Early Human Development, 30 (1992) 183-191
183
E1se.vie.rScientific Publishers Ireland Ltd. EHD 01317
Long chain polyunsaturated fatty acids and fetal growth Alison A. Leafa, Martin J. Leighfieldb, Kate L. Costeloea and Michael A. Crawford b ‘Joint Academic Department of Child Health, Medical College of St Bartholomew, London ECIA, and bInstitute of Brain Chemistry and Human Nutrition, Hackney Hospital, London E9 (UK)
(Received 24 January 1992; revision received 24 April 1992; accepted 7 May 1992)
Long chain polyunsaturated fatty acid composition of plasma choline phosphoglycerides has been measured at birth in 22 preterm infants. Positive correlations were found between both n-6 and n-3 fatty acids and measurements of growth and maturation. 20:4(n-6) and the sum of 20:3(n-6) + 20:4(n-6) correlated most strongly with weight and head circumference, while 22:6(n-3) showed strongest correlation with length of gestation. These findings are of relevance to understanding the role of nutrition in fetal growth and in establishing the group of infants most at risk of postnatal deficiency of essential fatty acids. Key words: n-6; n-3; long chain polyunsaturated
fatty acids; preterm infant; growth
Intmductlon
Arachidonic (20:4(n-6)) and docosahexaenoic (22:6(n-3)) acids are important structural components of cell membrane phospholipids. Arachidonic acid is quantitatively the most important long chain polyunsaturated fatty acid (LCPUFA) and is widespread throughout the body, although concentration is particularly high in brain and vascular endothelium. Docosahexaenoic acid, on the other hand has a highly specific distribution, representing the predominant membrane fatty acid of synaptosomes, retinal photoreceptors, mitochondria and spermatozoa, but being scarce in other tissues [ 11. Correspondence to: Alison A. Leaf, Senior Registrar, Newborn Centre, Monash Medical Centre, Locked bag 29, Clayton, Victoria 3168, Australia. 0378-3782l9USO5.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
184
During fetal life LCPUFAs are actively accumulated across the placenta to the fetus, in preference to their precursors [2] and the concentration of circulating LCPUFAs in plasma phospholipids is higher in newborn infants than in their mothers [3,4]. There is considerable variation in the LCPUFA composition of plasma phospholipids at birth in preterm infants [4,5]. Linoleic acid( 18:2(n-6)) and alpha-linolenic acid (18:3(n-3)) are both 18-carbon essential fatty acids (EFAs) and are the substrates for synthesis of LCPUFAs. Each gives rise to a separate group of long-chain derivatives by a series of chain elongation and desaturation reactions. The predominant n-6 LCPUFAs, derived from linoleic acid, are dihomo-gamma linolenic acid (20:3(n-6)) and arachidonic acid (20:4(n-6)) and the predominant n-3 LCPUFAs, derived from alpha-linolenic acid are eicosapentaenoic acid (20:5(n-3)) and docosahexaenoic acid (22:6(n-3)). As well as their structural role 20:3(n-6), 20:4(n-6) and 20:5(n-3) have an important metabolic function as precursors of prostaglandins and other eicosanoids. Choline phosphoglycerides are the predominant phospholipids in plasma, representing approximately 70% of the total [6]. We have studied the relationships between LCPUFA composition of this phospholipid class and measurements of fetal growth and maturity. Methods Znfants Samples were obtained from 22 infants with weight below 2000 g at birth. The only criterion for exclusion was obvious congenital anomaly and the group included one pair of non-identical twins. Blood was taken from the umbilical vein immediately after delivery of the placenta, or from the infant on arrival at the neonatal unit. Gestational age ranged from 24-36 weeks and was assessed by maternal menstrual history and ultrasound scan before twenty weeks gestation. If this information was not available gestational age was determined by clinical examination of the infant at birth [7]. Weight was measured by midwives at delivery using electronic scales and recorded to the nearest 5 g. Head circumference (HC) was recorded within the first 48 h of life by the same observer (AL), using a fresh paper tape for each infant. Eight infants were considered small for gestational age (SGA) with birthweight below the tenth percentile and of these eight five had “proportionate” growth retardation, with HC also below the tenth percentile and three had disproportionate growth, with HC above the tenth percentile [8]. Clinical details are given in Table I. Biochemical methods Blood samples were centrifuged for 15 rnin at 5000 rev./mm and plasma separated and stored at -20°C. Lipids were extracted by the method initially described by Folch et al. [9], using chloroform/methanol 2:1, with 100 mg 1-l of BHT (2,[6]-ditert-Butyl-p-cresol). Phosphoglyceride classes were separated by thin-layer chromatography on silica plates. The solvent used contained chloroform, methanol and water in the ratio: 60:30:4, with added BHT, 100 mg 1-t. Phospholipid classes were detected using dichlorofluorescein and identified using standards. The methylating
185 TABLE I Clinical details. Means with ranges in parentheses shown except for percentage fatty acid composition where latter is standard deviation. Infant group
n
GA (weeks)
Birth weight (g)
Head circumference (cm)
20:4(n-6) (%)
22:6(n-3) (%)
All infants
22
AGAa
14
SGAb
8
SGA-dispC
3
SGA-propd
5
30.5 (24-36) 29.4 (24-33) 32.5 (26-36) 34.0 (31-36) 31.6 (26-35)
1367 (600-1985) 1379 (600-1985) 1346 (680-1730) 1431 (1145-1730) 1295 (680- 1705)
27.6 (21.2-31.2) 27.3 (21.2-31.2) 28.0 (22.5-30.9) 29.9 (28.4-30.9) 26.8 (22.5-29.3)
16.52 (3.15) 16.70 (3.62) 16.21 (2.29) 17.58 (0.62) 15.39 (2.59)
4.49 (1.82) 3.93 (1.29) 5.48 (2.26) 5.51 (2.09) 5.47 (2.80)
‘AGA, appropriate for gestational age weight > 10th percentile. bSGA, small for gestational age weight < 10th percentile. ‘SGA-disp, HC > 10th percentile. dSGA-prop, weight and HC c 10th percentile.
