Effect of fatty acid status in cord blood serum on children's behavioral difficulties at 10 y of age: results from the LISAplus Study.

BACKGROUND: Little is known about the effect of fatty acid (FA) concentrations in cord blood on long-term behavioral outcomes. OBJECTIVE: We assessed the effect of FAs in cord blood serum on children's behavioral difficulties at the age of 10 y. DESIGN: A longitudinal study of 416 children from the population-based Influences of Lifestyle-Related Factors on the Immune System and the Development of Allergies in Childhood (LISAplus) birth cohort from Munich was conducted. Individual glycerophospholipid FAs in blood were analyzed in venous cord blood. Data on children's behavior were collected with a parent-reported Strength and Difficulties Questionnaire at 10 y of age. Zero-inflated Poisson regression models were applied and adjusted for sex, parental income, smoking during pregnancy, and dietary intake of arachidonic acid (AA) and DHA at 10 y. RESULTS: A 1% increase in DHA in cord blood serum was found to decrease total difficulties by (exp)ß(adj) = 0.93 (SE = 0.02, P < 0.0001) and hyperactivity or inattention by (exp)ß(adj) = 0.94 (SE = 0.03, P < 0.04). Higher long-chain (LC) PUFA concentrations in cord blood serum were associated with fewer emotional symptoms [(exp)ß(adj) = 0.95, SE = 0.03, P = 0.01], and similarly higher AA concentrations were associated with fewer emotional symptoms [(exp)ß(adj) = 0.94, SE = 0.03, P = 0.03]. CONCLUSION: Increased concentrations of DHA, LC-PUFAs, and AA in cord blood serum were associated with lower scores on a parent-completed behavioral screen. An appropriate FA supply to the developing fetus may be essential for optimal long-term behavioral outcomes in children.

Reversed phase LC/MS/MS method for targeted quantification of glycerophospholipid molecular species in plasma.

The relationship between lipid status and metabolism, infant development and health has widely been studied, but the importance of individual glycerophospholipid species for biological functions in infants has hardly been considered. We developed a method for quantitative analyses of plasma glycerophospholipids from small sample volume. Proteins were precipitated with methanol, which eliminated further sample preparation. The supernatant was analysed by reversed-phase HPLC using a gradient of water, methanol and isopropanol as mobile phase. Electrospray ionisation in negative mode in combination with tandem mass spectrometry enabled detection of specific fatty acids as fragments of glycerophospholipid species. With this combination of chromatography and mass spectrometry, PC, lyso-PC, PE and lyso-PE species and their relevant isobaric compounds were quantified. Method validation showed a linear working range between 0.05 µmol/L and 10 µmol/L in diluted plasma samples. The intra-assay coefficients of variation (n=6) ranged from 1.1% to 13.9%. Results were comparable with data of the human metabolome database and gas chromatographic fatty acid analyses. All quantitatively important PE and PC species are covered. The method can be applied for investigating dietary effects on plasma GP composition from small plasma volumes.

Influence of FADS polymorphisms on tracking of serum glycerophospholipid fatty acid concentrations and percentage composition in children.

BACKGROUND: Tracking of fatty acid (FA) contribution to plasma or serum lipids over time was shown in children and adults. However, the potential role of FADS gene variants has not been investigated. METHODS AND PRINCIPAL FINDINGS: Serum GP FA composition of 331 children aged 2 and 6 years, participating in an ongoing birth cohort study, was analyzed. Correlation coefficients were estimated to describe FA tracking over 4 years and to assess the influence of FADS variants on tracking. We found low to moderate tracking (r = 0.12-0.49) of FA compositions and concentration between 2 and 6 years. Concentration changes of total monounsaturated FA and total saturated FA over time correlated closely (r = 0.79) but percentage values were unrelated (r = -0.02). Tracking for n-6 long chain polyunsaturated fatty acid (LC-PUFA) concentrations was lower in subjects homozygous for the major allele of FADS variants and higher in carriers of at least one minor allele, whereas for total n-3 LC-PUFA concentrations and compositions this was vice versa. For individual n-3 PUFA inconsistent results were found. CONCLUSIONS AND SIGNIFICANCE: Serum GP FA composition shows low to moderate tracking over 4 years with a higher tracking for LC-PUFA metabolites than for their precursor FA. Serum PUFA levels and their tracking seem to be more influenced by lipid and lipoprotein metabolism than by FA specific pathways.

