10/9 68.4/ (378) 31.6/ /84.2 CF, control; DHA, docoshexenoic cid; AA, rchidonic cid. Mentl Development nd Long-chin Polyunsturtes in Term Infnts Eileen E Birch et l. 175

3 lerning disility in two silings which ws not provided y the prents efore the 18-month visit, nd the second ws not included ecuse she ws uncoopertive with testing due to concurrent fmily crisis. Smple sizes t 18 months were 20 in the control formul group, 17 in the DHA-supplemented formul group, nd 19 in the DHA+AA-supplemented formul group. BAYLEY SCALES OF DEVELOPMENT The Byley Scles of Infnt Development, 2nd edition (BSID-II) (Byley 1993) were used to ssess mentl nd psychomotor development. This test consists of three scles. The MDI ssesses memory, prolem solving, discrimintion, clssifiction, lnguge, nd socil skills. The PDI ssesses control of gross nd fine muscle groups, including wlking, running, jumping, prehension, use of writing implements, nd imittion of hnd movements. Rw scores for the mentl nd psychomotor scles, sed on the numer of items successfully completed, re converted to the MDI nd PDI stndrd scores sed on the child s ge t the time of testing. In ddition, performnce on the mentl nd motor scles t 18 months cn e nlyzed y scoring developmentl ge on three fcets of the test (cognitive, lnguge, nd motor) to provide profile of reltive strengths nd weknesses. Men scores for the MDI nd PDI re with stndrd devition of 15. The Behvior Rting Scle (BRS) ssesses qulittive spects of the child s test-tking ehvior, including orienttion, emotionl regultion, nd qulity of movement. All BRS items re scored pproprite to the child s ge nd reported s centile scores; verge performnce is t the 50th centile, performnce elow the 25th centile is clssified s questionle, performnce elow the 10th centile is clssified s non-optiml. SWEEP VEP ACUITY VEP cuity ws ssessed ccording to the swept prmeter protocol developed y Norci nd collegues (Norci nd Tyler 1985, Norci 1993) using squre-wve grtings phsereversing t 6.6 Hz. Detils of the protocol hve een descried previously (Birch et l. 1998). Briefly, two ipolr plcements of O z versus O 1 nd O 2 were used to record (gin = to , 3dB cut-off t 1 nd Hz) the EEG which ws dptively filtered in rel time to isolte the VEP (397 Hz smpling rte). Amplitude nd phse of the response t the second hrmonic of the stimultion frequency ws clculted for ech chnnel. Noise ws mesured y determining the mplitude nd phse of the two djcent non-hrmonic frequencies. Grting cuity ws estimted with n utomted lgorithm which exmines signl-tonoise rtio nd phse coherence nd performs liner regression for the finl descending lim of the vector verged function (minimum of three trils; typiclly five trils) relting VEP second hrmonic mplitude (mplitude t the reversl frequency of 13.2 Hz) to sptil frequency. Sweep VEP cuities were expressed in logmar (log of the minimum ngle of resolution, e.g. 20/20 corresponds to minimum ngle of resolution of 1 minute rc nd logmar of 0.0 while 20/200 corresponds to minimum ngle of resolution of 10 minutes rc nd logmar of 1.0). FPL ACUITY Behviorl cuity ws estimted using inoculr two-lterntive forced-choice preferentil looking protocol (Birch nd Hle 1988). Detils of the protocol hve een descried previously (Birch et l. 1998). Briefly, ll test sessions egn with low sptil frequency (0.4 cycles/deg), with pproximte 0.5 octve steps etween successive stimuli. A two-down one-up Rndomized ptients N =79 Control formul N =26 DHA-supplemented formul N =26 DHA+AA-supplemented formul N =27 Withdrwn efore 6-week visit N =3 three formul intolernce Withdrwn efore 6-week visit N =4 two formul intolernce; one restfeeding; one illness unrelted to formul Withdrwn efore 6-week visit N =1 one formul intolernce Withdrwn fter 6-week visit N =3 three lost to follow-up Withdrwn fter 6-week visit N =5 three lost to follow-up; two excluded Withdrwn fter 6-week visit N =7 four lost to follow-up; three illnesses unrelted to formul Completed tril N =20 Completed tril N =17 Completed tril N =19 Figure 1: Flow chrt for enrollment in nd progress through the rndomized tril. 176 Developmentl Medicine & Child Neurology 2000, 42:

4 stircse procedure ws used to converge on the cuity threshold in 10 reversls nd cuity ws estimted y mximum likelihood estimtion (Swnson nd Birch 1992). FPL cuities were expressed in logmar. GROWTH Weight ws mesured using Helthometer peditric strin guge scle (Helthometer, Bridgeview, IL, USA) ccurte to 1 g. Length ws mesured using Ellrd Length Bords (Ellrd Instrumenttion Ltd., Settle, Wshington, USA) ccurte to 0.1 cm. Growth dt were normlized y expressing them s z scores for term infnts of the pproprite ge nd sex using the nutritionl nthropometry softwre ville t the Ntionl Center for Helth Sttistics (NCHS) wesite ( BLOOD LIPIDS Blood smples (2.0 ml) were collected in ethylene dimine tetrcetic cid (EDTA) microtiner tues t 4 nd 12 months of ge vi heel stick, ided y infnt heel wrming pcks. Plsm nd RBCs were seprted y centrifugtion, lipids extrcted, trnsmethylted with oron trifluoride/ methnol, nd methylesters nlyzed y cpillry gs chromtogrphy using flme ioniztion detection. Results were otined s mss concentrtion (µg/ml of pcked RBCs) sed on the ddition of n internl stndrd (10 µg 23:0 ftty cid). Fttycid peks were identified y comprison to GLC68+11 stndrd nd using custom softwre which semiutomted dt processing (Hoffmn nd Uuy 1992). STATISTICAL ANALYSES Comprisons mong diet groups for MDI nd BRS scores were conducted vi prmetric one-wy ANOVA; multiple comprisons mong groups were conducted using the Bonferroni method (Rosner 19). As PDI scores were not normlly distriuted, comprisons mong diet groups were conducted vi one-wy ANOVA of rnks; multiple comprisons mong groups were mde using Dunn s method (Rosner 19). As developmentl-ge scores on the cognitive, lnguge, nd motor fcets represent ordinl dt, comprisons mong diet groups were conducted vi onewy ANOVA of rnks; multiple comprisons mong groups were mde using Dunn s method. Correltions etween Byley Scles scores nd lood lipids, visul cuities, nd growth were clculted y Person product-moment correltions (r) if oth vriles pssed normlity tests (Kolmogorov Smirnov) or y Spermn rnk correltions (r s ) if one or oth vriles filed normlity tests. Results MENTAL DEVELOPMENT INDEX (MDI) The verge MDI score for ech diet group reltive to the normtive score of is presented in Figure 2 nd Tle II. Long-chin polyunsturted ftty cids (LCP) dietry supply during the first 4 months of life hd significnt effect on MDI scores t 18 months of ge (F 2,53 =3.18, P<0.05). Pirwise multiple comprisons showed tht the DHA+AA diet group hd significntly higher MDI scores thn the control formul group (t=2.51, P<0.05). Only one infnt in the control formul group nd one infnt in the DHA diet group showed ccelerted performnce on the MDI, i.e. hd score 115. Accelerted performnce on the MDI ws noted in five infnts in the DHA+AA diet group. Two of the infnts in the control formul group showed delyed performnce on the MDI, i.e. hd score 85. None of the infnts in the DHA or DHA+AA diet groups showed delyed performnce. As shown in Figure 3, the MDI score t 18 months of ge ws positively correlted with plsm DHA t 4 months of ge (r=0.32, P=0.016). There ws no significnt correltion etween plsm LA, LNA, AA, or EPA t 4 months nd the MDI score t 18 months of ge. As shown in Figure 3, RBC-DHA t 4 months ws positively correlted with the MDI score t 18 months (r=0.29, P=0.030). In ddition, the MDI score t 18 months ws negtively correlted with oth RBC-LA t 4 months (r= 0.30, P=0.023) nd RBC-LNA t 4 months of ge (r= 0.45, P=0.0004). There ws no significnt correltion etween RBC-AA or RBC-EPA t 4 months nd the MDI score t 18 months of ge. There were no significnt correltions etween plsm or RBC-LA, LNA, AA, EPA, or DHA t 12 months nd the MDI score t 18 months of ge. As shown in Figure 4, MDI score t 18 months ws negtively correlted with VEP cuity t 4 months of ge (r= 0.37, MDI PDI , Control DHA DHA+AA Diet group Control DHA DHA+AA Diet group Figure 2: Mens nd stndrd errors of MDI scores nd PDI scores reltive to the normtive score of t 18 months for three rndomized diet groups: control, DHA supplemented, nd DHA+AA supplemented. Significnt differences etween diet groups re indicted y different letter codes (, ). Mentl Development nd Long-chin Polyunsturtes in Term Infnts Eileen E Birch et l. 177