reagent used was methanol and acetyl chloride, 15: 1 solution, at 80°C for 3 h, under nitrogen. Fatty acid methyl esters were separated by gas-liquid chromatography on a 25 m superox 0.53 mm i.d. column (Thames Chromatography) at 190°C using hydrogen as carrier gas and a flame ionisation detector. Signals were quantified by automatic integrator and fatty acids identified by relative chain length and comparison with standards. Individual fatty acids have been quantified as percentages of the total fatty acids in choline phosphoglycerides. Statistical
methods
The relationships between fatty acid composition and continuous variables: birthweight, HC and gestational age were analysed by linear regression. The relative importance of significant variables was measured using a stepwise multiple regression model. Comparisons between AGA and SGA infants were made using Student’s two-tailed t-test and between proportionate and disproportionate SGA using Mann-Whitney U Test. Calculations were made using “Microstat” software, Ecosoft Inc. 1984. This study was approved by the Ethical Committee of City and Hackney Health Authority. Results A summary of polyunsaturated fatty acid composition of plasma choline phosphoglycerides at birth is listed in Table II. The predominant n-6 fatty acids are
186 TABLE II Fatty acid composition of plasma choline phosphoglycerides, infants expressed as percentage of total fatty acids
mean (SD.), at birth in all 22 preterm
n-6 18:2 18:3 20:2 20:3 20:4 2214 225 Total LCPUFA 18:2/20:4
8.31 (3.27) 0.21 (0.17) 1.18 (1.17) 3.43 (1.28) 16.52 (3.15) 0.52 (0.24) 0.53 (0.34) 22.19 (4.43) 0.53 (0.28)
n-3 18:3 20:5 2215 2216 Total LCPUFA
0.07 (0.03) 0.68 (0.49) 0.33 (0.30) 4.49 (1.82) 5.51 (2.11)
TABLE III Correlation coefficients for the relationships between measurements of growth and maturation at birth and polyunsaturated fatty acids of plasma choline phosphoglycerides. Fatty acid
Birthweight
HC
Gestation
n-6 18:2 20~3 20:4 20:3+20:4 Total LCPUFA
0.217 0.443. 0.564** 0.576** 0.569**
0.193 0.515” 0.597*** 0.643*** 0.625***
0.200 0.394 0.371 0.411 0.393
n-3 18:3 22~6 Total LCPUFA ?? P < 0.05. **P < 0.01. ***p < 0.005.
-0.090 0.496* 0.405
-0.024 0.589** 0.530**
-0.153 0.638*** 0.586**
187
20 : 3n-6 + 20 : 4n-6 and HEAD CIRCUMFERENCE Plasma choline phosphoglyceride 30 28
.
r I 0.643 p ? ? 0.001
20
22
24
26
Head Clrcumfennce
26
30
32
(cm)
Fig. I. Relationship between 20:3(n-6) + 20:4(n-6) and head circumference: 0, AGA; SGA; 0, disproportionate SGA.
?? , proportionate
18:2(n-6), 20:3(n-6) and 20:4(n-6) and the only n-3 fatty acid present in significant amount is 22:6@3). Correlation coefficients for the relationship between specific fatty acids and growth variables are shown in Table III. Significant positive correlations were found between 20:3(n-6) and 20:4(n-6) and both weight and HC. Even stronger relationships were found between sum of 20:3(n-6) + 20:4(n-6) and total n-6 LCPUFAs and growth measurements (Fig. 1). There was no statistically significant relationship between linoleic acid and growth measurements, nor between n-6 fatty acids and gestational age. 22:6(n-3) showed a strong correlation with gestational age (Fig.2) and also with HC and birthweight. The sum of all n-3 LCPUFAs (20:5(n-3), 22:5(n-3) and 22:6(n-3)) also showed a positive correlation with both head circumference and gestational age. There were no significant differences between mean birthweight and HC in SGA and AGA infants. The SGA group were however significantly more mature at birth, P < 0.05.
