European Journal of Clinical Nutrition (2000) 54, 50±56 ß 2000 Macmillan Publishers Ltd. All rights reserved 0954±3007/00 $15.00 www.nature.com/ejcn

Arachidonic and docosahexaenoic acids are strongly associated in maternal and neonatal blood

K Ghebremeskel1, MA Crawford1*, C Lowy2, Y Min1, B Thomas1, I Golfetto1, D Bitsanis1 and K Costeloe3

1The Institute of Brain Chemistry and Human Nutrition, The University of North London, London; 2Department of Endocrinology, Diabetes and Metabolic Medicine, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, University of London, London; and 3Joint Academic Department of Child Health, Medical College of St Bartholomew's and Homerton Hospital, London, UK

Background: The red cell membrane fatty acid composition has frequently been used as an index of essential fatty acid (EFA) nutrition. After birth there is a decline in plasma arachidonic acid (AA) and docosahexaenoic (DHA) acids in babies fed on conventional formula which contains only the parent linoleic and a-linolenic acids. In human studies, the red cell phosphoglyceride composition appears to be more constant than that of plasma. In infants fed ®sh oil without AA, the AA proportions fall in the plasma but much less so in the red cells. This result might be considered to mean that there is no need for preformed AA. On the other hand, in a study where the levels of AA fell there was reduction of infant growth. Indeed, where cell membrane composition does change there is often an associated alteration in physiological functions of membranes. We therefore felt it worth investigating the balance between AA and DHA in a physiological situation where plasma levels are known to change, namely in pregnancy. Purpose: The aim of the study was to investigate a relationship between blood phosphoglyceride AA and DHA in pregnant women and neonates. Subjects: Health pregnant women from London, England (n ˆ 193) and their term babies n ˆ 45†; healthy pregnant women from Seoul, South Korea n ˆ 40† and their term babies n ˆ 40); and preterm neonates n ˆ 72† from London. Method: Blood samples were taken from British and Korean pregnant women during the third trimester, and from term and preterm babies at birth. These samples were taken for routine monitoring purposes in Korea and were a part of a study on pregnancy outcome for which ethical permission was granted from the East London and The City Health Authority and Lambeth, Southwark and Lewisham Health Authority. Approval was also obtained from the Ethical Committee of the Asan Medical Centre, Seoul, South Korea. Results: AA and DHA correlated in plasma choline phosphoglycerides (CPG) of the British mothers (r ˆ 0.52 P < 0.0001). The correlation coef®cients and signi®cance were much stronger in the red cell CPG and even more so in the term and preterm infant red cell CPGs (r ˆ0.75, 0.80 and 0.88, respectively). Similarly, AA and DHA correlated in red cell CPGs of the Korean women and their term babies. There was also a signi®cant relationship between the two fatty acids in red cell ethanolamine phosphoglycerides in the mothers and their babies. Both linoleic (LA) and -linolenic acids (ALA) were inversely associated with AA and DHA in some of the phosphoglyceride fractions of the mothers and babies. Conclusions: Although AA and DHA have different primary dietary origins, there were signi®cant relationships between AA and DHA in the phosphoglycerides of the red cell membrane. This ®nding seems surprising if the red cell composition is determined by diet. These results suggest a physiological mechanism which attempts to maintain an appropriate balance between AA and DHA. It is plausible that there is an optimum balance between AA and DHA for membrane stability, deformability, enzyme and receptor function. Sponsorship: The British Diabetic Association, March of Dimes Birth Defects Foundation and The Christopher H.R. Reeves Charitable Trust. Descriptors: linoleic; a-linolenic; arachidonic; docosahexaenoic; mothers; neonates European Journal of Clinical Nutrition (2000) 54, 50±56

