Studies suggest that dietary changes, especially an increased consumption of n-6 polyunsaturated fatty acids (PUFAs) and a decreased intake of n-3 PUFA, may contribute to the increasing prevalence of allergic diseases in children observed in recent decades [1, 2].
Arachidonic acid (20:4n-6, AA), a product of the essential n-6 PUFA linoleic acid (18:2n-6, LA), can act as substrate and be converted to pro-inflammatory eicosanoids . Using the same enzymatic pathway, the essential n-3 PUFA α-Linolenic acid (18:3n-3, ALA) is transformed to the long-chain PUFA (LC-PUFA) eicosapentaenoic acid (20:5n-3, EPA), and then docosahexaenoic acid (22:6n-3, DHA) . EPA can also be metabolized into eicosanoids; however, this form has less potent inflammatory effects compared to that derived from AA . Additionally, E-series resolvins can be generated from EPA. D-series resolvins and protectins can be derived from DHA. Protectins and resolvins have anti-inflammatory and inflammation resolving effects .
Pregnancy has been suggested to be a critical period for developmental programming, which may influence the later development of allergic diseases in childhood . Maternal diet, especially maternal PUFA intake, during pregnancy may modify neonatal immune response through epigenetic mechanisms and therefore alter disease susceptibility and predisposition [4, 5]. It is proposed that a higher intake of n-3 PUFA, for example, by fish oil supplementation, may have protective effects against the development of atopic diseases in the offspring, although not all studies show conclusive results [5-7].
Cord blood (CB) LC-PUFA composition reflects the LC-PUFA supply of the offspring around birth .
Therefore, we hypothesize that higher n-3 LC-PUFA, lower n-6 LC-PUFA concentrations, and a lower n-6/n-3 ratio in CB serum are associated with lower prevalence of doctor-diagnosed asthma, eczema, hay fever/allergic rhinitis, as well as aeroallergen sensitization up to 10 yr of age, even after accounting for later LC-PUFA status.
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Information on CB serum n-3, n-6 LC-PUFAs, or the n-6/n-3 ratio, at least one of the diseases entities and LC-PUFA composition in blood at one or more follow-ups at 2, 6, or 10 yr of age was available for 436 children. The participant flow is illustrated in Fig. S1.
Basic characteristics of the study population are presented in Table 1. Of the 436 children with CB serum fatty acid measurements, 280 children were examined at 2 yr, 277 children at 6 yr, and 243 children at 10 yr. The period prevalence of eczema was constantly high up to age 6 and decreased afterwards, whereas the prevalence of hay fever/allergic rhinitis, asthma, and aeroallergen sensitization increased after age 6 yr. The mean percentage of n-3 as well as n-6 LC-PUFA was higher in CB serum than in blood at later follow-ups, but the n-6/n-3 ratio remained stable.
Table 1. Basic characteristics of the study population. Values are presented as mean (SD) or percentage
| ||Birth||2 yr||6 yr||10 yr|
| N ||436||280||277||243|
|Eczema, %|| ||15||14||7|
|Asthma, %|| || ||3||5|
|Hay fever/allergic rhinitis, %|| || ||3||13|
|Aeroallergen sensitization, %|| || ||24||43|
|n-6 LC-PUFA a||25.3 (1.5)||13.3 (1.5)||13.6 (1.6)||15 (1.6)|
|n-3 LC-PUFA a||8.1 (1.6)||4.5 (0.8)||4.5 (0.9)||4.8 (1.1)|
|n-6/n-3 ratio||3.3 (0.7)||3.0 (0.5)||3.1 (0.6)||3.2 (0.6)|
|Total sum of fatty acids (mg/l)||644.7 (103.3)||1146.5 (160.9)||1198.8 (182.4)||1257.4 (202.5)|
|Parental education (% high)||82|| || || |
|Sex (% female)||43|| || || |
|Age (yr)|| ||2.01 (0.03)||6.1 (0.2)||10.2 (0.2)|
|Maternal age (yr)||32.7 (3.9)|| || || |
|Parental atopy (% yes)||64|| || || |
All investigated variables were tested for differences between children who participated in this analysis with the remaining children from the Munich study center who were not included (Table S1). There was a lower percentage of girls, and a higher parental education level in study participants (p = 0.0394 and p = 0.0229, respectively).