There was no difference in n-6 LCPUFAs between SGA and AGA infants. 22:6(n-3) was higher in the SGA group, P = 0.051. Using a multiple regression
22 : 6n-3 and GESTATIONAL AGE Plasma choline phosphoglyceride 12 11 -
r = 0.638 p = 0.001
10 -
98765432 -HO
0
lo-
’
24
I
26
I
I
I
26 30 32 Gestational Age (wks)
I 34
Fig. 2. Relationship between 22:6(n-3) and gestational age: 0, AGA; disproportionate SGA.
I
36 ?? , proportionate
SGA; 0,
model to study the inter-related effects of length of gestation, birthweight and intrauterine growth retardation, the latter two became insignificant. Partial r-squared for the three variables were 0.41, P = 0.001, 0.002 and 0.03, respectively. No significant differences between fatty acid composition of proportionate and disproportionate SGA infants was found. Discussion These data show the lowest percentages of LCPUFAs in the smallest and least mature infants and identify differences between the n-6 and n-3 fatty acids. n-6 Fatty acids related particularly to growth measurements, both birthweight and head circumference. In the initial description of n-6 fatty acid deficiency growth failure was a major feature [lO,l l] and has subsequently been shown to result in decreased cell division and reduced weight of body and brain in rats [ 12,131. A correlation between n-6 LCPUFAs and birthweight has recently been described in plasma triglycerides of preterm infants at four days of age [14]. In addition to weight our data show a strong correlation between n-6 LCPUFA and HC and also between n-3 fatty acids and growth.
189
The strongest correlation of 22:6(n-3), however, was with gestational age. A relationship between long chain n-3 fatty acids and gestational age has previously been reported [15]. Women in the Faroe Islands, where intake of n-3 LCPUFAs in fish is high, had a higher content of DHA in their erythrocyte phospholipids than women from The Netherlands or Canada. Faroese women give birth to infants with higher than average birthweight and longer gestation. The authors propose that the underlying mechanism may be an alteration in the balance of prostaglandin synthesis. High circulating n-3 fatty acids may inhibit production of the dienoic prostaglandins derived from arachidonic acid which stimulate uterine contraction, thus prolonging gestation. Our data may reflect the opposite end of the spectrum: with low 22:6(n-3) in the infants of shortest gestation. A strong correlation was found between 22:6(n-3) and HC, which is of interest as the highest concentration of 22:6(n-3) is found in the brain. Relationships between fatty acid composition and fetal growth may have a physiological or pathological basis. The highest requirement for cell membrane components would be expected to coincide with the time of most rapid growth. In the human fetus increase in body mass and brain size is greatest during the latter half of pregnancy. It is possible that at the time of birth of the smallest, least mature infants in our study the peak of LCPUFA requirement has not yet occurred. Friedman et al, 1978, described an increase in all LCPUFA in cord blood plasma phospholipids from 24 to 44 weeks gestation [4]. It was suggested that this was due to an increased demand for brain growth in the third trimester, supporting a physiological explanation for the gradual increase. Alternatively, the lower LCPUFA concentration seen in the very low-birthweight infants may have a pathological basis. Three factors might be involved: maternal circulating fatty acids which reflect dietary intake, placental function and fetal metabolism. Inadequate maternal diet has been associated with low birthweight [16]. Low intake of EFAs in all trimesters of pregnancy was shown to correlate with low birthweight. Furthermore significantly lower plasma and red cell content of arachidonic acid was found in both the mothers and infants where weight was below 2.5 kg at birth, compared with those in whom birthweight was above 3.0 kg [ 171. In this study no distinction was made between prematurity and IUGR as the cause for low birthweight. Even in normal low-risk pregnancies low levels of maternal LCPUFA have recently been described [ 181, suggesting that current western dietary habits may not provide the optimum amount or proportions of EFAs for reproduction. Placental function is important in transfer of LCPUFAs from mother to fetus [2]. The placenta also has complex metabolic and endocrine roles, including the control of vasomotor tone and myometrial contractility, mediated by prostaglandins. Intrauterine growth retardation (IUGR) associated with placental dysfunction tends to occur later in pregnancy and to spare head growth, resulting in disproportionate SGA infants, whereas IUGR with an earlier onset tends to affect head growth and result in proportionate SGA infants, Despite showing a relationship between head circumference and both n-6 and n-3 LCPUFAs, our data did not reveal any significant differences in LCPUFAs between SGA and AGA infants, nor between propor-