Introduction segments and has a crucial function in normal signal transduction and ampli®cation (Jones et al, 1997). The polyunsaturated fatty acids, arachidonic (AA) and AA and DHA composition of neural cell membranes in docosahexaenoic (DHA), are vital structural lipid compo- adults seems to be relatively well conserved. It is, however, nents in bio-membranes. AA is widely distributed in high responsive to some change during the period of growth. In proportions in cell membranes of all tissues. In contrast, experimental animals, membrane structural alterations DHA is found in synaptic and sperm membranes and to a resulting from changes in the AA and DHA composition lesser extent in non-neural tissues. DHA accounts for about lead to loss of cellular integrity and functions (Clausen & 60% of the structural fatty acids in the photoreceptor outer Moller 1967; Crawford & Sinclair 1972; Galli & Socini 1983). De®cits of the essential n-3 fatty acids cause *Correspondence: MA Crawford, The Institute of Brain Chemistry and cerebral haemorrhage in chicks (Budowski et al 1987), Human Nutrition, The University of North London, Holloway Road, London N7 8DB, UK. impaired learning in rats (Lamptey & Walker 1976, Bourre Received 30 October 1998; revised 21 April 1999; accepted 29 July 1999. et al, 1989), and delayed visual acuity maturation in non- Relationship between AA and DHA in pregnant women K Ghebremeskel et al 51 human primates (Connor et al, 1992). It also seems that the The genetic adrenoleucodystrophies are characterized by balance between AA and DHA is important for full severe mental retardation, ¯opiness control and blindness. behavioural performance (Wainwright et al, 1997). The failure in neurogenesis and loss of function is thought The foetus is normally provided with high proportions of to be due to the peroxisomal de®cit resulting in loss of AA and DHA obtained from the material circulation DHA and a distortion of neural cell membranes. Martinez through placental enrichment. This placental supply and Vazquez (1998) have reported improved myelination would be reduced if the mother had impaired fatty acid as seen by magnetic resonance imaging from treating metabolism or placental dysfunction, and denied if the baby peroxisomal disorders with DHA. However, our knowledge was born prematurely. Preterm and low birthweight term of the relationship between AA and DHA in general babies are reported to have lower AA and DHA at birth membrane lipids in mothers or babies is limited. None- compared with fully grown term babies. Previous studies theless, as early as 1978 the FAO=WHO expert consulta- have shown that a reduced umbilical cord and plasma AA tion on dietary fats recommend a balance for the !3 and !6 level is associated with low birthweight and pre-eclampsia, families in pregnancy and for infants (FAO=WHO, 1978). with plasma DHA concentration being found to be directly Innis (1992) found that diet-induced changes are slower related to gestational age (Crawford et al, 1989; Koletzko to occur in the red cells than in plasma and may not & Braun, 1991; Leaf et al, 1992; Velzing-Aarts et al, necessarily re¯ect levels in the brain and other internal 1999). organs. In a study of the membrane lipid composition in The plasma levels of AA decrease rapidly after birth in several different mammalian species, it was found that, the preterm infant, despite a three-fold rise in the propor- whilst there were wide differences in hepatic phosphogly- tions of its precursor linoleic acid (LA); a similar fall in cerides, there was no difference in the brain: it seemed that DHA has also been observed (Carlson et al, 1986; Leaf et brain size was sacri®ced rather than composition (Crawford al, 1992). This suggests that the rate of synthesis of AA and et al, 1976). All of this evidence suggests that membrane DHA from the parent compounds is not able to meet the composition may be of some signi®cance to physiological demand for growth and development. A relationship has function. Such evidence led Maurage et al (1998) to been reported between growth and plasma AA during the conclude that the balance between AA and DHA is likely postnatal period (Carlson et al, 1993; Carlson, 1996). to be important for proper brain development. It is therefore Special interest has been focused on the AA and DHA important to know whether accessible membranes like nutrition because they are present in breast milk, and breast those of the erthrocytes are in any way regulated. milk fed babies appear to have better mental performance As part of our study into the fatty acid status of mothers and health statistics, and fewer gastro-intestinal infections and babies, we investigated the association between AA and other form of morbidity (Oski, 1993; Scariati et al, and DHA and their parent compounds LA and -linolenic 1997). Babies fed on formula milk devoid of AA and DHA acid (ALA) in pregnant women and term and preterm during the postnatal period have reduced plasma AA and neonates and report our results, which imply that the red DHA status (Putnam et al, 1982; Innis et al, 1990; Gheb- cell membrane composition is not as variable as might have remeskel et al, 1995; Farquharson et al, 1993, 1995; been assumed. Makrides et al, 1994). Some studies have described lower transient visual and cognitive abilities (Jorgensen et al, Materials and methods 1994; Carlson et al, 1994, 1996a; Uauy et al, 1992; Birch et al, 1993; Makrides et al, 1993). Studies on term infants Subjects have come to similar conclusions (Carlson et al, 1996b; The mothers were part of a study on pregnancy outcome in Birch et al, 1998), except for one (Auestad et al, 1997), which blood was being drawn for clinical monitoring and a which found no difference in the supplemented compared portion reserved for biochemistry. The women were non- with those fed unsupplemented milk. In the latter case, smokers, non-vegetarians, normoglycaemic and normoten- DHA was supplemented at a level which was just above the sive non-diabetics with no previous complicated pregnancy lowest in the range reported in human milk (0.12% used in or family history of diabetes. Exclusions from the study a range of 0.1 ± 0.9%). Willats et al (1998) make the point were young women under the age of 16, and those who that many of the tests done are not predictive of later were mentally ill or handicapped, or for different reasons outcomes. Basic visual and neuronal pathways are laid not able to fully give competent written consent. Those down well before birth; so nutritional interventions at a who developed hypertension or diabetes during their preg- later time are unlikely to induce major changes in brain nancy were excluded. In order to compare women from a composition and function. However, they claim that pro- quite different ecological and nutritional environment, a blem solving at 10 months of age, which develops after parallel study was done in Korea. birth, is predictive and they found better performance if Healthy pregnant women from London n ˆ 193† and infants had previously been fed formula containing AA and the term babies n ˆ 45† of 45 of them, healthy pregnant DHA. women from Seoul, South Korea n ˆ 40† and their term Blood levels of AA decline not only in babies fed on babies n ˆ 40†, and preterm neonates n ˆ 72† from conventional formula but also in those fed formula enriched London were studied. The women were aged between 20 with ®sh oil but devoid of AA (Carlson et al, 1991; and 40 y. Gestational ages of the term babies were between Hoffman & Uauy 1992). Similarly, plasma and red cell 36 and 42, and the preterm less than 33 weeks. Blood DHA levels in babies decrease when they are fed on a specimens (5 ml) were taken from the pregnant mothers in formula milk which is either devoid of DHA or does not the third trimester (weeks 28 ± 36) and from the term and contain adequate amounts of the fatty acid. A change in the preterm babies at birth (cord blood). Plasma and red blood balance between AA and DHA is considered to disturb cells were separated by cold centrifugation at 1000 g for synaptic (Suzuki et al, 1998), hypocampal (Young et al, 15 min. After washing the red cells twice with an equal 1998) and red blood cell function (Escudero et al, 1998). volume of saline (0.85% NaCl), both blood fractions were