Table 2 shows mean and SD of n-3 LC-PUFA, n-6 LC-PUFA concentrations, and n-6/n-3 ratio in CB serum in children with (cases) and without (controls) eczema, asthma, hay fever/allergic rhinitis, or aeroallergen sensitization, respectively. It was tested whether the mean of n-3 LC-PUFA, n-6 LC-PUFA concentrations, and n-6/n-3 ratio in CB serum differs significantly between cases and controls. Children with aeroallergen sensitization at 10 yr had lower n-6 LC-PUFA concentrations in CB serum (mean in controls: 25.6 vs. mean in cases: 25.2, p = 0.0326).
Table 2. Mean and SD of n-3 LC-PUFA, n-6 LC-PUFA concentrations, and n-6/n-3 ratio in CB serum in children with (cases) and without (controls) eczema, asthma, hay fever/allergic rhinitis or aeroallergen sensitization, respectively. Differences were tested using t-test
| ||n-3 LC-PUFA in CBa||n-6 LC-PUFA in CBa||n-6/n-3 ratio in CB|
|2 yr||8.1 (1.6)||8.0 (1.6)||0.4831||25.2 (1.5)||25.4 (1.4)||0.2721||3.2 (0.7)||3.3 (0.7)||0.2717|
|6 yr||8.1 (1.6)||8.2 (1.5)||0.5270||25.3 (1.5)||25.2 (1.2)||0.7360||3.2 (0.7)||3.2 (0.6)||0.2808|
|10 yr||8.1 (1.6)||7.7 (1.3)||0.1316||25.3 (1.4)||25.4 (1.6)||0.7504||3.2 (0.7)||3.4 (0.6)||0.2449|
|6 yr||8.1 (1.6)||7.5 (1.3)||0.0817||25.3 (1.5)||25.7 (1.2)||0.1175||3.2 (0.7)||3.4 (0.5)||0.1325|
|10 yr||8.1 (1.6)||8.4 (1.3)||0.4137||25.3 (1.4)||25.5 (1.2)||0.3980||3.2 (0.7)||3.1 (0.6)||0.4917|
|Hay fever or allergic rhinitis|
|6 yr||8.1 (1.6)||8.3 (1.6)||0.4957||25.3 (1.4)||24.9 (1.8)||0.2940||3.2 (0.7)||3.1 (0.6)||0.1888|
|10 yr||8.1 (1.6)||8.0 (1.4)||0.7138||25.3 (1.5)||25.2 (1.3)||0.5704||3.2 (0.7)||3.2 (0.6)||0.8977|
|6 yr||8.1 (1.7)||8.0 (1.5)||0.8713||25.3 (1.4)||25.1 (1.5)||0.3079||3.3 (0.8)||3.2 (0.6)||0.6872|
|10 yr||8.0 (1.7)||8.1 (1.5)||0.5423||25.6 (1.4)||25.2 (1.5)||0.0326||3.3 (0.8)||3.2 (0.6)||0.1701|
Table 3 shows mean and SD of n-3 LC-PUFA, n-6 LC-PUFA concentrations and n-6/n-3 ratio cross-sectional at each follow-up in children with (cases) and without (controls) eczema, asthma, hay fever/allergic rhinitis, or aeroallergen sensitization, respectively. Children with eczema at 2 yr have significant lower concentrations of n-3 LC-PUFA (4.6 vs. 4.4, p = 0.0337) and a significant higher n-6/n-3 ratio (2.9 vs. 3.1, p = 0.0074). Additionally, lower n-3 LC-PUFA concentrations were observed in children with asthma at 6 yr (4.5 vs. 4.1, p = 0.0032) and aeroallergen sensitization at age 6 yr (4.5 vs. 4.3, p = 0.0205).
