Early Human Development. 31 (1992) 41-51 Elsevier Scientific Publishers Ireland Ltd.
41
EHD 01346
Feeding preterm infants a formula containing CzO and Cz2 fatty acids simulates plasma phospholipid fatty acid composition of infants fed human milk M. Thomas Clandininapb’“, Arlene Parrottaab Johny E. Van AerdeaTd Arturo R. Hervadae and Eric Liene “Nutrition and Metabolism Research Group, University of Alberta, Edmonton, Alberta T6G 2C2, bDepartment of Foocis and Nutrition, University of Alberta, tionton. Alberta T6G 2C2, CDepartment of Medicine, University of Alberta, Edmonton, Alberta T6G 2C2. dDepartment of Pediatrics, University of Alberta, Ehonton. Alberta T6G 2C2 (Canada) and eWyeth Ayerst Research, Radnor. Pennsylvania (USA)
(Received 14 April 1992; revision received 14 July 1992; accepted 15 July 1992)
Summary Thirty-four premature infants weighing less than 1500 grams at birth were fed preterm formula (formula), preterm infant formula manufactured to contain a balance of CSOand Cz2 06 and w3 fatty acids within the range characteristic of human milk (LCPE-formula) or their mothers’ expressed breast milk (EBM). Blood samples were obtained during the first week of life and after 28 days of feeding to determine the effect of feeding CzOand Cz2 w6 and 03 fatty acids on plasma lipids. Fatty acid analyses of red blood cell phospholipids indicated few differences between dietary treatment and age. Fatty acid content of plasma cholesterol esters indicated a high plasma cholesterol linoleate level for infants fed formula and a reduced content of CzO and Cz2 w6 and w3 fatty acids. For infants fed the modified formula (LCPE-formula) the levels of 20:4w6, 20:5w3 and 22:6w3 were higher than observed for the formula group and similar to those observed for infants fed EBM. By the fifth week of life, feeding the modified formula resulted in plasma phospholipid levels of Czo and Cz2 w6 and w3 fatty acids similar to levels of C2s and Cz2 06 and w3 fatty acids found in infants fed EBM and significantly higher than levels characteristic of infants fed formula. It is concluded that infants fed LCPE-formula illustrate an overall balance between CzOand C22 06 to w3 fatty acids in the plasma similar to that characteristic of infants fed human milk. Correspondence to: M.T. Clandinin, Nutrition and Metabolism Research Group, 533 Newton Research Building, University of Alberta, Edmonton, Alberta T6G 2C2, Canada. 0378-3782/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
42 Key words:
infant; formula; plasma; phospholipid; essential fatty acid
Introduction The focus for composition of fats for infant feeding has been on optimizing fatty acid balance for energy and for provision of essential fatty acids, often in excessive proportions. The biological advantage and compositional model provided by feeding human milk has been examined [l] and indicates that human milk provides an optimal balance of fatty acids, particularly with respect to essential fatty acid balance [ 1,2]. During fetal development CzOand CZ206 and 03 fatty acids are utilized for synthesis of new tissue membranes [l-3]. Research has emphasized a need to provide these essential membrane components by addition of CZOand Cl2 fatty acids of either w6 or w3 type to infant formulas [1,3-61. Previous studies have supplemented low birth weight infant formulas with only CZOand Cz2 03 fatty acids [5,7-lo] and indicate that these fatty acids are incorporated into red blood cell membrane [5,7]. It has been questioned whether these changes in red blood cell membrane reflect change occurring in fatty acid composition in brain or retina membrane
t111. Metabolism of long chain polyunsaturated fatty acids derived from 18:2w6 and 18:3w3 is essential for synthesis of complex structural lipids, leukotrienes, thromboxanes and prostaglandins. Fetal accretion of long chain metabolites of w6 and w3 fatty acids may result from maternal or placental synthesis and transfer [ 12,131, or alternatively, the fetus may be capable of metabolizing 18:206 and 18:3w3 to longer chain homologues [14]. After birth the infant must synthesize fatty acids of CZOand CX2type derived from 18:2w6 and 18:303 or be fed these fatty acids 131. Limitation of this metabolic capability would be most critical to the very low birth weight infant due to the very low fatty acid reserves. Metabolism of 06 and 03 fatty acids is competitive for the same enzyme system such that alteration of w3 and 06 Cts fatty acid levels in the diet changes levels of metabolites of these precursor fatty acids in specific brain membranes in a manner that also influences activity of membrane lipid dependent functions [ 15- 181. Thus, it is logical to infer that the balance between the long chain w3 versus 06 fatty acids fed has considerable potential to result in an increase in brain membrane 03 fatty acid content when 20:5w3 is fed, while decreasing membrane fatty acid content for the homologous series of competing fatty acids (i.e. the w6 series). As 20:4w6 is also a quantitatively and qualitatively important fatty acid of brain phospholipid [21, significant reduction in brain levels of 20:4w6 may be less than optimal. The present study reports the effect of feeding low birth weight infants a triglyceride mixture providing a balanced fatty acid profile within the range of the CT0 and CZ2 06 and 1.03fatty acid content that is characteristic of human milk. Methods All procedures concerning the use of human subjects were approved by the Human Experimentation Review Committee of the University of Alberta Hospitals.
43
Study population and feeding design
Thirty-four premature infants who were appropriate for gestational age and weighing less than 1500 grams at birth were selected from admissions to the Neonatal Intensive Care Unit of the University of Alberta Hospitals. Informed written parental consent was obtained. As it is unethical to withhold breast milk because of randomization, allocation into human milk or formula feeding was based on maternal preference. Infants receiving oral nasogastric feedings within the first days of life and who were medically stable, without major respiratory or metabolic illness or severe congenital malformations were studied. Gestational age was confirmed by Dubowitz score [ 191and also by early ultrasound dating. All premature infants were considered healthy on enrolment. Infants attained full feeds by intermittent gavage feeding by day 5 postpartum on average. Seven male and five female infants received their own mother’s milk (180-200 ml/kg per day). Preterm mother’s milk (EBM) was expressed in hospital, or at home, frozen and delivered to the neonatal unit within 24 h. Five male and five female infants were fed a commercial preterm formula (formula; SMA-24; WyethAyerst), while six male and six female infants received modified preterm formula manufactured to contain CT0 and CZ2 w6 and w3 fatty acids (LCPE-formula; Table I). Infants receiving formula were fed apparent intakes (150 ml/kg per day) approximately similar to that for infants fed human milk. All infants were strictly maintained on their particular feeding protocol. Introduction of any other source of fat intake resulted in loss of that infant from the study. Infants requiring transfusions were excluded from the study. Infants also received routine daily supplements of 1500 IU Vitamin A, 400 IU Vitamin D and 30 mg Vitamin C. Supportive therapy (aminophylline, phototherapy and in some cases oxygen) was provided as required. Infants were nursed predominantly on their sides in an elevated position, in temperature controlled incubators at thermoneutral temperature with 60% humidity. The commercial formula (SMA-24) was chosen because it provides a fatty acid profile most similar to that of human milk. The formula was provided by WyethAyerst Laboratories, Radnor, Pennsylvania. A second formulation (LCPE SMA-24) of similar overall composition was prepared to contain Czo and CZ2w6 and 03 fatty acids (Table II). This formulation was made by Wyeth-Ayerst Laboratories by incorporating a fish oil triglyceride preparation containing CZOand CZ2w6 and 03 fatty acids into the fat blend used to prepare the formula (Table II). This formulation was subjected to the usual product control measures and analysis of the fatty acid content before and after feeding. Crown-heel length and occipitofrontal head circumference were measured on entry to the study and weekly. Body weight was measured daily. Measurements were made by one research nurse. A blood sample (1 .Oml) was obtained from a small arm vein of each infant during the first week of life as soon as parental consent could be obtained and after 4 weeks of feeding. Plasma and red blood cells were immediately separated and stored at -70°C under nitrogen until analysis. Lipid analysis
Lipid analysis was completed within a few days of sample collection. Lipids were
32.5 1530 40.6 28.8
12 zt 2.6 sz 150 f 2.2 f 240 i 2.1 f 2.6
10 31.0 1312 5.6 1260 38.9 21.2
zlz 1.2 f 150 f 2.2 f 169 ?? 1.9 f 2.9
12 30.9 1383 4.8 1300 39.5 28.1
12 30.1 1193 5.1 1120 37.1 25.7 2.6 257 2.2 238 2.7 1.7
EBM
Formula
LCPE-formula
EBM
zt f f f zt +
Fifth
First
Values represent the mean f standard deviation. Values with different superscripts are significantly different at P < 0.05.