European Journal of Clinical Nutrition Relationship between AA and DHA in pregnant women K Ghebremeskel et al 52 ¯ushed with nitrogen and stored at 70C until required Table 1 Correlations between linoleic (LA), a-linolenic (ALA), for analysis. arachidonic (AA) and docosahexaenoic (DHA) acids in plasma choline Ethical approval was obtained from the East London and phosphoglyceride and red blood cell choline and ethanolamine phosphoglyceride lipid fractions of pregnant British women The City Health Authority and Lambeth, Southwark and Lewisham Health Authority. Approval was also obtained ALA AA DHA from the Ethical Committee of the Asan Medical Centre, Plasma choline phosphoglycerides n ˆ 126† Seoul, South Korea. LA r ˆ 0:023,ns r ˆ0:526, r ˆ0:500, P < 0:0001 P < 0:0001 Fatty acid analysis ALA r ˆ0:421, r ˆ0:187, Total plasma and red cell lipid was extracted by the method P < 0:0001 P < 0:05 of Folch et al (1957) by homogenizing the samples in AA r ˆ 0:520, P < 0:0001 chloroform and methanol (2:1v=v) containing 0.01% buty- RBC choline phosphoglycerides n ˆ 193† lated hydroxytoluene (BHT) as an antioxidant under N2. LA r ˆ 0:393, r ˆ0:078,ns r ˆ0:119,ns Phosphoglyceride classes were separated by thin-layer P < 0:001 chromatography on silica gel plates by the use of the ALA r ˆ0:54,ns r ˆ0:025,ns AA r ˆ 0:753, developing solvents chloroform, methanol and water P < 0:0001 (60:30:4v=v), containing 0.01% BHT in a nitrogen atmo- RBC ethanolamine phosphoglycerides n ˆ 135† sphere. The bands for the choline and ethanolamine phos- LA r ˆ 0:185, r ˆ0:182, r ˆ0:122,ns phoglycerides, being the main polar phosphoglycerides, P < 0:05 P < 0:05 were detected by spraying with a methanolic solution of ALA r ˆ0:340, r ˆ0:253, P < 0:0001 P < 0:01 2, 7-dichloro¯urescein (0.01% w=v) and identi®ed by the AA r ˆ 0:595, use of standards as used previously (Ghebremeskel et al, P < 0:0001 1995). Fatty acid methyl esters (FAME) were prepared by heating the lipid fractions with 5 ml of 15% acetyl chloride Table 2 Correlations between linoleic (LA), a-linolenic (ALA),  in methanol in a sealed vial at 70 C for 3 h under N2. arachidonic (AA) and docosahexaenoic (DHA) acids in plasma choline FAME were separated by a gas ± liquid chromatograph phosphoglyceride and red blood cell choline and ethanolamine (HRGC MEGA 2 Series, Fisons Instruments, Italy) ®tted phosphoglyceride lipid fractions of British term babies at birth with a capillary column (25 mÂ0.32 mm i.d., 0.25  ®lm, ALA AA DHA BP20). Hydrogen was used as a carrier gas, and the injector, oven and detector temperatures were 235, 210 Plasma choline phosphoglycerides n ˆ 37†  LA r ˆ 0:90, r ˆ0:782, r ˆ0:488, and 260 C. The FAME were identi®ed by comparison of P < 0:0001 P < 0:0001 P < 0:01 retention times with authentic standards and calculation of ALA r ˆ0:816, r ˆ0:312,ns equivalent chain length values. Peak areas were quanti®ed P < 0:0001 by a computer chromatography data system (EZChrom AA r ˆ 0:301,ns Chromatography Data System, Scienti®c Software Inc., RBC choline phosphoglycerides n ˆ 45† San Ramon, CA). LA r ˆ 0:578, r ˆ0:226, r ˆ0:019,ns P < 0:0001 P < 0:01 Data analysis ALA r ˆ0:328,ns r ˆ 0:024,ns The strength of association (Pearson Correlation Coef®- AA r ˆ 0:805, P < 0:0001 cient, r) between LA, ALA, AA and DHA was calculated. RBC ethanolamine phosphoglycerides n ˆ 44† A statistical package, SPSS for Windows (Release 7), was LA r ˆ 0:164,ns r ˆ0:317, r ˆ0:159,ns used to analyse the data. Signi®cance was considered if P < 0:05 P < 0:05. ALA r ˆ0:366, r ˆ0:265,ns P < 0:05 AA r ˆ 0:762, Results P < 0:0001 The data showed an interesting and rather an unexpected inverse relationship between the parent compounds (LA and ALA) and their metabolites (AA and DHA), and a P < 0:05). Similarly, LA was negatively correlated with direct relationship between AA and DHA (Tables 1 ± 5), DHA in plasma choline phosphoglycerides of the British which was especially strong in the red cell phosphoglycer- pregnant women r ˆ0:500, P < 0:0001) and term ide fractions. babies r ˆ0:488, P < 0:01), and red cell choline r ˆ0:331, P < 0:05) and ethanolamine of the Korean Relationship of linoleic acid (LA) to AA and DHA term babies r ˆ0:544, P < 0:05) and British preterm Linoleic acid showed a negative correlation with AA in the babies r ˆ0:473, P < 0:01). plasma choline phosphoglycerides of the British pregnant women r ˆ0:526, P < 0:0001†, British term babies r ˆ0:782, P < 0:0001), the Korean pregnant women Relationship of a-linolenic acid (ALA) to AA and DHA r ˆ0:892, P < 0:0001) as well as the red cell choline There was a signi®cant negative correlation between ALA r ˆ0:226, P < 0:01) and ethanolamine r ˆ0:317, and AA in plasma choline and red cell ethanolamine P < 0:05) phosphoglycerides of the British term babies, phosphoglycerides of the British pregnant women r ˆ red blood cell choline phosphoglycerides of the Korean 0:421, P < 0:0001; r ˆ0:340, P < 0:0001), Korean term babies r ˆ0:337, P < 0:05) and ethanolamine pregnant women r ˆ0:872, P < 0:0001; r ˆ0:315, phosphoglycerides of the preterm babies r ˆ0:356, P < 0:05), British term babies (r ˆ0:816, P < 0:0001,