Fatty acid status determination by cheek cell sampling combined with methanol-based ultrasound extraction of glycerophospholipids.

Tissue and blood fatty acid compositions are used as biological markers of fatty acid intakes to improve dietary assessments. These approaches are invasive and not well accepted, particularly in infants and young children. We developed a sensitive method for the analysis of fatty acids in cheek cell glycerophospholipids, which includes significant improvements of cell sampling, non-chromatographic isolation of glycerophospholipids and base-catalyzed synthesis of fatty acid methyl esters. Sphingophospholipids are not detected by this method. This enables a highly accurate determination of cheek cell glycerophospholipid fatty acids, even if cell numbers are limited. Coefficients of variation for fatty acids contributing more than 0.3% to total glycerophospholipid fatty acids are below 10% in samples with more than 10(5) cells. Good correlations were shown between docosahexaenoic and eicosapentaenoic acid percentages in cheek cell and plasma glycerophospholipids (r = 0.83 and 0.64, respectively; P < 0.001) with a linear relationship over the whole concentration range observed in adult study participants (n = 29). Cheek cell sampling is non-invasive and can easily be applied in infants. The accuracy and reliability of this new method is comparable to conventional invasive methods for the determination of the n-3 fatty acid status in humans, and it is well applicable in interventional or epidemiological studies.

Genetic variation in polyunsaturated fatty acid metabolism and its potential relevance for human development and health.

Blood and tissue contents of polyunsaturated fatty acid (PUFA) and long-chain PUFA (LC-PUFA) are related to numerous health outcomes including cardiovascular health, allergies, mental health and cognitive development. Evidence has accumulated to show that in addition to diet, common polymorphisms in the fatty acid desaturase (FADS) gene cluster have very marked effects on human PUFA and LC-PUFA status. Recent results suggest that in addition to fatty acid desaturase 1 and fatty acid desaturase 2, the gene product of fatty acid desaturase 3 is associated with desaturating activity. New data have become available to show that FADS single nucleotide polymorphisms (SNPs) also modulate docosahexaenoic acid status in pregnancy as well as LC-PUFA levels in children and in human milk. There are indications that FADS SNPs modulate the risk for allergic disorders and eczema, and the effect of breastfeeding on later cognitive development. Mechanisms by which FADS SNPs modulate PUFA levels in blood, breast milk and tissues should be explored further. More studies are required to explore the effects of FADS gene variants in populations with different ethnic backgrounds, lifestyles and dietary habits, and to investigate in greater depth the interaction of gene variants, diet and clinical end points, including immune response and developmental outcomes. Analyses of FADS gene variants should be included into all sizeable cohort and intervention studies addressing biological effects of PUFA and LC-PUFA in order to consider these important confounders, and to enhance study sensitivity and precision.

Variants of the FADS1 FADS2 gene cluster, blood levels of polyunsaturated fatty acids and eczema in children within the first 2 years of life.

BACKGROUND: Association of genetic-variants in the FADS1-FADS2-gene-cluster with fatty-acid-composition in blood of adult-populations is well established. We analyze this genetic-association in two children-cohort-studies. In addition, the association between variants in the FADS-gene-cluster and blood-fatty-acid-composition with eczema was studied. METHODS AND PRINCIPAL FINDINGS: Data of two population-based-birth-cohorts in The Netherlands and Germany (KOALA, LISA) were pooled (n = 879) and analyzed by (logistic) regression regarding the mutual influence of single-nucleotide-polymorphisms (SNPs) in the FADS-gene-cluster (rs174545, rs174546, rs174556, rs174561, rs3834458), on polyunsaturated fatty acids (PUFA) in blood and parent-reported eczema until the age of 2 years. All SNPs were highly significantly associated with all PUFAs except for alpha-linolenic-acid and eicosapentaenoic-acid, also after correction for multiple-testing. All tested SNPs showed associations with eczema in the LISA-study, but not in the KOALA-study. None of the PUFAs was significantly associated with eczema neither in the pooled nor in the analyses stratified by study-cohort. CONCLUSIONS AND SIGNIFICANCE: PUFA-composition in young children's blood is under strong control of the FADS-gene-cluster. Inconsistent results were found for a link between these genetic-variants with eczema. PUFA in blood was not associated with eczema. Thus the hypothesis of an inflammatory-link between PUFA and eczema by the metabolic-pathway of LC-PUFAs as precursors for inflammatory prostaglandins and leukotrienes could not be confirmed by these data.