Gestational age (weeks) Birth weight (g) Age (days) Body weight (g) Crown-heel length (cm) Head circumference (cm)
n
Characteristic
Subject age, body weight, length and head circumference at time of blood sampling.
TABLE I
f zt zt f
4.8 320a 2.4 2.0
34.2 2130 45.1 31.1
12
f 2.93 & 247b i 2.3 f 1.7
LCPE-formula
33.3 f 8.6 2040 ?? 345b 43.5 f 2.2 30.6 ?? 1.9
10
Formula
45 TABLE II Fatty acid composition of formula and formula modified to contain C20 and C22 06 and w3 fatty acids. Fatty acid (“/w/w) 6:0 8:0 lo:0 12:o 14:o 160 16:lw7 18:0 18:lw9 182~06 18:3w3 2o:o 2O:lw9 20~2~6 20.3~6 20:4w6 204~3 20~5~3 22:o 22:1w9 221206 22:4& 225~6 22:5w3 226~3 24:0 24:109 Ratio=
LCPE-formula 0
5.1 3.3 9.1 5.1 13.2 2.0 3.6 35.0 14.1 1.6 0.2 0.8 0.2 0.3 0.21 0.04 0.2 0.1 0.06 0.04 0.04 0.07 0.16 0.35 0.04 0.07 1.14
Formula 0.2 8.3 4.3 13.2 5.5 11.5 0.7 6.3 35.4 14.9 1.6 0.2 0.1 -
sRatio of Czs and Czz w6 to w3; it is approximately 1.4 in human milk [1,2]. For quantitation data on infant fatty acid intake see Ref. 2.
extracted from erythrocytes [20] and plasma lipids [21] in the presence of antioxidant (l-2 c(g of 2-ethoxyquin). Internal standards were added to enable quantitation of fatty acid content in each lipid class. Cholesterol pentadecanoate, tripentadecanoic acid and pentadecanoic acid were added as standards for determination of fatty acid content of plasma cholesterol ester, triglyceride and free fatty acid, respectively. Heptadecanoic acid was used to quantitate the separated phospholipid fraction. Phospholipids were separated [22], sprayed with 0.03% w/v 2 ’7 ’ dichlorofluorescein in 0.01 M NaOH for detection by comparison under UV light with appropriate standards. Plasma neutral lipid classes were separated and identified on silica gel G plates (Analtech Inc.) using petroleum:diethyl ether:acetic acid (80:20: 1 by volume) as solvent systems. Individual lipid classes were analyzed for fatty acid content after formation of fatty acid methyl esters [23].
46
Fatty acid methyl esters were stored at -70°C in a nitrogen atmosphere until analysis. Fatty acid methyl esters were separated and quantitated by automated gasliquid chromatography [18]. These analytical conditions are suitable for quantitation of all saturated-, mono-, di- and polyunsaturated fatty acids from Cs to Cz4 carbons in chain length [ 181. Statistical analysis Statistical analyses included preliminary oneway analysis of variance, multiple regression analysis and repeated measures analysis of variance to assess the effects of feeding and postnatal age. Significant differences were subsequently compared t241. RfSUltS
There were no significant differences in birth weight, length, head circumference or gestational age between EBM and formula-fed infants entering the study (Table I). The incidence of phototherapy, sodium bicarbonate or antibiotic therapy and oxygen requirements were similar for the three feeding groups. Clinical laboratory studies such as acid-base status, plasma and urine electrolytes and total plasma proteins and calcium remained within normal limits. The initial sample of blood was drawn as soon as parental consent was obtained (Table I). At this post
TABLE III Polyunsaturated fatty acid composition of red blood cell phospholipids. Fatty acid
Week one on entry (n = 34)
Phosphatidylethanolamine 18:2w6 3.03 * 0.21 2014~6 22.11 ?? 0.13 205~3 0.33 + 0.01 226~3 5.30 ?? 0.31 Phosphatidylcholine 18:206 12.10 f 0.90 20:406 9.35 f 0.52 20~5~3 0.24 * 0.04 226~3 1.15 ?? 0.12 Phosphatidylinositol 18:206 4.46 f 0.30 201406 14.29 f 0.88 20~503 0.32 f 0.23 226~3 2.08 f 0.14
Week five EBM (n = 12)
LCPE-formula (n = 12)
Formula (n = 10)
(“lo w/w) 3.8 f 0.4 21.8 ?? 0.9 0.68 f 0.08 5.5 ?? 0.4
4.9 20.1 0.81 5.0
f * * f
0.1 0.5 0.09 0.4
6.8 ?? 0.1 23.1 f 1.4 0.31 ?? 0.06 5.1 f 0.1
16.3 f 0.6 1.2 f 0.4 0.30 ?? 0.04 1.1 f 0.1
19.1 5.5 0.62 1.4
f f f f
0.5 0.3 0.09 0.1
23.4 5.8 0.25 0.8
f f f f
1.3 0.8 0.06 0.2
5.3 13.2 0.53 1.1
1.3 13.6 0.19 1.1
f f f f
0.1 1.1 0.08 0.1
1.1 19.1 0.19 3.4
f * f f
1.3 2.4 0.08 0.1
f 0.5 ZIZ1.5 f 0.24 f 0.3
Values illustrated represent the mean ?? S.E.M. While all fatty acid constituents were analyzed, only major essential fatty acid constituents are summarized above.