European Journal of Clinical Nutrition Relationship between AA and DHA in pregnant women K Ghebremeskel et al 53 Table 3 Correlations between linoleic (LA), a-linolenic (ALA), arachidonic (AA) and docosahexaenoic (DHA) acids in choline and ethanolamine phosphoglyceride fractions in red blood cells of British preterm babies at birth

ALA AA DHA ALA AA DHA

Choline phosphoglycerides (n ˆ 72) Ethanolamine phosphoglyceride (n ˆ 42)

LA r ˆ 0:109, r ˆ0:088, r ˆ0:101, r ˆ 0:599, r ˆ0:356, r ˆ0:473, ns ns ns P < 0:01 P < 0:019 P < 0:01 ALA r ˆ0:051, r ˆ0:074, r ˆ0:470, r ˆ0:456, ns ns P < 0:05 P < 0:05 AA r ˆ 0:869, r ˆ 0:883, P < 0:0001 P < 0:0001

Table 4 Correlations between linoleic (LA), a-linolenic (ALA), the Korean babies r ˆ0:545, P < 0:05; r ˆ0:850, arachidonic (AA) and docosahexaenoic (DHA) acids in plasma choline P < 0:01† and red cell ethanolamine phosphoglycerides phosphoglyceride and red blood cell choline and ethanolamine of the preterm babies (r ˆ0:456, P < 0:05). phosphoglyceride lipid fractions of Korean pregnant women

ALA AA DHA Relationship between AA and DHA Plasma choline phosphoglycerides n ˆ 20† LA r ˆ 0:709, r ˆ0:892, r ˆ0:377,ns AA and DHA correlated signi®cantly in plasma choline P < 0:001 P < 0:0001 phosphoglycerides of the British mothers r ˆ 0:520, ALA r ˆ0:872, r ˆ0:604, P < 0:0001†, and in red cell choline and ethanolamine P < 0:0001 P < 0:001 phosphoglycerides of the British mothers (r ˆ 0:753, AA r ˆ 0:332,ns P < 0:0001; r ˆ 0:595, P < 0:0001), Korean pregnant RBC choline phosphoglycerides n ˆ 40† LA r ˆ 0:253,ns r ˆ0:298,ns r ˆ0:172,ns women (r ˆ 0:654, P < 0:0001; r ˆ 0:790 , P < 0:0001), ALA r ˆ 0:317,ns r ˆ 0:333,ns British term babies r ˆ 0:805, P < 0:0001; r ˆ 0:762, AA r ˆ 0:654, P < 0:0001), Korean term babies (r ˆ 0:711, P < 0:0001, P < 0:0001 r ˆ 0:630, P < 0:01) and preterm babies (r ˆ 0:869, RBC ethanolamine phosphoglycerides n ˆ 40† LA r ˆ 0:275,ns r ˆ0:90,ns r ˆ 0:045,ns P < 0:0001; r ˆ 0:883, P < 0:0001). ALA r ˆ0:315, r ˆ0:115,ns These results show that, although there is a relationship P < 0:05 between AA and DHA in the plasma CPG fraction, a AA r ˆ 0:790, comparison of the red cell phosphoglyceride revealed a P < 0:0001 much stronger relationship in the same photphoglyceride fraction, which was even stronger in the term and preterm neonates. Table 5 Correlations between linoleic (LA), a-linolenic (ALA), arachidonic (AA) and docosahexaenoic (DHA) acids in plasma choline phosphoglyceride and red blood cell choline and ethanolamine phosphoglyceride lipid fractions of Korean term babies at birth Discussion