5 P=0.008), i.e. etter VEP cuity (lower logmar score) ws ssocited with etter MDI score. There ws no significnt correltion etween MDI score t 18 months nd FPL cuity t 4 months of ge (Figure 4). There ws no significnt correltion etween VEP cuity nor FPL cuity t 12 months nd the MDI score t 18 months. Tle II: Men nd stndrd errors for the Byley Scles of Infnt Development, 2nd edition Index Control DHA DHA+AA ANOVA MDI (men normtive score=) Men (SE) 1.7 (1.94) 2.4 (1.81) 5.6 (2.7) F 2,53 =3.18, P<0.05 Medin th to 75th centile 8.0 to to to 14.5 PDI (men normtive score=) Men + SE 1.4 (1.34) 0.6 (0.97) 1.7 (0.69) H 2 =4.05, P=0.13 Medin th to 75th centile 7.0 to to to 6.0 BRS (men normtive score=50) Men + SE 7.3 (23.7) 6.4 (20.9) 8.1 (24.6) F 2,53 =1.22, P=0.30 Medin th to 75th centile 7.0 to to to 33.0 * Groups with different superscripts (, ) re significntly different using the Bonferroni method r =0.32 P = r = 0.37 P =0.008 MDI MDI Plsm DHA t 4 months of ge (µg/ml) r =0.29 P =0.03 MDI MDI VEP cuity t 4 months of ge (logmar) r = 0.02 P = RBC-DHA t 4 months of ge (µg/ml) FPL cuity t 4 months of ge (logmar) Figure 3: Reltion etween plsm DHA () nd RBC-DHA () t 4 months of ge nd MDI stndrd score t 18 months of ge. The solid line shows the est fit to the dt y liner regression. Figure 4: Reltion etween sweep VEP cuity () nd FPL cuity () t 4 months of ge nd MDI stndrd score t 18 months of ge. The solid line shows the est fit to the dt y liner regression. 178 Developmentl Medicine & Child Neurology 2000, 42:

6 The MDI score t 18 months ws not correlted with normlized height, weight, or weight-for-length z scores t 4 months or 12 months of ge. PSYCHOMOTOR DEVELOPMENT INDEX (PDI) The verge PDI score for ech diet group reltive to the normtive score of is presented in Figure 2 nd Tle II. LCP dietry supply during the first 4 months of life filed to hve significnt effect on PDI scores t 18 months of ge (H 2 =4.05, P=0.13). None of the infnts in the control formul group or in the DHA diet group showed ccelerted performnce on the PDI. Two infnts showed ccelerted performnce on the PDI in the DHA+AA diet group. One infnt in the control formul group nd one infnt in the DHA+AA diet group showed delyed performnce on the PDI. None of the infnts in the DHA diet group showed delyed performnce. PDI score t 18 months of ge ws not significntly correlted with plsm or RBC-LA, LNA, AA, EPA, or DHA t 4 months of ge or t 12 months of ge. PDI score t 18 months ws negtively correlted with VEP cuity t 4 months of ge (see Fig. 5; r= 0.33, P=0.017), i.e. etter VEP cuity (lower logmar score) ws ssocited with etter PDI score. There ws no significnt correltion etween PDI score t 18 months nd FPL cuity t 4 months of ge (Fig. 5). There ws no significnt correltion etween VEP cuity nor FPL cuity t 12 months nd PDI score t 18 months. PDI score t 18 months ws not correlted with normlized height, weight, or weight-for-length z scores t 4 months. PDI score t 18 months ws positively correlted Tle III: Men nd stndrd errors for developmentl ge scores (in months) on the suscles of the Byley Scles of Infnt Development, 2nd edition Suscle Control DHA DHA+AA Men (SE) Men (SE) Men (SE) Cognitive 16.1 (0.32) 17.6 (0.24) 17.7 (0.18) Lnguge 16.0 (0.33) 16.8 (0.20) 17.1 (0.19) Motor 16.0 (0.24) 17.6 (0.21) 17.9 (0.16) * Groups with different superscripts (, ) re significntly different using Dunn s test for multiple comprisons. r = 0.33 P =0.017 r = 0.38 P =0.005 PDI BRS VEP cuity t 4 months of ge (logmar) VEP cuity t 4 months of ge (logmar) r = 0.04 P =0.73 r =0.04 P =0.76 PDI BRS FPL cuity t 4 months of ge (logmar) Figure 5: Reltion etween sweep VEP cuity () nd FPL cuity () t 4 months of ge nd PDI stndrd score t 18 months of ge. The solid line shows the est fit to the dt y liner regression FPL cuity t 4 months of ge (logmar) Figure 6: Reltion etween sweep VEP cuity () nd FPL cuity () t 4 months of ge nd BRS centile score t 18 months of ge. The solid line shows the est fit to the dt y liner regression. Mentl Development nd Long-chin Polyunsturtes in Term Infnts Eileen E Birch et l. 179