47
partum age, body weight, length and head circumference was similar between the three groups of infants (Table I). The final blood sample was drawn during the fifth week of life, after 4 further weeks of feeding (Table I). By this age formula-fed infants exhibited greater rates of growth in body weight compared to infants fed EBM (Table I). Linear growth was not apparently different between infants fed formula and LCPE-formula. As expected, fatty acid analyses of red blood cell phospholipids during the first and fifth week of life indicated few differences between dietary treatment and age in fatty acid composition (Table III). We found it difficult to conclude how the fatty acid composition of the red blood cell membrane phospholipids reflects diet and the infant’s overall essential fatty acid status during this period of life. Analysis of the quantitative fatty acid content of plasma cholesterol esters indicated an early effect of diet resulting in a high plasma cholesterol linoleate level for infants fed formula and a reduced content of CzOand Cz2 w6 and w3 fatty acids in the cholesterol ester fraction (Table IV). By the fifth week of life, infants fed EBM exhibited the highest plasma cholesterol ester content for all CzOand Cz2 w6 and w3 fatty acids. For infants fed the LCPE-formula the level of 20:4w6,20:5ti3 and 22:603 was higher than observed for the formula group and was similar to that observed for infants fed EBM (Table IV). The fatty acid composition of plasma triglyceride was also examined and reflected to some extent the overall fatty acid composition of the fats fed (data not shown). The quantitative fatty acid content of the plasma phospholipid fraction was determined (Table V) and indicated a high content of 18:2w6 in plasma phospholipids for infants fed formula. This finding is remarkable in that the 18:2w6 content of formula
TABLE IV Fatty acid content of infant plasma cholesterol ester fraction. Fatty acid (&ml)
C18:2w6 C20&6 C20:5w3 C22:603 C20 and 2206 C20 and 22~3 Total fatty acid
Week one (n = 34)
208.47 46.31 2.33 3.41 55.04 6.45 748
f 30.34 f 3.56 f 0.56 * 0.32 ?? 4.07 * 0.76 f 52.26
Week five EBM (n = 12)
LCPE-formula (n = 12)
Formula (n = 10)
263 53.5 2.7 3.9 61.6 8.8 794
277 33.7 3.9 3.8 41.7 8.8 650
351 f 44.8;’ 25.0 f 3.6*** 1.6 f 0.4** 1.6 f 0.5*** 29.9 f 4.1** 3.4 f 0.8** 810 f 106*
f 34.6 f 6.3 f 0.5 f 0.6 f 6.8 f 1.8 ?? 68.0
?? 21.1 f 4.6 f 0.8 f 0.6 f 5.1 f 1.3 f 67
Values illustrated represent the mean f S.E.M. in pg of fatty acid per ml of plasma. While all fatty acid constituents were determined only major essential fatty acid constituents are summarized above.. *Significant effect of feeding formula within week at P < 0.05. **Significant effect of feeding formula within week at P < 0.025. ***Significant effect of feeding formula within week at P < 0.001.
48 TABLE V Fatty acid content of infant plasma phospholipid fraction. Fatty acid (aglmt)
Week one (n = 34)
Week five EBM (n = 12)
Cl8:2w6 C20:406 C20:5w3 C22:603 C20 and 22~6 C20 and 22~3 Total Fatty Acid
221 * 150 * 4.57 f 30.51 * 198.19 * 37.64 * 1217.47 f
26.20 8.28 1.02 2.82 9.91 3.37 65.46
270 140 8.2 40.8 197 56.9 1230
f f * f f * f
3.5 40.4 3.8 3.2 9.6 4.6 83.7
LCPE-formula (n = 12)
Formula (n = 10)
285 f 102 f 13.2 f 38.0 * 163 * 57.6 * 1230 +
435 + 34.8* 82.2 + 9.0* 3.6 f 0.6* 13.2 * 2.3* 119 f 14.1* 19.5 ?? 3.2; 1448 * 104*
26.4 10.7 1.2 2.5 13.7 4.0 80.1
Values illustrated represent the mean f S.E.M. While all fatty acid constituents were analyzed, only major essential fatty acid constituents are summarized above. *Significant effects of feeding formula within week at P < 0.001.
is similar to that found in human milk and moreover, by modifying the fatty acid profile of formula by incorporation of long chain polyenoic essential fatty acids, this high level of 182~6 in the plasma phospholipid fraction is apparently reduced to be similar to that found in infants fed human milk. By the fifth week of life, feeding LCPE-formula normalized plasma phospholipid levels of Czs and C& w6 and w3 fatty acids to be similar to levels of those fatty acids found in infants fed EBM, significantly higher than levels characteristic of infants fed formula. Discussion In North America, marketed infant formulas do not contain homologues of 18:206 and 18:3w3. Inclusion of these longer chain homologues in formula fat blends has proven problematic as most sources of oil high in CZOand CZ2 w3 fatty acids and prepared in a manner acceptable for use in infant formulas contain little or no 204~6 or other CZOand CZ2w6 fatty acids. Thus, previous feeding studies have emphasized addition of a fish oil, MAXEPA [5,9]. Our search for a triglyceride source having potential to provide an optimal balance of long chain polyenoic fatty acids ruled out most marine oil sources on the basis of being too high in w3 fatty acids. Cultivation of fresh water cat fish provides a source of high quality oil for purification of a triglyceride that contains an appropriate balance of long chain polyenoic essential fatty acids when combined with the appropriate levels of other triglyceride sources used in the normal fat components of formula (modified-SMA; Table II). This modified SMA formula contained approximately half the 204~6 level typical of human milk, significant levels of other C 20 and Cz2 w6 fatty acids, an approximately equal balance between 20:4w6 and 205~3 and an overall ratio of C20 and CZ2w6 fatty acids to C2,-,and CZ2 w3 fatty acids of approximately 1.14 (Table I).
49
Feeding a long chain polyenoic essential fatty acid balance as similar as possible to that of human milk (LCPE-formula) results in synthesis of major plasma lipid fractions with similar fatty acid content to that observed for infants fed human milk. Based on the plasma cholesterol ester and phospholipid content of C20 and CZ2w6 and 03 fatty acids, it is apparent that the small intestine and liver of infants fed EBM or LCPE-formula produce lipids of fundamentally different composition compared to those fed formula. It is apparent that feeding the balance of CZOand ($2 06 and w3 fatty acids found in EBM or LCPE-formula results in incorporation of these fatty acids into phospholipids derived from the liver or perhaps small intestine. Whether or not tissues, such as developing brain and retina, utilize the essential fatty acid constituents derived from the plasma compartment and liver for structural membrane synthesis in a manner that also reflects similar clear diet-induced differences in fatty acid composition remains to be determined. In this regard, animal studies have indicated that dietary intakes which produce physiological change in membrane structure and function in the intestinal mucosa and liver [25,26] also result in differences in membrane composition and in transitions in the function of other tissues including specific brain membranes [reviewed by 15,16,25] and retina [26]. Such differences in function have recently been demonstrated for infants [27] and may be beneficial. To fully evaluate if the premature infant requires nutritional support with CZO and CZ206 and w3 fatty acids several parameters need to be evaluated. First, it is clear that feeding versus not feeding very long chain polyenoic essential fatty acids results in differences in the composition of a variety of fatty acid pools available for tissue uptake. Recent research in our laboratory indicates that the enterocyte in the brush border has desaturase activity [28] that responds by adaptation to physiological state and diet intake [29]. Thus, second, the role of enterocyte A6 and A5 desaturase activity in the infant’s small bowel in meeting the infant’s requirements for C,, and Cz2 w6 and 03 fatty acids must be examined. It is conceivable that while neural tissues may not synthesize the required CZOand Cz2 homologues of 18:2w6 and 18:3w3, the enterocyte may have capability to supply the required chain elongated desaturated essential fatty acids. Third, definition of fatty acid pools for uptake into neural and excitable tissues should be determined to enable provision of enrichment of CZOand C22 w6 and w3 constituents into the most appropriate pool for uptake. Finally, as structural membrane composition affects function, meaningful functional assessments will need to be defined to assess if differences in nutritional intake of CZOand CZ2w6 and w3 fatty acids alters functions at a physiological level [30]. It is concluded that infants fed LCPE-formula illustrate an overall balance between C2,, and CZ2w6 to 03 fatty acids in the plasma similar to that characteristic of infants fed human milk. Acknowledgements
The support of Wyeth Ayerst Research and the capable assistance of A. Wierzbicki (Technologist) and Brenda Young (Research Nurse) are gratefully acknowledged. J. Van Aerde is a Clinical Investigator and M.T. Clandinin is a Scholar of the Alberta Heritage Foundation for Medical Research.
