AJCN Tufts Nutrition Symposium, Boston & Online Sept 2009
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American Journal of Clinical Nutrition, Vol. 87, No. 5, 1170-1180, May 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Correlation between changes in blood fatty acid composition and visual sustained attention performance in children with inattention: effect of dietary n–3 fatty acids containing phospholipids1,2,3

Nachum Vaisman, Nehemia Kaysar, Yahalomit Zaruk-Adasha, Dori Pelled, Gérard Brichon, Georges Zwingelstein and Jacques Bodennec

1 From the Clinical Nutrition Unit (NV and YZ-A) and the Child and Adolescent Psychiatry Unit (NK), Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel; Enzymotec Ltd, Migdal-HaEmeq, Israel (DP); Université de Lyon, Université Lyon 1, Institut Michel Pacha (GB, GZ, and JB), UMR CNRS 5123 (JB), and CTRS-IDÉE, Hospices Civiles de Lyon (JB), Lyon, France

2 Supported by Enzymotec Ltd, Migdal-HaEmeq, Israel.

3 Address reprint requests to N Vaisman, Clinical Nutrition Unit, Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel-Aviv, 64239, Israel. E-mail: vaisman{at}tasmc.health.gov.il.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background:Increasing evidence supports n–3 fatty acid (FA) supplementation for patients with psychiatric disorders, such as attention deficit hyperactivity disorder. However, the exact metabolic fate of dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on different glyceride carriers remains unclear.

Objective:We investigated whether conjugation of EPA and DHA to phospholipid (PL-n–3) or to triacylglycerol (fish oil; FO) affects their incorporation in blood compartments and influences executive functioning.

Design:Children aged 8–13 y with impaired visual sustained attention performance received placebo, 250 mg/d EPA + DHA esterified to PL-n–3 (300 mg/d phosphatidylserine), or FO for 3 mo in a randomized double-blind manner. Main outcome measures included plasma and erythrocyte FA profile and continuous performance test results (Test of Variables of Attention; TOVA).

Results:Sixty of the 83 children enrolled completed the interventions (n = 18–21 per group). There was an enrichment of EPA (1.5–2.2-fold), docosapentaenoic acid (DPA; 1.2-fold), and DHA (1.3-fold) in the PL fraction in the plasma of FO- and PL-n–3–fed children. In erythrocytes, only PL-n–3 resulted in a significant reduction ({approx}30%) of very-long-chain saturated FAs (C20-24) and in an increase (1.2- and 2.2-fold, respectively) in linoleic acid and DPA. Total TOVA scores increased in the PL-n–3 (x ± SD: 3.35 ± 1.86) and FO (1.72 ± 1.67) groups but not in the placebo group (–0.42 ± 2.51) (PL-n–3 > FO > placebo; P < 0.001). A significant correlation between the alterations in FAs and increased TOVA scores mainly occurred in the PL-n–3 group.

Conclusion:Consumption of EPA+DHA esterified to different carriers had different effects on the incorporation of these FAs in blood fractions and on the visual sustained attention performance in children. This trial was registered at clinicaltrials.gov as NCT00382616.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The nervous system is the organ with the second largest concentration of lipids, exceeded only by adipose tissue. Approximately 35% of these lipids are long-chain polyunsaturated fatty acids (LC-PUFAs), such as arachidonic acid (AA; 20:4n–6) and docosahexaenoic acid (DHA; 22:6n–3) (1), which are known to play an essential role in brain development and functions (2). Both AA and DHA could be either provided in diet or synthesized in the body from the essential fatty acid (EFA) linoleic acid (LA; 18:2n–6) and {alpha}-linolenic acid (18:3n–3), respectively (3). Epidemiologic studies (4, 5) suggest that dietary consumption of n–3 LC-PUFAs, especially eicosapentaenoic acid (EPA; 20:5n–3) and DHA, affects neuropsychiatric disorders, presumably because of their structural and neurochemical involvement in pathophysiological processes (6-8). Moreover, interventions with n–3 LC-PUFAs ranging from 1 to 6.2 g/d EPA and from 0 to 3.4 g/d of DHA were associated with a therapeutic effect in a broad spectrum of psychiatric disorders (9-12). Similarly, supplementation of DHA-containing phospholipid (PL), such as the bovine brain cortex phosphatidylserine (PS), to animal models was shown to attenuate neuronal effects of aging (13) and to affect behavior as well (14, 15). In humans, administration of 300 mg/d PS for 3 mo to subjects with age-associated memory impairment resulted in a consistent improvement of performance in memory tests (16) and was also associated with beneficial effect on symptoms of depression in geriatric women (17).