7 with normlized weight-for-length z scores t 12 months (r s =0.325, P=0.020) ut not with normlized weight or length z scores t 12 months. COGNITIVE, LANGUAGE, AND MOTOR SUBSCALES As shown in Tle III, developmentl ges on the cognitive suscle were significntly different mong diet groups (H 2 =11.24, P=0.004). On multiple comprison testing (Dunn s method), the control formul group performed significntly poorer thn oth the DHA diet group (Q=2.94, P<0.05) nd the DHA+AA diet group (Q=2.84, P<0.05). No significnt differences were found mong diet groups for developmentl ges on the lnguge suscle (see Tle II); the control, DHA, nd DHA+AA diet groups performed t the 16.0-, 16.8-, nd 17.1-month levels, respectively. Also shown in Tle II re the significnt differences found mong diet groups for developmentl ges on the motor suscle (H 2 =15.00, P<0.001). On multiple comprison testing (Dunn s method), the control formul group performed significntly poorer thn oth the DHA diet group (Q=3.64, P<0.05) nd the DHA+AA diet group (Q=2.93, P<0.05). BEHAVIORAL RATING SCALE (BRS) As shown in Tle II, BRS scores did not differ significntly mong diet groups (F 2,53 =1.22, P=0.30). Three infnts in the control formul group, two infnts in the DHA diet group, nd five infnts in the DHA+AA diet group performed ove the th centile. None of the infnts scored in the lowest 10th centile. There were no significnt correltions etween BRS scores t 18 months nd plsm or RBC-LA, LNA, AA, EPA, or DHA t 4 or 12 months of ge. As shown in Figure 6, the BRS score t 18 months ws negtively correlted with VEP cuity t 4 months of ge (r= 0.4, P=0.015); i.e. etter VEP cuity (lower logmar score) ws ssocited with etter BRS score. There ws no significnt correltion etween BRS score t 18 months nd FPL cuity t 4 months of ge (Fig. 6). There were no significnt correltions etween VEP cuity nor FPL cuity t 12 months nd BRS score t 18 months. BRS score t 18 months ws not correlted with normlized height, weight, or weight-for-length z scores t 4 months. BRS score t 18 months ws positively correlted with normlized weight-for-length z scores t 12 months (r=0.375, P=0.008) ut not with normlized weight or length z scores t 12 months. Discussion Supplementtion of term infnt formul milk with 0.36% DHA nd 0.72% AA during the first 4 months of life ws ssocited with men increse of 7 points on the Mentl Development Index of the Byley Scles t 18 months of ge over the control formul group. Power clcultions for this tril were initilly sed on the smple size necessry to detect 15-point difference (1SD) in the MDI nd PDI scores etween groups. However, our smple sizes t 18 months exceeded 15-per-diet group nd the stndrd devitions of mesurement were smller thn nticipted (MDI SD rnged from 8 to 11 nd PDI interqurtile intervls rnged from 7 to 10). The smller thn expected stndrd devitions my hve resulted from the more restrictive inclusion nd exclusion criteri for norml term infnts used in the present study versus the Byley normtive smple (see Method section), the segregtion of infnts y erly diet (unlike the Byley norms), the use of single exminer for ll Byley tests (unlike the Byley norms), or other unknown fctors. Due to the smller thn expected stndrd devitions, we were le to detect smller differences etween groups thn nticipted. It should e noted tht, while 7-point increse in the MDI is smll shift for n individul child reltive to the stndrd devition of the normtive smple, 7 points