50
References 1 Clandinin, M.T., Chappell, J.E. and Heim, T. (1981): Do low birthweight infants require nutrition with chain elongation-desaturation products of essential fatty acids? Prog. Lip. Res., 20, 901-904. 2 Clandinin, M.T., Chappell, J.E., Heim, T., Swyer, P.R. and Chance, G.W. (1981): Fatty acid utilization in perinatal de novo synthesis of tissues. Early Hum. Dev., 5, 355-366. 3 Clandinin, M.T., Chappell, J.E. and Van Aerde, J.E.E. (1989): Requirements of newborn infants for long chain polyunsaturated fatty acids. Acta Paediatr. Stand. Suppl., 351, 63-71. 4 Putnam, J.C., Carlson, S.E., DeVoe, P.W. and Barness, L.A. (1982): The effect of variations in dietary fatty acids on the fatty acid composition of erythrocyte phosphatidylcholine and phosphatidylethanolamine in human infants. Am. J. Clin. Nutr., 36, 106-l 14. 5 Carlson, S.E., Rhodes, P.G., Rao, V.S. and Goldgar, D.E. (1987): Effect of fish oil supplementation on the n-3 fatty acid content of red blood cell membranes in preterm infants. Pediatr. Res., 21, 507-510. 6 Koletzko, B., Schmidt, E., Bremer, H.J., Haug, M. and Harzer, G. (1989): Effects of dietary longchain polyunsaturated fatty acids on the essential fatty acid status of premature infants. Eur. J. Pediatr., 148, 69-75. 7 Carlson, SE., Rhodes, P.G. and Ferguson, M.G. (1986): Docosahexaenoic acid status of preterm infants at birth and following feeding with human milk or formula. Am. J. Clin. Nutr., 4, 798-804. 8 Carlson, S.E., Cooke, R.J., Rhodes, P.G., Peeples, J.M., We&man, S.H. and Tolley, E.A. (1991): Long term feeding of formulas high in linolenic acid and marine oil to very low birth weight infants: Phospholipid fatty acids. Pediatr. Res., 30, 404-412. 9 Liu, C.F., Carlson, S.E., Rhodes, P.G., Rao, V.S. and Meydrech, E.F. (1987): Increase in plasma phospholipid docosahexaenoic and eicosapentaenoic acids as a reflection of their intake and mode of administration. Pediatr. Res., 22, 292-296. 10 Uauy, R.D., Birch, D.G., Birch, E.E., Tyson, J.E. and Hoffman, D.R. (1990): Effect of dietary omega-3 fatty acids on retinal function of very-low-birth-weight neonates. Pediatr. Res., 28, 485-492. 11 Innis, SM., Foote, K.D., MacKinnon, M.J. and King, D.J. (1990): Plasma and red cell fatty acids of low birthweight infants fed their mothers expressed breast milk or preterm infant formula. Am. J. Clin. Nutr., 51, 994-1000. 12 Koletzko, B. and Muller, J. (1990): C&and rrans-isomeric fatty acids in plasma lipids of newborn infants and their mothers. Biol. Neonate, 57, 172-178. 13 Chambaz, J., Ravel, D., Manier, M.-C., Pepin, D., Mulliez, N. and Bereziat, G. (1985): Essential fatty acids interconversion in the human fetal liver. Biol. Neonate, 47, 136-140. 14 Clandinin, M.T., Chappell, J.E., Leong, S., Heim, T., Swyer, P.R. and Chance, G.W. (1980): Intrauterine fatty acid accretion rates in human brain: Implications for fatty acid requirements. Early Hum. Dev., 4, 121-129. 15 Foot, M., Cruz, T.F. and Clandinin, M.T. (1983): Effect of dietary lipid on synaptosomal acetylcholinesterase activity. Biochem. J., 211, 507-509. 16 Hargreaves, K. and Clandinin, M.T. (1987): Phosphocholine transferase activity in plasma membrane: Effect of diet. Biochem. Biophys. Res. Commun., 145 (1) 309-315. 17 Hargreaves, K. and Clandinin, M.T. (1989): Coordinate control of CDP-choline and phosphatidylethanolamine methyltransferase pathways for phosphatidylcholine biosynthesis occurs in response to change in diet fat. B&him. Biophys. Acta, 1001, 262-267. 18 Hargreaves, K. and Clandinin, M.T. (1987): Phosphatidylethanolamine methyltransferase: evidence for influence of diet fat on selectivity of substrate for methylation in rat brain synaptic plasma membranes. B&him. Biophys. Acta, 918, 97-105. 19 Dubowitx, L.M., Dubowitz, V. and Goldberg, C. (1970): Clinical assessment of gestational age in the newborn infant. J. Pediatr., 77, l-10. 20 Dodge, J.T. and Phillips, G.B. (1967): Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells. J. Lipid Res., 8, 667-675. 21 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957): A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226, 497-509.
51 22
Touchstone, J.C., Chen, J.C. and Beaver, K.M. (1980): Improved separation of phosphohpids in thin layer chromatography. Lipids, 15, 61-62. 23 Morrison, W.R. and Smith, L.M. (1964): Preparation of fatty acid methylesters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res., 5, 600-608. 24 Steel, R.G.D. and Torrie, J.H. (1960): Principles and Procedures of Statistics. McGraw-Hill Book Company Inc., Toronto. 25 Clandinin, M.T., Field, C.J., Hargreaves, K., Morson, L.A. and Zsigmond, E. (1985): Role of diet fat in subcellular structure and function. Can. J. Physiol. Pharmacol., 63, 546-556. 26 Connor, W.E. and Neuringer, M. (1988): The effects of n-3 fatty acid deficiency and repletion upon the fatty acid composition and function of the brain and retina. In: Biological Membranes: Aberrations in Membrane Structure and Function, pp. 275-94. Editors: M.L. Kamovsky, A. Lequaf and L.C. Bolis. Alan R. Liss, New York. 27 Carbon, S.E., Cooke, R., Werkman, S. and Peeples, J. (1989): Docosahexaenoate and eicosapentaenoate supplementation of preterm infants: effects on phospholipid DHA and visual acuity. FASEB J., 3, A1056. 28 Garg, M.L., Keelan, M., Thomson, A.B.R. and Clandinin, M.T. (1988): Fatty acid desaturation in the intestinal mucosa. B&him. Biophys. Acta, 958, 139-141. 29 Garg, M.L., Keelan, M., Thomson, A.B.R. and Clandinin, M.T. (1992): Desaturation of hnoleic acid in the small bowel is increased by short-term fasting and by dietary content of hnoleic acid. Biochim. Biophys. Acta, in press. 30 Clandinin, M.T. (1992): Summary comments made at Third International Congress on Essential Fatty Acids and Eicosanoids, Adelaide, Australia, February 29-March 4, in press.