Table 3. Mean and SD of n-3 LC-PUFA, n-6 LC-PUFA concentrations and n-6/n-3 ratio at each follow-up in children with (cases) and without (controls) eczema, asthma, hay fever/allergic rhinitis or aeroallergen sensitization, respectively. Differences were tested using t-test
| ||n-3 LC-PUFA at follow-upsa||n-6 LC-PUFA at follow-upsa||n-6/n-3 ratio at follow-ups|
|2 yr||4.6 (0.9)||4.4 (0.8)||0.0337||13.2 (1.5)||13.5 (1.6)||0.0715||2.9 (0.5)||3.1 (0.5)||0.0074|
|6 yr||4.5 (0.9)||4.4 (0.8)||0.3148||13.6 (1.6)||13.6 (1.4)||0.9646||3.1 (0.5)||3.1 (0.6)||0.6738|
|10 yr||4.5 (1.0)||4.5 (1.0)||0.5952||14.4 (1.9)||14.2 (1.7)||0.4852||3.3 (0.6)||3.3 (0.6)||0.9266|
|6 yr||4.5 (0.9)||4.1 (0.5)||0.0032||13.7 (1.6)||13.1 (1.3)||0.1217||3.1 (0.5)||3.1 (0.6)||0.8204|
|10 yr||4.5 (1.0)||4.4 (1.1)||0.5148||14.4 (1.9)||14.5 (2.2)||0.6645||3.3 (0.6)||3.4 (0.6)||0.1650|
|Hay fever or allergic rhinitis|
|6 yr||4.5 (0.9)||4.4 (0.9)||0.6619||13.6 (1.6)||13.8 (1.6)||0.6287||3.1 (0.5)||3.2 (0.5)||0.3884|
|10 yr||4.5 (1.0)||4.4 (1.1)||0.3204||14.4 (1.9)||14.3 (1.9)||0.7181||3.3 (0.6)||3.3 (0.7)||0.2460|
|6 yr||4.5 (0.9)||4.3 (0.8)||0.0205||13.7 (1.5)||13.6 (1.6)||0.8680||3.1 (0.6)||3.2 (0.5)||0.1074|
|10 yr||4.5 (1.0)||4.6 (1.1)||0.3149||14.3 (1.9)||14.5 (1.9)||0.1193||3.3 (0.6)||3.3 (0.6)||0.9008|
The results of GEE on allergic diseases are presented in Table 4. There was no significant association between n-3 LC-PUFA, n-6 LC-PUFA concentrations, or the n-6/n-3 ratio in CB serum with any of the investigated outcomes. Thus, the CB serum LC-PUFA concentrations do not appear to have any effect on allergic disease risk at any time point investigated.
Table 4. Results of a GEE in which n-3, n-6 LC-PUFA concentrations, and n-6/n-3 ratio in CB serum are regressed on allergic diseases. ORs are adjusted for parental education, sex, time of follow-up (2 yr, 6 yr or 10 yr for eczema; 6 yr and 10 yr for asthma, hay fever/allergic rhinitis and aeroallergen sensitization), age, maternal age at birth, parental atopy, total sum of fatty acids, as well as LC-PUFA concentrations at follow-up (FU) were included
| || N ||n-3 LC-PUFAa|| N ||n-6 LC-PUFAa|| N ||n-6/n-3 ratio|
|Hay fever or allergic rhinitis|
Children with eczema at a given age was significantly associated with lower concentrations of n-3 LC-PUFA at this same age [OR = 0.71, 95%-CI = (0.55–0.92), p = 0.0085].
In an additional sensitivity analysis, the GEE models were restricted to children with complete information on eczema, asthma, hay fever/allergic rhinitis, or aeroallergen sensitization and fatty acid measurements at all time points (Table S2). This analysis showed similar results. Additionally, there was a borderline significant inverse association between n-6 LC-PUFA in CB serum and eczema [0.83 (0.68–1.02), p = 0.0784].