represents significnt shift in the men performnce of cohort of children. In ddition to higher MDI score in the DHA+AA-supplemented group, there ws trend towrd higher prevlence of ccelerted performnce on the MDI in the DHA+AA-supplemented group. Supplementtion of milk-sed term infnt formul with 0.36% DHA lone ws ssocited with men increse of 4 points on the MDI. Both the cognitive nd motor suscles of the MDI showed significnt developmentl ge dvntge for DHA nd DHA+AA-supplemented groups over the control group. While similr trend ws found for the lnguge suscle, it did not rech sttisticl significnce. These dt support long-term cognitive dvntge of infnt dietry DHA supply during the first 4 months of life. The significnt correltions etween plsm nd RBC-DHA t 4 months of ge ut not t 12 months of ge nd MDI t 18 months of ge support the hypothesis tht erly dietry supply of DHA ws significnt determinnt of improved performnce on the MDI. The significnt correltion etween sweep VEP cuity t 4 months of ge nd the MDI score t 18 months of ge provides support for the use of sweep VEP cuity s vlid outcome mesure for rin development during infncy in clinicl trils of infnt nutrition. FPL cuity t 4 months, on the other hnd, filed to show significnt correltion with MDI scores t 18 months. This could suggest tht FPL cuity is poor predictor of lter mentl development or, lterntely, it my e tht the higher vriility ssocited with FPL s compred with sweep VEP cuity mesurements limited our ility to detect correltion with the current smple sizes. Neither the PDI nor the BRS showed significnt differences mong diet groups, consistent with specific dvntge of DHA supplementtion on mentl development. Both scores were correlted, however, with sweep VEP cuity. As in erlier studies of dietry DHA supply in preterm infnts (Crlson 19, 1993), the PDI ws correlted with growth t 12 months of ge s ssessed y weight:length rtio z scores. A recent multicenter study of DHA supplementtion of infnt formul y Scott et l. (1998) reported no effect of providing dietry DHA during the first yer of life on the MDI scores t 12 months of ge. There re severl differences etween the study y Scott nd coworkers nd the present study tht my hve led to the different outcome. These include the lower level of DHA provided to infnts in the Scott study (0.1% to 0.2% of totl ftty cids versus 0.36% in the present study), different exclusion nd inclusion criteri, different ge t testing (12 months versus 18 months in the present study), the use of multiple exminers in the multicenter study y Scott et l. (which my increse vriility), nd the use of different versions of the Byley tests (the newer BSID-II hs more emphsis on cognitive tsks thn the originl version). While the Byley Scles of Infnt Development re stndrd clinicl ssessment tool, its predictive vlue for lter IQ nd scholstic chievement is high only if the study popultion includes children with delyed, norml, nd ccelerted 1 Developmentl Medicine & Child Neurology 2000, 42:

8 development. Our cohort contined only few children with delyed or ccelerted performnce ut most children were within norml limits (48 of 56). Thus, the predictive vlue for school-ge performnce my e limited. A reevlution of the cohort t 4 yers of ge, using the Wechsler Preschool nd Primry Scles of Intelligence Revised (WPPSI-R) (Wechsler 1989), is currently underwy to ddress the permnence of differences in cognitive function mong the three diet groups. Accepted for puliction 22nd Septemer Acknowledgements This project ws supported y NIH grnt HD223 nd supplementry funding ws provided y the Med Johnson Nutritionl Center (Evnsville, IN). Milk-sed infnt formuls were generously provided y the Med Johnson Nutritionl Center (Evnsville, IN). Importnt contriutions in the conduct of this study were mde y Yolnd Cstñed, BSN; Dinn Wheton, BS; Mrk Bne, PhD; Stephen Nusinowitz, PhD; Solnge Slomo, PhD; Deie Cornelius, BA; nd Jn Birenum, BS. 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