ALA AA DHA Arachidonic and docosahexaenoic acids are highly unsatu- rated, long chain derivatives of linoleic and -linolenic Plasma choline phosphoglycerides n ˆ 20† LA r ˆ0:121,ns r ˆ0:367,ns r ˆ0:381,ns acids. They are the parents of the !6 and !3 essential fatty ALA r ˆ0:270,ns r ˆ0:545, acid families. LA is primarily stored in plant seeds whilst P < 0:05 ALA accounts for a high proportion of the fatty acids AA r ˆ 0:392,ns associated with photosynthesis and is hence found in the RBC choline phosphoglycerides n ˆ 40† green parts of the plant. LA and ALA are not only LA r ˆ 0:382,ns r ˆ0:337, r ˆ0:331, P < 0:05 P < 0:05 segregated in the plant, but also in seasons as the seeds ALA r ˆ0:41,ns r ˆ 0:782,ns appear in the late and mature part of the growing season AA r ˆ 0:711, whilst the green leafy foods are most abundant earlier in the P < 0:0001 season. RBC ethanolamine phosphoglycerides n ˆ 20† LA r ˆ 0:655,ns r ˆ0:333,ns r ˆ0:544, AA and DHA are not found in any signi®cant amounts P < 0:05 in plant foods. Although they are synthesized by animals, ALA r ˆ0:378,ns r ˆ0:850, only small mammals (ca < 5 kg bodyweight) accumulate P < 0:01 high proportions of AA and DHA. In large land mammals, AA r ˆ 0:630, there is little DHA found in muscle and organ lipids with P < 0:01 the exception of the brain and testes. By contrast the marine food chain does not contain ¯owering plants bearing protected seeds, hence it is dominated by photosynthesis r ˆ0:366, P < 0:05) and red cell ethanolamine phospho- and is !3 rich. As the marine food chain is largely glycerides of the preterm babies r ˆ0:470, P < 0:05†. carnivorous, the larger marine animals that are higher in Similarly, ALA was inversely related to DHA in red cell the food chain concentrate DHA. This difference between ethanolamine phosphoglycerides of the British mothers land and marine food chains results in the !6 and !3 fatty (r ˆ0:253, P < 0:01), plasma choline phosphoglycerides acids as well as preformed AA and DHA occurring in of the Korean mothers (r ˆ0:604, P < 0:001), plasma contrasting aspects (Hilditch & Williams 1964, Crawford et choline and red cell ethanoalmine phosphoglycerides of al, 1976, 1988; Gunstone & Norris 1983; Gurr, 1984).

European Journal of Clinical Nutrition Relationship between AA and DHA in pregnant women K Ghebremeskel et al 54 The inclusion in this study of British and Korean women ordering the lipids in domain formation and hence compo- provided the opportunity to address the association between sition (Moore et al, 1999). LA, ALA, AA and DHA in blood in the face of widely The results of our study could be relevant to the varying dietary intakes of the two fatty acids but within a discussion on infant nutrition. Should formula milk include physiological context. Data obtained at birth from healthy AA and DHA in order to meet requirements and maintain term and preterm babies provided an indication of the balance between them? Or is it suf®cient to add the parent relative proportions of AA and DHA which are delivered compounds LA and ALA in the hope that the tight balance by the placenta to the foetus at two different time points of observed will be achieved indirectly? Competitive inhibi- gestation. Ruyle et al (1990) have presented evidence tion, the slow rate of conversion (Sprecher, 1993) and which indicates that erythrocytes play a major role in higher oxidation rates (Sinclair, 1975; Leyton et al, 1987) providing AA and DHA for the placenta to transport to of LA and ALA compared to AA and DHA, and the inverse the foetus. The fact that the correlations strengthened from relationship between precursor and product shown here and maternal plasma to red cells and were even stronger in the described by Galli and Socini (1983) suggest there could be term and more so with the preterm infant is consistent with an advantage to AA and DHA being preformed. Further- this view of a red cell participation in transfer. These data more it is now apparent that, in the brain, ALA is sub- suggest a prominent role for the placenta in the mainten- stantially and preferentially used more for cholesterol as ance of AA and DHA balance during foetal development. compared to DHA synthesis (Cunnane et al, 1994). If LA In comparing the women from London and Seoul; the and ALA are not converted in suf®cient amounts and data shows a surprisingly similar association between AA proportion to meet the requirements (Emken et al, 1994) and DHA in maternal red cell membrane phosphoglycer- expressed by the tight AA=DHA relationship, one needs to ides despite the different genetic and nutritional back- ask what price is being paid in cell membrane formation. grounds. Since there is no evidence of a high correlation Human breast milk universally contains signi®cant pro- between AA and DHA, and LA and ALA in foods (Tinoco, portions of AA and DHA. Whilst most European formula- 1982; Ackman, 1982; Gunstone & Norris, 1983; Gurr, tions have followed breast milk in this respect, there are some 1984), the strong membrane correlatoins between AA and who argue that the high level of LA in the formula will be DHA suggest the involvement of a physiological selection converted to AA suf®ciently to meet the requirements. The process rather than diet. negative relationship between LA and AA observed in our There was a signi®cant negative relationship between study suggests that this assumption may be incorrect. the parent compounds LA and ALA and their respective In a previous study of pareterm neonates, Leaf et al major metabolites AA and DHA in the mothers and the (1992) have reported that the plasma AA is reduced to a babies. This was unexpected and rather intriguing. AA and third of its initial value despite a three-fold increase in its DHA are synthesized from LA and ALA, respectively. precursor LA between birth and 3 weeks of age. Moreover, Hence, an increase in the availability of the parent com- Koletzko et al (1996), by the use of isotope balance pounds might be expected to result in a concomitant equations, have estimated that endogenous synthesis con- increase in their metabolites. This is not the case and our tributes only about 23% of total plasma arachidonic acid in observation is consistent with the ®nding of Galli and human infants born at full term. There is a considerable Socini (1983) who showed that membrane AA is replaced disparity in the incorporation into the developing brain by LA when the dietary intake of the latter is increased. between preformed AA and AA synthesized de novo from These results imply that cell membrane compositional LA (Sinclair, 1975). There is also evidence of a reduction target for essential fatty acids is not simply a product of in weight gain in preterm babies who were fed on formula the desaturation and chain elongation of LA and ALA. The milk enriched with ®sh-oil (as a source of EPA and DHA) combination of synthesis and direct intake for AA and but devoid of AA (Carlson et al, 1993; Carlson 1996; DHA will result in a pool available for cell membrane Ghebremeskel et al, unpublished data). synthesis. Indeed, the strong relationship between AA and DHA in maternal and foetal (cord) red cells suggest there is a quasi-homeostatic mechanism operating to maintain an Conclusion appropriate balance of these fatty acids in the red cell membrane. This study was conducted under a condition known to These conclusions raise a question on the general induce changes in the circulating plasma fatty acids, intrerpretation of red cell membrane fatty acid data. namely pregnancy. Whilst unphysiological, experimental Many circulating components are in fact regulation. changes in diets will alter plasma and red cell membrane There is evidence pointing to the possibility that de®cits composition, we studied women living under quite different of AA and DHA may contribute to the complications of but nonetheless physiological conditions. The study prematurity (Crawford et al, 1998) and Carlson et al (1998) revealed a strong relationship between red cell membrane has recently described a signi®cant preventive effect phosphoglyceride AA and DHA in maternal and in cord against necrotizing enterocolitis by supplementing preterm blood. This relationship held for both term and preterm infants with AA and DHA. Most studies on diet and lipids babies and their mothers. We also found an inverse asso- have been done on storage lipids, i.e. on neutral lipids. The ciation between the parent fatty acids LA and ALA and responsiveness of neutral lipids to dietary alterations may their metabolites AA and DHA. We conclude that there is a have been a false steer, as the membrane lipids are clearly regulatory mechanism maintaing the AA and DHA com- much less responsive within physiological dimensions of position of red cell membranes. diet and, from our data, appear to be regulated. Phospho- glycerides, especially those in the cell membrane, have a Acknowledgements ÐThe authors thank The British Diabetic Association, structural connotation in which the membrane protein will March of Dimes Birth Defects Foundation and The Christopher H.R. be a major determinant of the lipid protein interaction, Reeves Charitable Trust for providing funds for the study. The contribu-