Deficiencies of n–3 fatty acids (FAs) are associated with a wide range of neuropsychiatric and neurodevelopmental disorders, such as attention-deficit hyperactivity disorder (ADHD) (18-21), dyslexia (22), depression and aggression (6), and autism (23). The effect of DHA supplementation of 345 mg/d or 3.6 g/body wt, provided as ethyl esters (24) in capsules or as triacylglycerol in functional food (25), was tested in ADHD children. These interventions induced a pronounce increase in blood n–3 LC-PUFAs, but failed to affect ADHD symptoms. Results of recent studies, in which EPA- and DHA-enriched fish oils (FOs) were provided along with n–6 LC-PUFA containing oils and/or vitamins to ADHD children were conflicting (26-29).

To date, the role of the carrier in the metabolic fate of these FAs in schoolchildren, remains poorly studied. Current available reports, obtained mostly from infant formulas studies, compare DHA attached to the backbone of PL (eg, n–3–enriched egg yolk) to counter dietary intervention containing mostly fish or algal oils (30-32). Interestingly, early observations suggested that dietary LC-PUFAs esterified to PLs rather than triacylglycerols are more effective substrates for brain tissues accretion in term baboons (33).

In the current study, we investigated the differential incorporation of n–3 LC-PUFAs provided as either PL (mainly PS) or triacylglycerol (FO) into blood fractions compared with rapeseed oil in schoolchildren with impaired attention performance. The effect of these dietary supplements on the children's executive functions was also assessed.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
This study was conducted between July 2004 and January 2005 at Tel-Aviv Sourasky Medical Center (Tel-Aviv, Israel). The subjects were recruited during their summer vacation, when most schoolchildren with ADHD are voluntarily taken off stimulant medications. The 250 children or their surrogates who responded to a newspaper advertisement were first screened by telephone. Children were included if they were 8–13 y of age and had received a previous diagnosis of ADHD by a clinical psychiatrist, neurologist, or pediatrician. Children with significant sensory or neurological limitations, epilepsy, mental retardation, psychosis, or pervasive developmental disorder were excluded. Also excluded were children taking medications with known central nervous system effects, including stimulants or dietary supplements other than vitamins. Of these applicants (Figure 1Go), 102 (27 females) were screened with a continuous performance test (CPT), a Test of Variables of Attention (TOVA) (34) (see below), to determine their total TOVA score. A score lower than –1.8 (SD) from age- and sex-adjusted normal means suggests a high probability of impaired attention performance (34).


Figure 1
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  		FIGURE 1.. Flow diagram of subject (boys and girls) participation throughout the study.

 
All children were white, and no socioeconomic data were collected. The protocol was approved by the Institutional Review Board at Tel Aviv Sourasky Medical Center (TASMC-03-NV-220-CTIL), and written informed consent was obtained from all parents or legal guardians.

Protocol and diets
Eighty-three of the 102 eligible participants were randomly assigned in a double-blind manner to receive n–3 LC-PUFA–containing PLs (PL-n–3; Enzymotec LTD, Migdal HaEmeq, Israel), FO (Ocean Nutrition, NS, Canada), or placebo (rapeseed oil; Milumor, Kiryat Bialik, Israel) for 3 mo. The randomization sequence was a block randomization process (block size 3), ensuring equal numbers of male and female applicants in each arm of the trial after every 3 randomized subjects and approximately equal numbers overall (P = 0.8564; Figure 1Go).