190
tionate and disproportionate SGA infants. The numbers in these groups were, however, small. Fatty acid composition of umbilical arterial wall has however shown features of EFA deficiency which were most marked in the smallest growth retarded infant [ 171. It is possible that these differences between umbilical artery and cord blood reflect a latent deficiency, all available EFAs and LCPUFAs being taken for growth of essential organs such as brain, at the expense of less vital tissue, ie the umbilical artery. Also in umbilical artery, Ongari et al. observed lower 20:4(n-6), increased triene-tetraene ratio, suggestive of EFA deficiency and reduced prostacyclin synthesis, in infants who were SGA in association with maternal hypertension [19]. In agreement with our findings, low 20:4(n-6) and decreased prostacyclin were related to weight and not to gestational age. There is little information available on EFA metabolism in the human fetus. Some elongation and desaturation of l&carbon EFAs may occur in liver, or possibly in brain, as has been demonstrated in brain cells of fetal rats [20]. The principal source of LCPUFA, however, appears to be selective transfer across the placenta [2,21]. We have shown a relationship between fetal growth measurements and circulating LCPUFAs. The smallest, least mature infants are born with the lowest circulating levels of these essential fatty acids and yet are the infants in whom the requirement of LCPUFAs for brain growth will be greatest. Whether the differences are pathological or physiological requires further study of maternal levels and placental handling of EFAs in relation to fetal growth. The fact that levels are low and that n-3 and n-6 LCPUFAs relate to different aspects of growth and maturation has important implications for the understanding of developmental biology and also for the nutritional requirements of preterm infants. Acknowledgements
We wish to thank the staff of the Special Care Baby Unit at the Hornet-ton Hospital for their cooperation and the EC, Barclay Trust, Nestle and Vlaardingen Research, Unilever for financial support. References 1
2 3 4 5 6 7
Salem, N., Kim H.-Y. and Yergey, J.A. (1986): Docosahexaenoic acid: membrane function and metabolism. In: Health Effects of Polyunsaturated Fatty Acids in Seafood, pp. 263-317. Editors: A.P. Simopoulos, R.R. Kifer and R.E. Martin. Academic Press, Orlando, Florida. Kuhn, D. and Crawford, M. (1986): Placental Essential fatty acid transport and prostaglandin synthesis. Prog. Lipid Res., 23, 345-353. Olegard, R. and Svennerholm, L. (1970): Fatty acid composition of plasma and red cell phosphoglycerides in full term infants and their mothers. Acta Paediatr. Stand., 59, 637-647. Friedman, Z., Danon, A., Lamberth, E.L. and Mann, W.J. (1978): Cord blood fatty acid composition in infants and in their mothers during the third trimester. J. Pediatr., 92, 461-466. Shires, SE., Conway, S.P., Rawson, I., Dear, P.F.R. and Kelleher, J. (1986): Fatty acid composition of plasma and erythrocyte phospholipids in preterm infants. Early Hum. Dev., 13, 53-63. Phillips, G.B. and Dodge, J.T. (1967): Composition of phospholipids and phospholipid fatty acids of human plasma. J. Lipid Res., 8, 676-681. Dubowitz, L.M.S., Dubowitz, V. and Goldberg, C. (1970): Clinical assessment of gestational age in the newborn infant. J. Pediatr., 77, I-10.
191
13
14 15
20
21
Gairdner, D. and Pearson, J. (1971): Revised Gairdner-Pearson growth charts. Arch. Dis. Child., 60, 1202. Folch, J.L. and Sanley, G.H.S. (1957): A simple method for the isolation and purification of total lipids form animal tissues. J. Biol. Chem., 226, 497-509. Burr, G.O. and Burr, M. (1929): A new deficiency disease produced by the rigid exclusion of fat from the diet. J. Biol. Chem., 82, 345-367. Burr, G.O. and Burr, M. (1930): On the nature and role of the fatty acids essential in nutrition. J. Biol. Chem., 86, 587-621. Paoletti, R. and Galli, C. (1972): Effects of essential fatty acid deficiency on the central nervous system in the growing rat. In: Lipids, Malnutrition and the Developing Brain, pp. 9-19. Editors: K.M. Elliot and J. Knight. Ciba Found Symp No. 3. Elsevier Excerpta Medica, Amsterdam. Crawford, M.A. and Sinclair, A.J. (1972): Nutritional influences in the evolution of mammalian brain. In: Lipids, Malnutrition and the Developing Brain, pp. 267-287. Editors: K.M. Elliot and J. Knight. Ciba Found Symp No. 3. Elsevier Excerpta Medica, Amsterdam. Koletzko, B. and