European Journal of Clinical Nutrition Relationship between AA and DHA in pregnant women K Ghebremeskel et al 55 tion of the staff of the Special Care Baby Unit at the Homerton Hospital Crawford MA, Costeloe K, Ghebremeskel K & Phylactos A (1998): The and The Diabetic Day Centre, St Thomas' Hospital is gratefully acknowl- inadequacy of the essential fatty acid content in preterm infant. Eur. J. edged. Pediatr. 157(Suppl 1), S23 ± S27. Cunnane SC, Williams SCR, Bell JD, Brookes S, Craig K, Iles RA & References Crawford MA (1994): Utilization of uniformly labelled 13C-polyunsa- turated fatty acids in the synthesis of long chain fatty acids and Ackman RG (1982): Fatty acid composition of ®sh oils. In: Nutritional cholesterol accumulating in the neonatal rat brain. J. Neurochem. 62, Evaluation of Long-chain Fatty Acids in Fish Oils, eds. SM Barlow & 2429 ± 2436. ME Stansby, pp 25 ± 88. London: Academic Press. Emken EA, Adlof RO & Gulley RM (1994): Dietary linoleic acid Auestad N, Montalto MB, Hall RT, Fitzgerald KM, Wheeler RE, Connor in¯uences desaturation and acylation of deuterium-labelled linoleic WE, Neuringer M, Connor SL, Taylor JA & Hartmann EE (1997): and alpha linolenic acids in young adult males. Biochim. Biophys. Visual acuity, erythrocyte fatty acid composition, and growth in term Acta 1213, 277 ± 288. infants fed formulas with long chain polyunsaturated fatty acids for one Escudero A, Montilla JC, Garcia JM, Sanchez-Quevedo MC, Periago JL, year. Ross Pediatric Lipid Study. Pediatr. Res. 41, 1 ± 10. Hortelano P, Suarez MD (1998): Effect of dietary (n-9), (n-6) and (n-3) Birth EE, Birth DG, Hoffman D & Uauy R (1993): Dietary essential fatty fatty acids on membrane lipid composition and morphology of rat acid supply and visual acuity development. Invest. Ophthal. Visual Sci. erythrocytes. Biochim. Biophys. Acta 1394, 65 ± 73. 33, 3242 ± 3253. FAO=WHO (1978): The role of dietary fats and oils in human nutrition.A Birch EE, Hoffman DR, Uauy R, Birch DG & Prestidge C (1998): Visual Joint Expert Consultation, Nutrition Report no. 3. Rome: FAO. acuity and the essentiality of docosahexaenoic acid and arachidonic acid Farquharson J, Cockburn F, Patrick AW, Jamieson E & Logan RW (1993): in the diet of term infants. Pediatr. Res. 44, 201 ± 209. Infant cerebral cortex Phospholipid fatty acid composition and diet. Bourre JM, Marianne FJ, Youyou A, Dumont O, Picotti M, Pascal G & Lancet 340, 810 ± 813. Durand G (1989): The effects of dietary alpha-linolenic acid on the Farquharson J, Jamieson EC, Logan RW, Patrick WJA, Howatson AG & composition of nerve membranes, enzymatic activity, amplitude of Cockburn F (1995): Age- and dietary-related distribution of hepatic electrophysiological parameter, resistance to poisons and performance arachidonic and docosahexaenoic acid in early infancy. Pediatr. Res. of learning tasks in rats. J. Nutr. 58, 511 ± 520. 38(3), 361 ± 365. Budowski P, Leigh®eld MJ & Crawford MA (1987): Nutritional encepha- Folch J, Lees M & Slone-Stanley GH (1957): A simple method for the lomalacia in the chick: an exposure of the vulnerable period for isolation and puri®cation of total lipids from animal tissue. J. Biol. cerebellar development and the possible need for both o6 and o3 Chem. 226, 497 ± 507. fatty acids. Br. J. Nutr. 56, 511 ± 520. Galli C & Socini A (1983): Dietary lipids in pre- and post-natal develop- Carlson SE (1996): Arachidonic acid status of human infants: in¯uence of ment. In: Dietary Fats and Health, eds. EG Perkins & WJ Visek, vol. gestational age at birth and diets with very long chain n-3 and n-6 fatty 16, pp. 278 ± 301. Proceedings of American Oil Chemists' Society acids. J. Nutr. 126(4), 1092S ± 1098S. Conference, Chicago, USA. Carlson SE, Rhodes PG & Ferguson MG (1986): Docosahexaenoic acid Ghebremeskel K, Leigh®eld M, Leaf A, Costeloe K & Crawford MA status of preterm infants at birth and following feeding with human milk (1995): Fatty acid composition of plasma red cell phospohilipids of or formula. Am. J. Clin. Nutr. 44, 798 ± 804. preterm babies fed on breast milk and formulae. Eur. J. Pediatr. 