41
EHD 01346
Feeding preterm infants a formula containing CzO and Cz2 fatty acids simulates plasma phospholipid fatty acid composition of infants fed human milk M. Thomas Clandininapb’“, Arlene Parrottaab Johny E. Van AerdeaTd Arturo R. Hervadae and Eric Liene “Nutrition and Metabolism Research Group, University of Alberta, Edmonton, Alberta T6G 2C2, bDepartment of Foocis and Nutrition, University of Alberta, tionton. Alberta T6G 2C2, CDepartment of Medicine, University of Alberta, Edmonton, Alberta T6G 2C2. dDepartment of Pediatrics, University of Alberta, Ehonton. Alberta T6G 2C2 (Canada) and eWyeth Ayerst Research, Radnor. Pennsylvania (USA)
(Received 14 April 1992; revision received 14 July 1992; accepted 15 July 1992)
Summary Thirty-four premature infants weighing less than 1500 grams at birth were fed preterm formula (formula), preterm infant formula manufactured to contain a balance of CSOand Cz2 06 and w3 fatty acids within the range characteristic of human milk (LCPE-formula) or their mothers’ expressed breast milk (EBM). Blood samples were obtained during the first week of life and after 28 days of feeding to determine the effect of feeding CzOand Cz2 w6 and 03 fatty acids on plasma lipids. Fatty acid analyses of red blood cell phospholipids indicated few differences between dietary treatment and age. Fatty acid content of plasma cholesterol esters indicated a high plasma cholesterol linoleate level for infants fed formula and a reduced content of CzO and Cz2 w6 and w3 fatty acids. For infants fed the modified formula (LCPE-formula) the levels of 20:4w6, 20:5w3 and 22:6w3 were higher than observed for the formula group and similar to those observed for infants fed EBM. By the fifth week of life, feeding the modified formula resulted in plasma phospholipid levels of Czo and Cz2 w6 and w3 fatty acids similar to levels of C2s and Cz2 06 and w3 fatty acids found in infants fed EBM and significantly higher than levels characteristic of infants fed formula. It is concluded that infants fed LCPE-formula illustrate an overall balance between CzOand C22 06 to w3 fatty acids in the plasma similar to that characteristic of infants fed human milk. Correspondence to: M.T. Clandinin, Nutrition and Metabolism Research Group, 533 Newton Research Building, University of Alberta, Edmonton, Alberta T6G 2C2, Canada. 0378-3782/92/$05.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
42 Key words:
infant; formula; plasma; phospholipid; essential fatty acid
Introduction The focus for composition of fats for infant feeding has been on optimizing fatty acid balance for energy and for provision of essential fatty acids, often in excessive proportions. The biological advantage and compositional model provided by feeding human milk has been examined [l] and indicates that human milk provides an optimal balance of fatty acids, particularly with respect to essential fatty acid balance [ 1,2]. During fetal development CzOand CZ206 and 03 fatty acids are utilized for synthesis of new tissue membranes [l-3]. Research has emphasized a need to provide these essential membrane components by addition of CZOand Cl2 fatty acids of either w6 or w3 type to infant formulas [1,3-61. Previous studies have supplemented low birth weight infant formulas with only CZOand Cz2 03 fatty acids [5,7-lo] and indicate that these fatty acids are incorporated into red blood cell membrane [5,7]. It has been questioned whether these changes in red blood cell membrane reflect change occurring in fatty acid composition in brain or retina membrane
t111. Metabolism of long chain polyunsaturated fatty acids derived from 18:2w6 and 18:3w3 is essential for synthesis of complex structural lipids, leukotrienes, thromboxanes and prostaglandins. Fetal accretion of long chain metabolites of w6 and w3 fatty acids may result from maternal or placental synthesis and transfer [ 12,131, or alternatively, the fetus may be capable of metabolizing 18:206 and 18:3w3 to longer chain homologues [14]. After birth the infant must synthesize fatty acids of CZOand CX2type derived from 18:2w6 and 18:303 or be fed these fatty acids 131. Limitation of this metabolic capability would be most critical to the very low birth weight infant due to the very low fatty acid reserves. Metabolism of 06 and 03 fatty acids is competitive for the same enzyme system such that alteration of w3 and 06 Cts fatty acid levels in the diet changes levels of metabolites of these precursor fatty acids in specific brain membranes in a manner that also influences activity of membrane lipid dependent functions [ 15- 181. Thus, it is logical to infer that the balance between the long chain w3 versus 06 fatty acids fed has considerable potential to result in an increase in brain membrane 03 fatty acid content when 20:5w3 is fed, while decreasing membrane fatty acid content for the homologous series of competing fatty acids (i.e. the w6 series). As 20:4w6 is also a quantitatively and qualitatively important fatty acid of brain phospholipid [21, significant reduction in brain levels of 20:4w6 may be less than optimal. The present study reports the effect of feeding low birth weight infants a triglyceride mixture providing a balanced fatty acid profile within the range of the CT0 and CZ2 06 and 1.03fatty acid content that is characteristic of human milk. Methods All procedures concerning the use of human subjects were approved by the Human Experimentation Review Committee of the University of Alberta Hospitals.
43
Study population and feeding design
Thirty-four premature infants who were appropriate for gestational age and weighing less than 1500 grams at birth were selected from admissions to the Neonatal Intensive Care Unit of the University of Alberta Hospitals. Informed written parental consent was obtained. As it is unethical to withhold breast milk because of randomization, allocation into human milk or formula feeding was based on maternal preference. Infants receiving oral nasogastric feedings within the first days of life and who were medically stable, without major respiratory or metabolic illness or severe congenital malformations were studied. Gestational age was confirmed by Dubowitz score [ 191and also by early ultrasound dating. All premature infants were considered healthy on enrolment. Infants attained full feeds by intermittent gavage feeding by day 5 postpartum on average. Seven male and five female infants received their own mother’s milk (180-200 ml/kg per day). Preterm mother’s milk (EBM) was expressed in hospital, or at home, frozen and delivered to the neonatal unit within 24 h. Five male and five female infants were fed a commercial preterm formula (formula; SMA-24; WyethAyerst), while six male and six female infants received modified preterm formula manufactured to contain CT0 and CZ2 w6 and w3 fatty acids (LCPE-formula; Table I). Infants receiving formula were fed apparent intakes (150 ml/kg per day) approximately similar to that for infants fed human milk. All infants were strictly maintained on their particular feeding protocol. Introduction of any other source of fat intake resulted in loss of that infant from the study. Infants requiring transfusions were excluded from the study. Infants also received routine daily supplements of 1500 IU Vitamin A, 400 IU Vitamin D and 30 mg Vitamin C. Supportive therapy (aminophylline, phototherapy and in some cases oxygen) was provided as required. Infants were nursed predominantly on their sides in an elevated position, in temperature controlled incubators at thermoneutral temperature with 60% humidity. The commercial formula (SMA-24) was chosen because it provides a fatty acid profile most similar to that of human milk. The formula was provided by WyethAyerst Laboratories, Radnor, Pennsylvania. A second formulation (LCPE SMA-24) of similar overall composition was prepared to contain Czo and CZ2w6 and 03 fatty acids (Table II). This formulation was made by Wyeth-Ayerst Laboratories by incorporating a fish oil triglyceride preparation containing CZOand CZ2w6 and 03 fatty acids into the fat blend used to prepare the formula (Table II). This formulation was subjected to the usual product control measures and analysis of the fatty acid content before and after feeding. Crown-heel length and occipitofrontal head circumference were measured on entry to the study and weekly. Body weight was measured daily. Measurements were made by one research nurse. A blood sample (1 .Oml) was obtained from a small arm vein of each infant during the first week of life as soon as parental consent could be obtained and after 4 weeks of feeding. Plasma and red blood cells were immediately separated and stored at -70°C under nitrogen until analysis. Lipid analysis
Lipid analysis was completed within a few days of sample collection. Lipids were
32.5 1530 40.6 28.8
12 zt 2.6 sz 150 f 2.2 f 240 i 2.1 f 2.6
10 31.0 1312 5.6 1260 38.9 21.2
zlz 1.2 f 150 f 2.2 f 169 ?? 1.9 f 2.9
12 30.9 1383 4.8 1300 39.5 28.1
12 30.1 1193 5.1 1120 37.1 25.7 2.6 257 2.2 238 2.7 1.7
EBM
Formula
LCPE-formula
EBM
zt f f f zt +
Fifth
First
Values represent the mean f standard deviation. Values with different superscripts are significantly different at P < 0.05.