Braun, M. (1991): Arachidonic acid and early human growth: is there a relation? Ann. Nutr. Metab., 35, 128-131. Olsen, S.J., Sorensen, J.D., Secher, N.J., Hedegaard, M., Henriksen, T.B., Hansen, S. and Grant, A. (1986): Random&d controlled trial of the effect of fish oil supplementation on pregnancy duration. Lancet, ii (8503), 367-369. Doyle, W., Crawford, M.A., Laurence, B.M. and Drury, P. (1982): Dietary survey during pregnancy in a low-socioeconomic group. Hum. Nutr. Appl. Nutr., 36A, 95-106. Crawford, M.A., Doyle, W., Drury, P., Lennon, A., Costeloe, K. and Leighfield, M. (1989): n-6 and n-3 fatty acids during early human development. J. Int. Med., 225 (Suppl. l), 159-169. Holman, R.T., Johnson, S.B. and Ogbum, P.L. (1991): Deficiency of essential fatty acids and membrane fluidity during pregnancy and lactation. Proc. Natl. Acad. Sci. USA, 88, 4835-4839. Ongari, M.A., Ritter, J.M., Orchard, M.A., Waddell, K.A., Blair, IA. and Lewis, P.J. (1984): Correlation of prostacyclin synthesis by human umbilical artery with status of essential fatty acid. Am. J. Obstet. Gynecol., 149, 455-460. Cook, H.W. (1978): In vitro formation of polyunsaturated fatty acids by desaturation in rat brain: some properties of the enzymes in developing brain and comparisons with liver. J. Neurochem., 30, 1327-1334. Crawford, M.A., Hassan, A.G. and Williams, G. (1976): Essential fatty acids and fetal brain growth. Lancet, i, 452-453.
183
E1se.vie.rScientific Publishers Ireland Ltd. EHD 01317
Long chain polyunsaturated fatty acids and fetal growth Alison A. Leafa, Martin J. Leighfieldb, Kate L. Costeloea and Michael A. Crawford b ‘Joint Academic Department of Child Health, Medical College of St Bartholomew, London ECIA, and bInstitute of Brain Chemistry and Human Nutrition, Hackney Hospital, London E9 (UK)
(Received 24 January 1992; revision received 24 April 1992; accepted 7 May 1992)
Long chain polyunsaturated fatty acid composition of plasma choline phosphoglycerides has been measured at birth in 22 preterm infants. Positive correlations were found between both n-6 and n-3 fatty acids and measurements of growth and maturation. 20:4(n-6) and the sum of 20:3(n-6) + 20:4(n-6) correlated most strongly with weight and head circumference, while 22:6(n-3) showed strongest correlation with length of gestation. These findings are of relevance to understanding the role of nutrition in fetal growth and in establishing the group of infants most at risk of postnatal deficiency of essential fatty acids. Key words: n-6; n-3; long chain polyunsaturated
fatty acids; preterm infant; growth
Intmductlon
Arachidonic (20:4(n-6)) and docosahexaenoic (22:6(n-3)) acids are important structural components of cell membrane phospholipids. Arachidonic acid is quantitatively the most important long chain polyunsaturated fatty acid (LCPUFA) and is widespread throughout the body, although concentration is particularly high in brain and vascular endothelium. Docosahexaenoic acid, on the other hand has a highly specific distribution, representing the predominant membrane fatty acid of synaptosomes, retinal photoreceptors, mitochondria and spermatozoa, but being scarce in other tissues [ 11. Correspondence to: Alison A. Leaf, Senior Registrar, Newborn Centre, Monash Medical Centre, Locked bag 29, Clayton, Victoria 3168, Australia. 0378-3782l9USO5.00 0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
184
During fetal life LCPUFAs are actively accumulated across the placenta to the fetus, in preference to their precursors [2] and the concentration of circulating LCPUFAs in plasma phospholipids is higher in newborn infants than in their mothers [3,4]. There is considerable variation in the LCPUFA composition of plasma phospholipids at birth in preterm infants [4,5]. Linoleic acid( 18:2(n-6)) and alpha-linolenic acid (18:3(n-3)) are both 18-carbon essential fatty acids (EFAs) and are the substrates for synthesis of LCPUFAs. Each gives rise to a separate group of long-chain derivatives by a series of chain elongation and desaturation reactions. The predominant n-6 LCPUFAs, derived from linoleic acid, are dihomo-gamma linolenic acid (20:3(n-6)) and arachidonic acid (20:4(n-6)) and the predominant n-3 LCPUFAs, derived from alpha-linolenic acid are eicosapentaenoic acid (20:5(n-3)) and docosahexaenoic acid (22:6(n-3)). As well as their structural role 20:3(n-6), 20:4(n-6) and 20:5(n-3) have an important metabolic function as precursors of prostaglandins and other eicosanoids. Choline phosphoglycerides are the predominant phospholipids in plasma, representing approximately 70% of the total [6]. We have studied the relationships between LCPUFA composition of this phospholipid class and measurements of fetal growth and maturity. Methods