In a further sensitivity analysis, general linear models for each follow-up were conducted (Table S3). The results from the GEE models were confirmed by this analysis. There was also no significant association between CB serum n-3, n-6 LC-PUFA concentrations, or the n-6/n-3 ratio with any of the allergic diseases. Children with eczema at the age of 6 yr had significantly lower concentrations of n-3 LC-PUFA at the same age [0.60 (0.38–0.97), p = 0.0364], but not at 2 yr [0.70 (0.45–1.09), p = 0.1189] or 10 yr [0.90 (0.51–1.59), p = 0.7212]. Aeroallergen sensitization at age 6 yr was inverse associated with n-3 LC-PUFA at the same age [0.68 (0.47–0.98), p = 0.0407], but not at 10 yr [0.90 (0.69–1.18), p = 0.4619].
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This study investigated the association between n-3 and n-6 LC-PUFA composition as well as the n-6/n-3 ratio in CB serum and the development of eczema, asthma, hay fever/allergic rhinitis and allergic sensitization up to 10 yr of age, after accounting for LC-PUFA concentrations in blood using longitudinal data from the Munich LISAplus birth cohort.
We could not find a protective effect of elevated n-3 LC-PUFA concentrations in CB serum on any of the investigated disease entities. When fatty acid concentrations measured at the same time point were entered in the model, children with eczema exhibit lower levels of n-3 LC-PUFA.
To our knowledge, this is the first study in which repeated fatty acids measurements starting from birth to 10 yr of age were included using a longitudinal model.
The ALSPAC study is the only observational study which published data on fatty acid concentrations in CB serum and atopic outcomes, but without adjustment for fatty acids later in life. There was some evidence; however, the results were not significant any more after adjustment for multiple testing, and therefore, Newson et al. concluded that it is unlikely that CB serum fatty acid concentrations are an important risk factor for the later development of allergic diseases .
Several observational and interventional studies explored the association between atopic diseases and diet [7, 16-18] as well as maternal n-3 LC-PUFA status during pregnancy [19-24] or lactation [25-27].
In an earlier publication based on data from the LISAplus birth cohort, the association between maternal dietary intake during the last 4 wk of pregnancy and allergic diseases in the offspring up to the age of 2 yr was investigated . In this analysis, a higher intake of n-6 PUFA rich foods such as margarine and vegetable oils and a lower intake of n-3 PUFA rich foods such as fish were associated with eczema in the offspring. In our analysis, we used exact measurements of the n-3 and n-6 LC-PUFA concentrations in CB serum instead of food intake as proxy but could not replicate this finding. However, in this study, we focused on LC-PUFAs, whereas margarine is rich in linoleic acid, which may mediate the effect, but was not considered in the current analysis. Additionally, our study population was limited to the Munich study center only and the sample size was reduced due to limited blood sample availability.
In fact, the most consistent evidence was found in studies focusing on dietary intake, especially on dietary fish intake as a source of n-3 PUFA [7, 16-18]. Therefore, it has been hypothesized that a sufficient n-3 LC-PUFA supply may prevent from atopic diseases, especially during the pre- and early post-natal period when the immature immune system is developing [5, 28].
Some intervention studies have been conducted to investigate the association between n-3 LC-PUFA status and atopic diseases aiming to increase the n-3 LC-PUFA concentrations during pregnancy [19-24] or lactation [25-27]. The studies in which cytokines were measured, reported a more favorable immune response [19-21, 25, 27].
Nevertheless, it is not clear whether these immunological changes can avoid clinical manifestation of allergic diseases. The studies which investigated clinical outcomes, reported less or less severe atopic diseases in the intervention group [20, 22-24, 26], although most of these studies had a short follow-up period of 1 year [20, 23, 24, 26], a small sample size with few cases [20, 23] or the results were not significant anymore after adjustment for multiple comparisons [24, 26]. Olsen et al.  used registry data to follow-up children at 16 yr of age and found a strong reduction of asthma and especially atopic asthma risk in the intervention group.
The findings from observational studies investigating food intake could not be confirmed by interventional studies. This might be due to the above mentioned limitations or due to residual confounding in observational studies, whereas dietary intake is related to a certain life-style. Donahue et al. investigated the associations between n-3 and n-6 PUFAs in maternal diet, maternal blood, and CB plasma and found the strongest correlation for DHA+EPA. Therefore, the assessment of n-3 LC-PUFA in diet and blood should lead to similar results, whereas the exact measurement in blood is supposed to be more accurate.