Gestational age (weeks) Birth weight (g) Age (days) Body weight (g) Crown-heel length (cm) Head circumference (cm)
n
Characteristic
Subject age, body weight, length and head circumference at time of blood sampling.
TABLE I
f zt zt f
4.8 320a 2.4 2.0
34.2 2130 45.1 31.1
12
f 2.93 & 247b i 2.3 f 1.7
LCPE-formula
33.3 f 8.6 2040 ?? 345b 43.5 f 2.2 30.6 ?? 1.9
10
Formula
45 TABLE II Fatty acid composition of formula and formula modified to contain C20 and C22 06 and w3 fatty acids. Fatty acid (“/w/w) 6:0 8:0 lo:0 12:o 14:o 160 16:lw7 18:0 18:lw9 182~06 18:3w3 2o:o 2O:lw9 20~2~6 20.3~6 20:4w6 204~3 20~5~3 22:o 22:1w9 221206 22:4& 225~6 22:5w3 226~3 24:0 24:109 Ratio=
LCPE-formula 0
5.1 3.3 9.1 5.1 13.2 2.0 3.6 35.0 14.1 1.6 0.2 0.8 0.2 0.3 0.21 0.04 0.2 0.1 0.06 0.04 0.04 0.07 0.16 0.35 0.04 0.07 1.14
Formula 0.2 8.3 4.3 13.2 5.5 11.5 0.7 6.3 35.4 14.9 1.6 0.2 0.1 -
sRatio of Czs and Czz w6 to w3; it is approximately 1.4 in human milk [1,2]. For quantitation data on infant fatty acid intake see Ref. 2.
extracted from erythrocytes [20] and plasma lipids [21] in the presence of antioxidant (l-2 c(g of 2-ethoxyquin). Internal standards were added to enable quantitation of fatty acid content in each lipid class. Cholesterol pentadecanoate, tripentadecanoic acid and pentadecanoic acid were added as standards for determination of fatty acid content of plasma cholesterol ester, triglyceride and free fatty acid, respectively. Heptadecanoic acid was used to quantitate the separated phospholipid fraction. Phospholipids were separated [22], sprayed with 0.03% w/v 2 ’7 ’ dichlorofluorescein in 0.01 M NaOH for detection by comparison under UV light with appropriate standards. Plasma neutral lipid classes were separated and identified on silica gel G plates (Analtech Inc.) using petroleum:diethyl ether:acetic acid (80:20: 1 by volume) as solvent systems. Individual lipid classes were analyzed for fatty acid content after formation of fatty acid methyl esters [23].
46
Fatty acid methyl esters were stored at -70°C in a nitrogen atmosphere until analysis. Fatty acid methyl esters were separated and quantitated by automated gasliquid chromatography [18]. These analytical conditions are suitable for quantitation of all saturated-, mono-, di- and polyunsaturated fatty acids from Cs to Cz4 carbons in chain length [ 181. Statistical analysis Statistical analyses included preliminary oneway analysis of variance, multiple regression analysis and repeated measures analysis of variance to assess the effects of feeding and postnatal age. Significant differences were subsequently compared t241. RfSUltS
There were no significant differences in birth weight, length, head circumference or gestational age between EBM and formula-fed infants entering the study (Table I). The incidence of phototherapy, sodium bicarbonate or antibiotic therapy and oxygen requirements were similar for the three feeding groups. Clinical laboratory studies such as acid-base status, plasma and urine electrolytes and total plasma proteins and calcium remained within normal limits. The initial sample of blood was drawn as soon as parental consent was obtained (Table I). At this post
TABLE III Polyunsaturated fatty acid composition of red blood cell phospholipids. Fatty acid
Week one on entry (n = 34)
Phosphatidylethanolamine 18:2w6 3.03 * 0.21 2014~6 22.11 ?? 0.13 205~3 0.33 + 0.01 226~3 5.30 ?? 0.31 Phosphatidylcholine 18:206 12.10 f 0.90 20:406 9.35 f 0.52 20~5~3 0.24 * 0.04 226~3 1.15 ?? 0.12 Phosphatidylinositol 18:206 4.46 f 0.30 201406 14.29 f 0.88 20~503 0.32 f 0.23 226~3 2.08 f 0.14
Week five EBM (n = 12)
LCPE-formula (n = 12)
Formula (n = 10)
(“lo w/w) 3.8 f 0.4 21.8 ?? 0.9 0.68 f 0.08 5.5 ?? 0.4
4.9 20.1 0.81 5.0
f * * f
0.1 0.5 0.09 0.4
6.8 ?? 0.1 23.1 f 1.4 0.31 ?? 0.06 5.1 f 0.1
16.3 f 0.6 1.2 f 0.4 0.30 ?? 0.04 1.1 f 0.1
19.1 5.5 0.62 1.4
f f f f
0.5 0.3 0.09 0.1
23.4 5.8 0.25 0.8
f f f f
1.3 0.8 0.06 0.2
5.3 13.2 0.53 1.1
1.3 13.6 0.19 1.1
f f f f
0.1 1.1 0.08 0.1
1.1 19.1 0.19 3.4
f * f f
1.3 2.4 0.08 0.1
f 0.5 ZIZ1.5 f 0.24 f 0.3
Values illustrated represent the mean ?? S.E.M. While all fatty acid constituents were analyzed, only major essential fatty acid constituents are summarized above.