Znfants Samples were obtained from 22 infants with weight below 2000 g at birth. The only criterion for exclusion was obvious congenital anomaly and the group included one pair of non-identical twins. Blood was taken from the umbilical vein immediately after delivery of the placenta, or from the infant on arrival at the neonatal unit. Gestational age ranged from 24-36 weeks and was assessed by maternal menstrual history and ultrasound scan before twenty weeks gestation. If this information was not available gestational age was determined by clinical examination of the infant at birth [7]. Weight was measured by midwives at delivery using electronic scales and recorded to the nearest 5 g. Head circumference (HC) was recorded within the first 48 h of life by the same observer (AL), using a fresh paper tape for each infant. Eight infants were considered small for gestational age (SGA) with birthweight below the tenth percentile and of these eight five had “proportionate” growth retardation, with HC also below the tenth percentile and three had disproportionate growth, with HC above the tenth percentile [8]. Clinical details are given in Table I. Biochemical methods Blood samples were centrifuged for 15 rnin at 5000 rev./mm and plasma separated and stored at -20°C. Lipids were extracted by the method initially described by Folch et al. [9], using chloroform/methanol 2:1, with 100 mg 1-l of BHT (2,[6]-ditert-Butyl-p-cresol). Phosphoglyceride classes were separated by thin-layer chromatography on silica plates. The solvent used contained chloroform, methanol and water in the ratio: 60:30:4, with added BHT, 100 mg 1-t. Phospholipid classes were detected using dichlorofluorescein and identified using standards. The methylating
185 TABLE I Clinical details. Means with ranges in parentheses shown except for percentage fatty acid composition where latter is standard deviation. Infant group
n
GA (weeks)
Birth weight (g)
Head circumference (cm)
20:4(n-6) (%)
22:6(n-3) (%)
All infants
22
AGAa
14
SGAb
8
SGA-dispC
3
SGA-propd
5
30.5 (24-36) 29.4 (24-33) 32.5 (26-36) 34.0 (31-36) 31.6 (26-35)
1367 (600-1985) 1379 (600-1985) 1346 (680-1730) 1431 (1145-1730) 1295 (680- 1705)
27.6 (21.2-31.2) 27.3 (21.2-31.2) 28.0 (22.5-30.9) 29.9 (28.4-30.9) 26.8 (22.5-29.3)
16.52 (3.15) 16.70 (3.62) 16.21 (2.29) 17.58 (0.62) 15.39 (2.59)
4.49 (1.82) 3.93 (1.29) 5.48 (2.26) 5.51 (2.09) 5.47 (2.80)
‘AGA, appropriate for gestational age weight > 10th percentile. bSGA, small for gestational age weight < 10th percentile. ‘SGA-disp, HC > 10th percentile. dSGA-prop, weight and HC c 10th percentile.
reagent used was methanol and acetyl chloride, 15: 1 solution, at 80°C for 3 h, under nitrogen. Fatty acid methyl esters were separated by gas-liquid chromatography on a 25 m superox 0.53 mm i.d. column (Thames Chromatography) at 190°C using hydrogen as carrier gas and a flame ionisation detector. Signals were quantified by automatic integrator and fatty acids identified by relative chain length and comparison with standards. Individual fatty acids have been quantified as percentages of the total fatty acids in choline phosphoglycerides. Statistical
methods
The relationships between fatty acid composition and continuous variables: birthweight, HC and gestational age were analysed by linear regression. The relative importance of significant variables was measured using a stepwise multiple regression model. Comparisons between AGA and SGA infants were made using Student’s two-tailed t-test and between proportionate and disproportionate SGA using Mann-Whitney U Test. Calculations were made using “Microstat” software, Ecosoft Inc. 1984. This study was approved by the Ethical Committee of City and Hackney Health Authority. Results A summary of polyunsaturated fatty acid composition of plasma choline phosphoglycerides at birth is listed in Table II. The predominant n-6 fatty acids are
186 TABLE II Fatty acid composition of plasma choline phosphoglycerides, infants expressed as percentage of total fatty acids
mean (SD.), at birth in all 22 preterm
n-6 18:2 18:3 20:2 20:3 20:4 2214 225 Total LCPUFA 18:2/20:4
8.31 (3.27) 0.21 (0.17) 1.18 (1.17) 3.43 (1.28) 16.52 (3.15) 0.52 (0.24) 0.53 (0.34) 22.19 (4.43) 0.53 (0.28)
n-3 18:3 20:5 2215 2216 Total LCPUFA
0.07 (0.03) 0.68 (0.49) 0.33 (0.30) 4.49 (1.82) 5.51 (2.11)
TABLE III Correlation coefficients for the relationships between measurements of growth and maturation at birth and polyunsaturated fatty acids of plasma choline phosphoglycerides. Fatty acid
Birthweight
HC
Gestation
n-6 18:2 20~3 20:4 20:3+20:4 Total LCPUFA
0.217 0.443. 0.564** 0.576** 0.569**
0.193 0.515” 0.597*** 0.643*** 0.625***
0.200 0.394 0.371 0.411 0.393
n-3 18:3 22~6 Total LCPUFA ?? P < 0.05. **P < 0.01. ***p < 0.005.