154, Carlson SE, Cooke RJ, Rhodes PG, Peeples JM, Werkman SH & Tolley 46 ± 52. EA (1991): Long term feeding of formulas high in linolenic acid and Gunstone F & Norris FA (1983): Lipids in Foods. Oxford: Pergamon marine ®sh oil to very low birth weight infants: phospholipid fatty acids. Press. Pediatr. Res. 30, 404 ± 412. Gurr MI (1984): Agricultural aspects of lipids. In:. The Lipid Handbook, Carlson SE, Werkman SH, Peeples JM, Cooke RJ & Tolley EA (1993): eds. Gunstone FD, Harewood JL & Padley FB. London: Chapman & Arachidonic acid status correlates with ®rst year growth in preterm Hall. infants. Proc. Natl Acad. Sci. USA 90, 1073 ± 1077. Hilditch TP & Williams PN (1964): The Chemical Composition of Natural Carlson SE, Werkman SH, Peeples JM & Wilson WM (1994): Long-chain Fats. London, Chapman & Hall. fatty acids and early visual and cognitive development of preterm Hoffman DR & Uauy R (1992): Essentially of dietary omega 3-fatty acids infants. Eur. J. Clin. Nutr. 48(2), S27 ± 30. for premature infants: plasma and red blood cell fatty acid composition. Carlson SE, Werkman SH & Tolley EA (1996a): Effect of long-chain Lipids 55, 395 ± 399. essential fatty acid supplementation on visual acuity and growth of Innis SM (1992): Plasma and red blood cell fatty acid values as indexes of preterm infants with and without bronchopulmonary dysplasia. Am. J. essential fatty acids in the developing organs of infants fed with milk or Clin. Nutr. 63, 687 ± 697. formulas. J. Pediatr. 120 , S78 ± 86. Carlson SE, Ford AJ, Werkman SH, Peeples JM & Koo WW (1996b): Innis SM, Foote KD, MacKinnon MJ & King DJ (1990): Plasma and red Visual acuity and fatty acid status of term infants fed human milk and blood cell fatty acids of low birthweight infants fed their mother's formulas with and without docosahexaenoate and arachidonate from expressed milk or preterm infant formula. Am. J. Clin. Nutr. 51, egg yolk lecithin. Pediatr. Res. 39(5), 882 ± 888. 994 ± 1000. Carlson SE, Montalto MB, Ponder DL, Werkman SH & Korones SB Jones CR, Arai T & Rapoport SI (1997): Evidence for the involvement of (1998): Lower incidence of necrotizing enterocolitis in infants docosahexaenoic acid in cholinergic stimulated signal transduction at fed a preterm formula with egg phospholipids. Pediatr. Res. 44, the synapse. Neurochem. Res. 22(6), 663 ± 670. 491 ± 498. Koletzko B & Braun M (1991): Arachidonic acid and early human growth. Clausen J & Moller D (1967): Allergic encephalomyelitis induced by brain Is there a relation? Ann. Nutr. Metab. 35, 128 ± 131. antigen after de®ciency in polyunsaturated fatty acids during myelina- Koletzko B, Decsi T & Demmelmair H (1996): Arachidonic acid supply tion. Acta Neurol. Scand. 43, 375 ± 388. and metabolism in human infants born at full term. Lipids 31(1), Connor WE, Neuringer M & Reisbick S (1992): Essential fatty acids: 79 ± 83. the importance of n-3 fatty acids in the retina and brain. Nutr. Rev. 50, Lamptey MS & Walker BL (1976): A possible essential role for dietary 21 ± 29. linolenic acid in the development of the young rat. J Nutr. 10, 86 ± 89. Crawford MA & Sinclair AJ (1972): Nutritional in¯uences in the evolution Leaf AA, Leigh®eld MJ, Costeloe KL & Crawford MA (1992): Factors of the mammalian brain. In: Lipids, Malnutrition and the Developing affecting the long-chain polyunsaturated fatty acid composition of Brain, eds. K Elliot & J Knight, pp 267 ± 292. Ciba Foundation plasma choline phospohglycerides in preterm infants. J. Pediatr. Gas- Symposium. Amsterdam: Elsevier. troenterol. Nutr. 14, 300 ± 308. Crawford MA, Casperd NM & Sinclair AJ (1976): The long chain Leyton J, Drury PJ & Crawford MA (1987): Differential oxidation of metabolites of linoleic and linolenic acids in liver and brain saturated and unsaturated fatty acids in vivo in the rat. Br. J. Nutr. 57, in herbivores and carnivores. Comp. Biochem. Physiol. 54B, 383 ± 393. 395 ± 401. Makrides M, Simmer K, Goggin M & Gibson RA (1993): Erthrocyte Crawford MA, Doyle W, Drury P, Ghebremeskel K, Harbige L, Leyton J docasahexaenoic acid correlates with visual response of healthy term & Williams G (1988): The food chain for n-6 and n-3 fatty acid. In: infants. Pediatr. Res. 34, 425 ± 427. Proceedings of NATO Advanced Workshop on Dietary !3 and !6 Fatty Makrides M, Neumann MA, Byard RW, Simmer K & Gibson RA (1994): Acids. Biological Effects and Nutritonal Essentiality, Belgirate, eds. C Fatty acid composition of brain, retina and erythrocytes in breast- and Galli, AP Simopoulos, pp 5 ± 19. New York: Plenum Press. formula-fed infants. Am. J. Clin. Nutr. 60, 189 ± 194. Crawford MA, Doyle W, Drury P, Lennon A, Costeloe K & Leigh®eld MJ Martinez M & Vazquez E (1998): MRI evidence that docosahexaenoic (1989): N-6 and n-3 fatty acids during early human development. acid ethyl ester improves myelination in generalized peroxisomal J. Intern. Med. 225(Suppl 1), 159 ± 169. disorders. Neurology 51, 26 ± 32.