47
partum age, body weight, length and head circumference was similar between the three groups of infants (Table I). The final blood sample was drawn during the fifth week of life, after 4 further weeks of feeding (Table I). By this age formula-fed infants exhibited greater rates of growth in body weight compared to infants fed EBM (Table I). Linear growth was not apparently different between infants fed formula and LCPE-formula. As expected, fatty acid analyses of red blood cell phospholipids during the first and fifth week of life indicated few differences between dietary treatment and age in fatty acid composition (Table III). We found it difficult to conclude how the fatty acid composition of the red blood cell membrane phospholipids reflects diet and the infant’s overall essential fatty acid status during this period of life. Analysis of the quantitative fatty acid content of plasma cholesterol esters indicated an early effect of diet resulting in a high plasma cholesterol linoleate level for infants fed formula and a reduced content of CzOand Cz2 w6 and w3 fatty acids in the cholesterol ester fraction (Table IV). By the fifth week of life, infants fed EBM exhibited the highest plasma cholesterol ester content for all CzOand Cz2 w6 and w3 fatty acids. For infants fed the LCPE-formula the level of 20:4w6,20:5ti3 and 22:603 was higher than observed for the formula group and was similar to that observed for infants fed EBM (Table IV). The fatty acid composition of plasma triglyceride was also examined and reflected to some extent the overall fatty acid composition of the fats fed (data not shown). The quantitative fatty acid content of the plasma phospholipid fraction was determined (Table V) and indicated a high content of 18:2w6 in plasma phospholipids for infants fed formula. This finding is remarkable in that the 18:2w6 content of formula
TABLE IV Fatty acid content of infant plasma cholesterol ester fraction. Fatty acid (&ml)
C18:2w6 C20&6 C20:5w3 C22:603 C20 and 2206 C20 and 22~3 Total fatty acid
Week one (n = 34)
208.47 46.31 2.33 3.41 55.04 6.45 748
f 30.34 f 3.56 f 0.56 * 0.32 ?? 4.07 * 0.76 f 52.26
Week five EBM (n = 12)
LCPE-formula (n = 12)
Formula (n = 10)
263 53.5 2.7 3.9 61.6 8.8 794
277 33.7 3.9 3.8 41.7 8.8 650
351 f 44.8;’ 25.0 f 3.6*** 1.6 f 0.4** 1.6 f 0.5*** 29.9 f 4.1** 3.4 f 0.8** 810 f 106*
f 34.6 f 6.3 f 0.5 f 0.6 f 6.8 f 1.8 ?? 68.0
?? 21.1 f 4.6 f 0.8 f 0.6 f 5.1 f 1.3 f 67
Values illustrated represent the mean f S.E.M. in pg of fatty acid per ml of plasma. While all fatty acid constituents were determined only major essential fatty acid constituents are summarized above.. *Significant effect of feeding formula within week at P < 0.05. **Significant effect of feeding formula within week at P < 0.025. ***Significant effect of feeding formula within week at P < 0.001.
48 TABLE V Fatty acid content of infant plasma phospholipid fraction. Fatty acid (aglmt)
Week one (n = 34)
Week five EBM (n = 12)
Cl8:2w6 C20:406 C20:5w3 C22:603 C20 and 22~6 C20 and 22~3 Total Fatty Acid
221 * 150 * 4.57 f 30.51 * 198.19 * 37.64 * 1217.47 f
26.20 8.28 1.02 2.82 9.91 3.37 65.46
270 140 8.2 40.8 197 56.9 1230
f f * f f * f
3.5 40.4 3.8 3.2 9.6 4.6 83.7
LCPE-formula (n = 12)
Formula (n = 10)
285 f 102 f 13.2 f 38.0 * 163 * 57.6 * 1230 +
435 + 34.8* 82.2 + 9.0* 3.6 f 0.6* 13.2 * 2.3* 119 f 14.1* 19.5 ?? 3.2; 1448 * 104*
26.4 10.7 1.2 2.5 13.7 4.0 80.1
Values illustrated represent the mean f S.E.M. While all fatty acid constituents were analyzed, only major essential fatty acid constituents are summarized above. *Significant effects of feeding formula within week at P < 0.001.
is similar to that found in human milk and moreover, by modifying the fatty acid profile of formula by incorporation of long chain polyenoic essential fatty acids, this high level of 182~6 in the plasma phospholipid fraction is apparently reduced to be similar to that found in infants fed human milk. By the fifth week of life, feeding LCPE-formula normalized plasma phospholipid levels of Czs and C& w6 and w3 fatty acids to be similar to levels of those fatty acids found in infants fed EBM, significantly higher than levels characteristic of infants fed formula. Discussion In North America, marketed infant formulas do not contain homologues of 18:206 and 18:3w3. Inclusion of these longer chain homologues in formula fat blends has proven problematic as most sources of oil high in CZOand CZ2 w3 fatty acids and prepared in a manner acceptable for use in infant formulas contain little or no 204~6 or other CZOand CZ2w6 fatty acids. Thus, previous feeding studies have emphasized addition of a fish oil, MAXEPA [5,9]. Our search for a triglyceride source having potential to provide an optimal balance of long chain polyenoic fatty acids ruled out most marine oil sources on the basis of being too high in w3 fatty acids. Cultivation of fresh water cat fish provides a source of high quality oil for purification of a triglyceride that contains an appropriate balance of long chain polyenoic essential fatty acids when combined with the appropriate levels of other triglyceride sources used in the normal fat components of formula (modified-SMA; Table II). This modified SMA formula contained approximately half the 204~6 level typical of human milk, significant levels of other C 20 and Cz2 w6 fatty acids, an approximately equal balance between 20:4w6 and 205~3 and an overall ratio of C20 and CZ2w6 fatty acids to C2,-,and CZ2 w3 fatty acids of approximately 1.14 (Table I).
49
Feeding a long chain polyenoic essential fatty acid balance as similar as possible to that of human milk (LCPE-formula) results in synthesis of major plasma lipid fractions with similar fatty acid content to that observed for infants fed human milk. Based on the plasma cholesterol ester and phospholipid content of C20 and CZ2w6 and 03 fatty acids, it is apparent that the small intestine and liver of infants fed EBM or LCPE-formula produce lipids of fundamentally different composition compared to those fed formula. It is apparent that feeding the balance of CZOand ($2 06 and w3 fatty acids found in EBM or LCPE-formula results in incorporation of these fatty acids into phospholipids derived from the liver or perhaps small intestine. Whether or not tissues, such as developing brain and retina, utilize the essential fatty acid constituents derived from the plasma compartment and liver for structural membrane synthesis in a manner that also reflects similar clear diet-induced differences in fatty acid composition remains to be determined. In this regard, animal studies have indicated that dietary intakes which produce physiological change in membrane structure and function in the intestinal mucosa and liver [25,26] also result in differences in membrane composition and in transitions in the function of other tissues including specific brain membranes [reviewed by 15,16,25] and retina [26]. Such differences in function have recently been demonstrated for infants [27] and may be beneficial. To fully evaluate if the premature infant requires nutritional support with CZO and CZ206 and w3 fatty acids several parameters need to be evaluated. First, it is clear that feeding versus not feeding very long chain polyenoic essential fatty acids results in differences in the composition of a variety of fatty acid pools available for tissue uptake. Recent research in our laboratory indicates that the enterocyte in the brush border has desaturase activity [28] that responds by adaptation to physiological state and diet intake [29]. Thus, second, the role of enterocyte A6 and A5 desaturase activity in the infant’s small bowel in meeting the infant’s requirements for C,, and Cz2 w6 and 03 fatty acids must be examined. It is conceivable that while neural tissues may not synthesize the required CZOand Cz2 homologues of 18:2w6 and 18:3w3, the enterocyte may have capability to supply the required chain elongated desaturated essential fatty acids. Third, definition of fatty acid pools for uptake into neural and excitable tissues should be determined to enable provision of enrichment of CZOand C22 w6 and w3 constituents into the most appropriate pool for uptake. Finally, as structural membrane composition affects function, meaningful functional assessments will need to be defined to assess if differences in nutritional intake of CZOand CZ2w6 and w3 fatty acids alters functions at a physiological level [30]. It is concluded that infants fed LCPE-formula illustrate an overall balance between C2,, and CZ2w6 to 03 fatty acids in the plasma similar to that characteristic of infants fed human milk. Acknowledgements
The support of Wyeth Ayerst Research and the capable assistance of A. Wierzbicki (Technologist) and Brenda Young (Research Nurse) are gratefully acknowledged. J. Van Aerde is a Clinical Investigator and M.T. Clandinin is a Scholar of the Alberta Heritage Foundation for Medical Research.