-0.090 0.496* 0.405
-0.024 0.589** 0.530**
-0.153 0.638*** 0.586**
187
20 : 3n-6 + 20 : 4n-6 and HEAD CIRCUMFERENCE Plasma choline phosphoglyceride 30 28
.
r I 0.643 p ? ? 0.001
20
22
24
26
Head Clrcumfennce
26
30
32
(cm)
Fig. I. Relationship between 20:3(n-6) + 20:4(n-6) and head circumference: 0, AGA; SGA; 0, disproportionate SGA.
?? , proportionate
18:2(n-6), 20:3(n-6) and 20:4(n-6) and the only n-3 fatty acid present in significant amount is 22:6@3). Correlation coefficients for the relationship between specific fatty acids and growth variables are shown in Table III. Significant positive correlations were found between 20:3(n-6) and 20:4(n-6) and both weight and HC. Even stronger relationships were found between sum of 20:3(n-6) + 20:4(n-6) and total n-6 LCPUFAs and growth measurements (Fig. 1). There was no statistically significant relationship between linoleic acid and growth measurements, nor between n-6 fatty acids and gestational age. 22:6(n-3) showed a strong correlation with gestational age (Fig.2) and also with HC and birthweight. The sum of all n-3 LCPUFAs (20:5(n-3), 22:5(n-3) and 22:6(n-3)) also showed a positive correlation with both head circumference and gestational age. There were no significant differences between mean birthweight and HC in SGA and AGA infants. The SGA group were however significantly more mature at birth, P < 0.05.
There was no difference in n-6 LCPUFAs between SGA and AGA infants. 22:6(n-3) was higher in the SGA group, P = 0.051. Using a multiple regression
22 : 6n-3 and GESTATIONAL AGE Plasma choline phosphoglyceride 12 11 -
r = 0.638 p = 0.001
10 -
98765432 -HO
0
lo-
’
24
I
26
I
I
I
26 30 32 Gestational Age (wks)
I 34
Fig. 2. Relationship between 22:6(n-3) and gestational age: 0, AGA; disproportionate SGA.
I
36 ?? , proportionate
SGA; 0,
model to study the inter-related effects of length of gestation, birthweight and intrauterine growth retardation, the latter two became insignificant. Partial r-squared for the three variables were 0.41, P = 0.001, 0.002 and 0.03, respectively. No significant differences between fatty acid composition of proportionate and disproportionate SGA infants was found. Discussion These data show the lowest percentages of LCPUFAs in the smallest and least mature infants and identify differences between the n-6 and n-3 fatty acids. n-6 Fatty acids related particularly to growth measurements, both birthweight and head circumference. In the initial description of n-6 fatty acid deficiency growth failure was a major feature [lO,l l] and has subsequently been shown to result in decreased cell division and reduced weight of body and brain in rats [ 12,131. A correlation between n-6 LCPUFAs and birthweight has recently been described in plasma triglycerides of preterm infants at four days of age [14]. In addition to weight our data show a strong correlation between n-6 LCPUFA and HC and also between n-3 fatty acids and growth.