European Journal of Clinical Nutrition Relationship between AA and DHA in pregnant women K Ghebremeskel et al 56 Maurage C, Guesnet P, Pinault M, Rochette de Lempdes J, Durand G, Suzuki H, Park SJ, Tamura M & Ando S (1998): Effect of the long-term Antoine J & Couet C (1998): Effect of two types of ®sh oil supple- feeding of dietary lipids on the learning ability, fatty acid composition mentation on plasma and erythrocyte phospholipids in formula-fed term of brain stem phospholipids and synaptic membrane ¯uidity in adult infants. Biol. Neonate 74, 416 ± 429. mice: a comparison of sardine oil diet with palm oil diet. Mech. Ageing Moore DJ, Gioioso S, Sills RH & Mendelsohn R (1999): Some relation- Devl. 101, 119 ± 128. ships between membrane phospholipids domains conformational order, Tinoco J (1982): Dietary requirements and functions of a-linolenic acid in and cell shape in intact human erythrocytes. Biochim. Biophys. Acta animals. Prog. Lipid Res. 20, 1 ± 45. 1415, 342 ± 438. Uauy R, Birch E & Birch D (1992): Visual and brain function measure- Oski FA (1993): Infant nutrition, physical growth, breatfeeding, and ments in study of n-3 fatty acid requirements of infants. J. Pediatr. 120, general nutrition. Curr. Opin. Pediatr. 5, 385 ± 388. S168 ± 180. Putnam JC, Carlson SE, Devoe PW & Barness LA (1982): The effect of Velzing-Aarts F, van der Klis FRM, van der Dijs FPL & Muskiet FAJ variations in dietary fatty acids on the fatty acid composition of (1999): Umbilical vessels of preeclamptic women have low contents of erythrocyte phosphatidylcholine and phosphatidylethanolamine in both n-3 and n-6 long chain polyunsaturated fatty acids. Am. J. Clin. human infants. Am. J. Clin. Nutr. 36, 106 ± 114. Nutr. 69, 293 ± 298. Ruyle M, Connor WE, Anderson GJ & Richard IL (1990): Placental Wainwright PE, Xing HC, Mutsaers L, McCutcheon D & Kyle D (1997): transfer of essential fatty acids in humans: venous ± arterial difference Arachidonic acid offsets the effects on mouse brain and behavior of a for docosahexaenoic acid in fetal umbilical erythrocytes. Proc. Natl diet with a low (n-6):(n-3) ratio and very high levels of docosahex- Acad. Sci. USA 87, 7902 ± 7906. aenoic acid. J. Nutr. 127, 184 ± 193. Scariati PD, Grummer-Strawn LM & Fein SB (1997)): A longitudinal Willatts P, Forsyth JS, DiModugno MK, Varma S & Colvin M (1998): analysis of infant morbidity and the extent of breastfeeding in the Effect of long chain polyunsaturated fatty acids in infant formula on United States. Pediatrics 99(6), E5. problem solving at 10 months of age. Lancet 352, 688 ± 691. Sinclair AJ (1975): Long chain polyunsaturated fatty acids in the mam- Young C, Gean PW, Wu SP, Lin CH & Shen YZ (1998): Cancellation of malian brain. Proc. Nutr. Soc. 34, 287 ± 291. low-frequency stimulation-induced long-term depression by docosahex- Sprecher H (1993): Interconversions between 20 and 22 carbon n-3 and n- aenoic acid in the rat hippocampus. Neurosci. Lett. 247, 198 ± 200. 6 fatty acids via 4-desaturase independent pathways. In: IIIrd Interna- tional Congress on Essential Fatty Acids and Eicosanoids, ed. AJ Sinclair & R Gibson, pp 18 ± 22. American Oil Chemists' Society.

European Journal of Clinical Nutrition