50
References 1 Clandinin, M.T., Chappell, J.E. and Heim, T. (1981): Do low birthweight infants require nutrition with chain elongation-desaturation products of essential fatty acids? Prog. Lip. Res., 20, 901-904. 2 Clandinin, M.T., Chappell, J.E., Heim, T., Swyer, P.R. and Chance, G.W. (1981): Fatty acid utilization in perinatal de novo synthesis of tissues. Early Hum. Dev., 5, 355-366. 3 Clandinin, M.T., Chappell, J.E. and Van Aerde, J.E.E. (1989): Requirements of newborn infants for long chain polyunsaturated fatty acids. Acta Paediatr. Stand. Suppl., 351, 63-71. 4 Putnam, J.C., Carlson, S.E., DeVoe, P.W. and Barness, L.A. (1982): The effect of variations in dietary fatty acids on the fatty acid composition of erythrocyte phosphatidylcholine and phosphatidylethanolamine in human infants. Am. J. Clin. Nutr., 36, 106-l 14. 5 Carlson, S.E., Rhodes, P.G., Rao, V.S. and Goldgar, D.E. (1987): Effect of fish oil supplementation on the n-3 fatty acid content of red blood cell membranes in preterm infants. Pediatr. Res., 21, 507-510. 6 Koletzko, B., Schmidt, E., Bremer, H.J., Haug, M. and Harzer, G. (1989): Effects of dietary longchain polyunsaturated fatty acids on the essential fatty acid status of premature infants. Eur. J. Pediatr., 148, 69-75. 7 Carlson, SE., Rhodes, P.G. and Ferguson, M.G. (1986): Docosahexaenoic acid status of preterm infants at birth and following feeding with human milk or formula. Am. J. Clin. Nutr., 4, 798-804. 8 Carlson, S.E., Cooke, R.J., Rhodes, P.G., Peeples, J.M., We&man, S.H. and Tolley, E.A. (1991): Long term feeding of formulas high in linolenic acid and marine oil to very low birth weight infants: Phospholipid fatty acids. Pediatr. Res., 30, 404-412. 9 Liu, C.F., Carlson, S.E., Rhodes, P.G., Rao, V.S. and Meydrech, E.F. (1987): Increase in plasma phospholipid docosahexaenoic and eicosapentaenoic acids as a reflection of their intake and mode of administration. Pediatr. Res., 22, 292-296. 10 Uauy, R.D., Birch, D.G., Birch, E.E., Tyson, J.E. and Hoffman, D.R. (1990): Effect of dietary omega-3 fatty acids on retinal function of very-low-birth-weight neonates. Pediatr. Res., 28, 485-492. 11 Innis, SM., Foote, K.D., MacKinnon, M.J. and King, D.J. (1990): Plasma and red cell fatty acids of low birthweight infants fed their mothers expressed breast milk or preterm infant formula. Am. J. Clin. Nutr., 51, 994-1000. 12 Koletzko, B. and Muller, J. (1990): C&and rrans-isomeric fatty acids in plasma lipids of newborn infants and their mothers. Biol. Neonate, 57, 172-178. 13 Chambaz, J., Ravel, D., Manier, M.-C., Pepin, D., Mulliez, N. and Bereziat, G. (1985): Essential fatty acids interconversion in the human fetal liver. Biol. Neonate, 47, 136-140. 14 Clandinin, M.T., Chappell, J.E., Leong, S., Heim, T., Swyer, P.R. and Chance, G.W. (1980): Intrauterine fatty acid accretion rates in human brain: Implications for fatty acid requirements. Early Hum. Dev., 4, 121-129. 15 Foot, M., Cruz, T.F. and Clandinin, M.T. (1983): Effect of dietary lipid on synaptosomal acetylcholinesterase activity. Biochem. J., 211, 507-509. 16 Hargreaves, K. and Clandinin, M.T. (1987): Phosphocholine transferase activity in plasma membrane: Effect of diet. Biochem. Biophys. Res. Commun., 145 (1) 309-315. 17 Hargreaves, K. and Clandinin, M.T. (1989): Coordinate control of CDP-choline and phosphatidylethanolamine methyltransferase pathways for phosphatidylcholine biosynthesis occurs in response to change in diet fat. B&him. Biophys. Acta, 1001, 262-267. 18 Hargreaves, K. and Clandinin, M.T. (1987): Phosphatidylethanolamine methyltransferase: evidence for influence of diet fat on selectivity of substrate for methylation in rat brain synaptic plasma membranes. B&him. Biophys. Acta, 918, 97-105. 19 Dubowitx, L.M., Dubowitz, V. and Goldberg, C. (1970): Clinical assessment of gestational age in the newborn infant. J. Pediatr., 77, l-10. 20 Dodge, J.T. and Phillips, G.B. (1967): Composition of phospholipids and of phospholipid fatty acids and aldehydes in human red cells. J. Lipid Res., 8, 667-675. 21 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957): A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226, 497-509.
51 22
Touchstone, J.C., Chen, J.C. and Beaver, K.M. (1980): Improved separation of phosphohpids in thin layer chromatography. Lipids, 15, 61-62. 23 Morrison, W.R. and Smith, L.M. (1964): Preparation of fatty acid methylesters and dimethylacetals from lipids with boron fluoride-methanol. J. Lipid Res., 5, 600-608. 24 Steel, R.G.D. and Torrie, J.H. (1960): Principles and Procedures of Statistics. McGraw-Hill Book Company Inc., Toronto. 25 Clandinin, M.T., Field, C.J., Hargreaves, K., Morson, L.A. and Zsigmond, E. (1985): Role of diet fat in subcellular structure and function. Can. J. Physiol. Pharmacol., 63, 546-556. 26 Connor, W.E. and Neuringer, M. (1988): The effects of n-3 fatty acid deficiency and repletion upon the fatty acid composition and function of the brain and retina. In: Biological Membranes: Aberrations in Membrane Structure and Function, pp. 275-94. Editors: M.L. Kamovsky, A. Lequaf and L.C. Bolis. Alan R. Liss, New York. 27 Carbon, S.E., Cooke, R., Werkman, S. and Peeples, J. (1989): Docosahexaenoate and eicosapentaenoate supplementation of preterm infants: effects on phospholipid DHA and visual acuity. FASEB J., 3, A1056. 28 Garg, M.L., Keelan, M., Thomson, A.B.R. and Clandinin, M.T. (1988): Fatty acid desaturation in the intestinal mucosa. B&him. Biophys. Acta, 958, 139-141. 29 Garg, M.L., Keelan, M., Thomson, A.B.R. and Clandinin, M.T. (1992): Desaturation of hnoleic acid in the small bowel is increased by short-term fasting and by dietary content of hnoleic acid. Biochim. Biophys. Acta, in press. 30 Clandinin, M.T. (1992): Summary comments made at Third International Congress on Essential Fatty Acids and Eicosanoids, Adelaide, Australia, February 29-March 4, in press.