High-throughput analysis of total plasma fatty acid composition with direct in situ transesterification.

BACKGROUND: Plasma fatty acid (FA) composition reflects dietary intake and endogenous turnover and is associated with health outcomes on a short and long term basis. The total plasma FA pool represents the composition of all FA containing lipid fractions. We developed a simplified and affordable high-throughput method for the analysis of total plasma FA composition, suitable for large studies. METHODOLOGY/PRINCIPAL FINDINGS: The total lipid FA from 100 microl plasma is transferred in situ into methyl esters, avoiding initial extraction and drying steps. The fatty acid methyl esters are extracted once and analyzed by gas chromatography. For the new direct in situ transesterification method optimal, reaction parameters were determined. Intra-assay analysis (n=8) revealed coefficients of variation below 4% for FA contributing more than 1% to total FA. CONCLUSIONS/SIGNIFICANCE: The results show good agreement with FA concentrations obtained by a reference method. The new direct in situ transesterification method is robust and simple. Sample preparation time and analysis costs are reduced to a minimum. This method is an economically and ecologically superior alternative to conventional methods for assessing plasma FA status in large studies.

Fatty acid composition of serum glycerophospholipids in children.

OBJECTIVE: To determine reference values for fatty acid (FA) composition of serum glycerophospholipids (GPs) in children with a new high-throughput method. STUDY DESIGN: The GP FA composition of 1326 serum samples obtained from a cohort of 951 children at 2 and 6 years, participating in the German Influences of Lifestyle Related Factors on the Immune System and the Development of Allergies in Childhood (LISA) study, was analyzed with a new high-throughput method. Only 2 simple preparation steps were necessary to obtain fatty acid methyl esters selectively from serum GPs. The FA status was determined by separating and quantifying the fatty acid methyl esters with high-resolution capillary gas chromatography. RESULTS: FA values in serum GPs were in very good agreement with other published values in serum or plasma phospholipids for most of the analyzed FAs. No major age and sex differences in GP FA composition were observed. CONCLUSION: The serum GP FA values obtained from children aged 2 and 6 years may serve as reference values in clinical practice (eg, for monitoring and improving therapeutic interventions). Furthermore, they can serve as a reference point for interpreting FA values in clinical and epidemiological studies.

Role of FADS1 and FADS2 polymorphisms in polyunsaturated fatty acid metabolism.

Tissue availability of polyunsaturated fatty acids (PUFAs) depends on dietary intake and metabolic turnover and has a major impact on human health. Strong associations between variants in the human genes fatty acid desaturase 1 (FADS1, encoding Delta-5 desaturase) and fatty acid desaturase 2 (FADS2, encoding Delta-6 desaturase) and blood levels of PUFAs and long-chain PUFAs (LC-PUFAs) have been reported. The most significant associations and the highest proportion of genetically explained variability (28%) were found for arachidonic acid (20:4n-6), the main precursor of eicosanoids. Subjects carrying the minor alleles of several single nucleotide polymorphisms had a lower prevalence of allergic rhinitis and atopic eczema. Therefore, blood levels of PUFAs and LC-PUFAs are influenced not only by diet, but to a large extent also by genetic variants common in a European population. These findings have been replicated in independent populations. Depending on genetic variants, requirements of dietary PUFA or LC-PUFA intakes to achieve comparable biological effects may differ. We recommend including analyses of FADS1 and FADS2 polymorphism in future cohort and intervention studies addressing biological effects of PUFAs and LC-PUFAs.

High-throughput analysis of fatty acid composition of plasma glycerophospholipids.

Plasma FA composition, a marker of FA status and dietary intake, is associated with health outcomes on a short- and long-term basis. Detailed investigation of the relationships between plasma FA composition and health requires the analysis of large numbers of samples, but manual sample preparation is very cumbersome and time consuming. We developed a high-throughput method for the analysis of FAs in plasma glycerophospholipids (GPs) with increased sensitivity. Sample preparation requires two simple steps: protein precipitation and subsequent base catalyzed methyl ester synthesis. Analysis of GP FAs is performed by gas chromatography. Coefficients of variation for FAs contributing more than 1% to total FAs are below 4%. Compared with the established reference method, results of the new method show good agreement and very good correlations (r > 0.9). The new method reduces the manual workload to about 10% of the reference method. Only 100 microl plasma volume is needed, which allows for the analysis of samples from infants. The method is well suitable for application in large clinical trials and epidemiological studies.