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The strongest correlation of 22:6(n-3), however, was with gestational age. A relationship between long chain n-3 fatty acids and gestational age has previously been reported [15]. Women in the Faroe Islands, where intake of n-3 LCPUFAs in fish is high, had a higher content of DHA in their erythrocyte phospholipids than women from The Netherlands or Canada. Faroese women give birth to infants with higher than average birthweight and longer gestation. The authors propose that the underlying mechanism may be an alteration in the balance of prostaglandin synthesis. High circulating n-3 fatty acids may inhibit production of the dienoic prostaglandins derived from arachidonic acid which stimulate uterine contraction, thus prolonging gestation. Our data may reflect the opposite end of the spectrum: with low 22:6(n-3) in the infants of shortest gestation. A strong correlation was found between 22:6(n-3) and HC, which is of interest as the highest concentration of 22:6(n-3) is found in the brain. Relationships between fatty acid composition and fetal growth may have a physiological or pathological basis. The highest requirement for cell membrane components would be expected to coincide with the time of most rapid growth. In the human fetus increase in body mass and brain size is greatest during the latter half of pregnancy. It is possible that at the time of birth of the smallest, least mature infants in our study the peak of LCPUFA requirement has not yet occurred. Friedman et al, 1978, described an increase in all LCPUFA in cord blood plasma phospholipids from 24 to 44 weeks gestation [4]. It was suggested that this was due to an increased demand for brain growth in the third trimester, supporting a physiological explanation for the gradual increase. Alternatively, the lower LCPUFA concentration seen in the very low-birthweight infants may have a pathological basis. Three factors might be involved: maternal circulating fatty acids which reflect dietary intake, placental function and fetal metabolism. Inadequate maternal diet has been associated with low birthweight [16]. Low intake of EFAs in all trimesters of pregnancy was shown to correlate with low birthweight. Furthermore significantly lower plasma and red cell content of arachidonic acid was found in both the mothers and infants where weight was below 2.5 kg at birth, compared with those in whom birthweight was above 3.0 kg [ 171. In this study no distinction was made between prematurity and IUGR as the cause for low birthweight. Even in normal low-risk pregnancies low levels of maternal LCPUFA have recently been described [ 181, suggesting that current western dietary habits may not provide the optimum amount or proportions of EFAs for reproduction. Placental function is important in transfer of LCPUFAs from mother to fetus [2]. The placenta also has complex metabolic and endocrine roles, including the control of vasomotor tone and myometrial contractility, mediated by prostaglandins. Intrauterine growth retardation (IUGR) associated with placental dysfunction tends to occur later in pregnancy and to spare head growth, resulting in disproportionate SGA infants, whereas IUGR with an earlier onset tends to affect head growth and result in proportionate SGA infants, Despite showing a relationship between head circumference and both n-6 and n-3 LCPUFAs, our data did not reveal any significant differences in LCPUFAs between SGA and AGA infants, nor between propor-
190
tionate and disproportionate SGA infants. The numbers in these groups were, however, small. Fatty acid composition of umbilical arterial wall has however shown features of EFA deficiency which were most marked in the smallest growth retarded infant [ 171. It is possible that these differences between umbilical artery and cord blood reflect a latent deficiency, all available EFAs and LCPUFAs being taken for growth of essential organs such as brain, at the expense of less vital tissue, ie the umbilical artery. Also in umbilical artery, Ongari et al. observed lower 20:4(n-6), increased triene-tetraene ratio, suggestive of EFA deficiency and reduced prostacyclin synthesis, in infants who were SGA in association with maternal hypertension [19]. In agreement with our findings, low 20:4(n-6) and decreased prostacyclin were related to weight and not to gestational age. There is little information available on EFA metabolism in the human fetus. Some elongation and desaturation of l&carbon EFAs may occur in liver, or possibly in brain, as has been demonstrated in brain cells of fetal rats [20]. The principal source of LCPUFA, however, appears to be selective transfer across the placenta [2,21]. We have shown a relationship between fetal growth measurements and circulating LCPUFAs. The smallest, least mature infants are born with the lowest circulating levels of these essential fatty acids and yet are the infants in whom the requirement of LCPUFAs for brain growth will be greatest. Whether the differences are pathological or physiological requires further study of maternal levels and placental handling of EFAs in relation to fetal growth. The fact that levels are low and that n-3 and n-6 LCPUFAs relate to different aspects of growth and maturation has important implications for the understanding of developmental biology and also for the nutritional requirements of preterm infants. Acknowledgements
We wish to thank the staff of the Special Care Baby Unit at the Hornet-ton Hospital for their cooperation and the EC, Barclay Trust, Nestle and Vlaardingen Research, Unilever for financial support. References 1
2 3 4 5 6 7
Salem, N., Kim H.-Y. and Yergey, J.A. (1986): Docosahexaenoic acid: membrane function and metabolism. In: Health Effects of Polyunsaturated Fatty Acids in Seafood, pp. 263-317. Editors: A.P. Simopoulos, R.R. Kifer and R.E. Martin. Academic Press, Orlando, Florida. Kuhn, D. and Crawford, M. (1986): Placental Essential fatty acid transport and prostaglandin synthesis. Prog. Lipid Res., 23, 345-353. Olegard, R. and Svennerholm, L. (1970): Fatty acid composition of plasma and red cell phosphoglycerides in full term infants and their mothers. Acta Paediatr. Stand., 59, 637-647. Friedman, Z., Danon, A., Lamberth, E.L. and Mann, W.J. (1978): Cord blood fatty acid composition in infants and in their mothers during the third trimester. J. Pediatr., 92, 461-466. Shires, SE., Conway, S.P., Rawson, I., Dear, P.F.R. and Kelleher, J. (1986): Fatty acid composition of plasma and erythrocyte phospholipids in preterm infants. Early Hum. Dev., 13, 53-63. Phillips, G.B. and Dodge, J.T. (1967): Composition of phospholipids and phospholipid fatty acids of human plasma. J. Lipid Res., 8, 676-681. Dubowitz, L.M.S., Dubowitz, V. and Goldberg, C. (1970): Clinical assessment of gestational age in the newborn infant. J. Pediatr., 77, I-10.
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