In our study, we could not find a protective effect of n-3 LC-PUFA concentration in CB serum on any of the allergic diseases, as seen for the ALSPAC study . CB serum fatty acid composition represents the fatty acid supply of the fetus at birth. The models were adjusted for the fatty acid composition in blood at each follow-up to rule out confounding by lifestyle or dietary factors, which may be reflected in fatty acid concentrations in blood. The longitudinal study design increased this study's statistical power, as the maximal number of observations available was included in the models, and allows modeling the effect of LC-PUFA in CB serum as well at the follow-ups at several time points up to 10 yr of age. Due to the constraints of the longitudinal model, the variables for asthma, eczema, and hay fever/allergic rhinitis which were asked yearly had to be combined into the periods between the blood measurements to obtain the same time points. In a sensitivity analysis, general linear models were conducted for each follow-up. The results were similar for all follow-ups, and we could not identify a time-varying effect of CB serum fatty acids. Another limitation of this study is the potential bias associated with non-random loss to follow-up. A comparison of all investigated variables between participants who were included in this analysis and non-participants showed a significantly higher percentage of parents with high education level in the participants group, but no significant differences in all other investigated variables. However, a sensitivity analysis restricted to children with complete data for all times of follow-up showed similar results, but, only in this analysis, there was a borderline significant inverse association between n-6 LC-PUFA in CB serum and eczema. A further limitation is the relatively low habitual fish and seafood intake in Germany, compared with other European countries , leading to a limited range of n-3 LC-PUFA intakes, whereas some of the intervention studies found effects on immune response with high dosages of fish oil supplementation.
As observational and interventional studies on dietary intake of fatty acids and allergic diseases are inconclusive , we thought that prospectively collected and longitudinally analysed data on fatty acid composition in CB serum could elucidate the protective association. However, this study with data on repeated measurements of fatty acids during a period of 10 yr failed to show strong and consistent associations.
Clearly, further interventional studies with adequate sample size, long-term follow-up, and repeated measurements are required to confirm the role of n-3 and n-6 PUFAs in the development of atopic diseases.
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This work was supported by grants 01 EG 9732, 01 EG 9705/2, and 01 EE 9401-4 from the Federal Ministry for Education, Science, Research, and Technology; FKZ 20462296 from the Federal Ministry of Environment; Ludwig-Maximilians-Universities’ innovative research priority Project MC-Health; the Commission of the European Communities, within the 7th Framework Programme, research grant no. FP7/2007-13 (EarlyNutrition Project), and the European Research Council, grant agreement n° 3226059. This abstract does not necessarily reflect the views of the Commission and in no way anticipates the future policy in this area. We thank all the families who participated in the LISAplus Study. Furthermore, we thank all members of the LISAplus Study group for their excellent work. We also acknowledge Larissa Piniek-Tzimourtas (University of Munich Medical Center, Dr von Hauner Children's Hospital, Division of Metabolic and Nutritional Medicine, Munich, Germany) for her excellent contribution to the measurements of fatty acid composition. The LISAplus Study group consists of the following: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Epidemiology I, Munich (J Heinrich, HE Wichmann, S Sausenthaler, CM Chen, and M Schnappinger); Department of Pediatrics, Municipal Hospital ‘St. Georg,’ Leipzig (M Borte and U Diez); Marien-Hospital Wesel, Department of Pediatrics, Wesel (A von Berg, C Beckmann, and I Groß); Pediatric Practice, Bad Honnef (B Schaaf); Helmholtz Centre for Environmental Research–UFZ, Department of Environmental Immunology/Core Facility Studies, Leipzig (I Lehmann, M Bauer, C Gräbsch, S Röder, and M Schilde); University of Leipzig, Institute of Hygiene and Environmental Medicine, Leipzig (O Herbarth, C Dick, and J Magnus); IUF–Institut für Umweltmedizinische Forschung, Düsseldorf (U Krämer, E Link, and C Cramer); Technical University Munich, Department of Pediatrics, Munich (CP Bauer and U Hoffmann); and ZAUM–Center for Allergy and Environment, Technical University, Munich (H Behrendt, J Grosch, and F Martin).