Supplements were formulated by emulsifying the different lipid preparations to a dairy chocolate-flavored spread (Hashachar Chocolate Flavored Spread; Hashachar Haoleh, Haifa, Israel) with 4–7 mg/d citrus oil extract (Ganir, Sde Gat, Israel) that was added to buffer taste discernment (total 25 g/d). Tocopherol mixtures (0.2% by wt) were added to the base stock FO (control FO treatment) by the manufacturer (Ocean Nutrition Ltd, Halifax, Canada). No antioxidant other than the original preparation was introduced to the formulated matrix. The PL-n–3 was synthesized by Enzymotec Ltd (Migdal HaEmeq, Israel) using different sources of marine species. Rosemary extract, ascorbyl palmitate, and mixed natural tocopherols (0.8% by wt) were added to the final formulation. The participant's parents or guardians were instructed to spread a daily aliquot of enriched chocolate-flavored paste on a single slice of bread. Compliance was monitored weekly by phone calls. In addition, following the baseline visit, 14 identically appearing spreads containers were allocated and collected at 10–12-d intervals. The chocolate spread containers were weighted, and the dosage was determined according to the container weight. The daily PL and FA composition of the different treatments are given in Table 1Go. The parents were instructed to not provide the spreads to their children until after the blood samples were collected.


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  		TABLE 1. Phospholipid and main fatty acid composition of the study interventions

 
The subjects' parents were regularly asked to instruct their children to maintain their usual level of physical activity and diet throughout the study and to report any symptoms, disease onset, medication or dietary supplement consumption, or change in usual habits. Concomitant use of prescription or nonprescription agents with potent psychotropic properties, including ADHD treatments and dietary supplements (except for vitamins), was prohibited within 4 wk before the initial evaluation and during the entire intervention. Noncompliance was defined as poor consumption (<70%) or discontinuation of the assigned experimental diet or a parallel use of medications and/or dietary supplements as specified under exclusion criteria.

Blood lipid analysis
Nonfasting blood samples (5 mL) were drawn from a peripheral vein into heparinized tubes at baseline and at the end of the 3-mo intervention during the early morning hours. Erythrocytes were separated from plasma by Ficoll-Hypaque density-gradient centrifugation at 1260 x g for 30 min at 4 °C. The plasma and erythrocytes were immediately collected and stored at –80 °C until analyzed for lipids. The lipid analysis in plasma and erythrocytes, respectively, for subjects in the PL-n–3 (n = 1, 4), FO (n = 3, 3), and placebo (n = 2, 2) groups could not be performed at baseline or after 3 mo because of insufficient sample material.

Lipids were extracted from plasma and erythrocytes according to a modified Folch procedure (35). Briefly, the samples were homogenized in methanol and, after addition of chloroform [final chloroform –methanol ratio of (2:1, vol:vol)], extraction was allowed to occur at room temperature. The samples were filtered and washed twice with water containing 0.25% KCl (wt:vol). The chloroform lower phase evaporated, and the total lipid content was determined by weight. Total PLs were quantified after mineralization by using sulfuric acid:perchloric acid (2:1, vol:vol) containing 0.1% V2O4, followed by phosphate determination (36). Individual PLs were separated by 2-dimensional thin-layer chromatography (TLC) (silica gel plates G60; Merck, Darmstadt, Germany) by using tetrahydrofuran-acetone-methanol-water (50:20:40:6, vol:vol:vol:vol) as the first developing solvent and chloroform-acetone-methanol-acetic acid-water (50:20:10:15:5, vol:vol:vol:vol) as the second developing solvent (37). The TLC plate was subsequently dried, and the PLs were visualized with the Dittmer and Lester reagent (38) and quantified as described above.

Determinations of the PL and neutral lipid FA profiles from the indicated blood fractions were performed as follows: PLs were separated from neutral lipids by TLC using diisopropylether as the developing solvent, and FAs were converted to methyl esters using acetyl chloride (39). Neutral lipids were resolved from each other on TLC using hexane-diisopropylether–acetic acid (80:20:1, vol:vol:vol) as the developing solvent. Triacylglycerol and cholesterol esters plasma fractions, identified according to authentic standards, were transmethylated as described for PLs or first submitted to saponification and then methylated with acetyl chloride. FA methyl esters were quantified by a gas chromatography apparatus (Chrompack CP9000, Les Ulis, France) equipped with a flame ionization detector. Separation was achieved on a 30-m Omegawax 250 capillary column with an internal diameter of 0.25 mm and a film thickness of 0.25 µm (Supelco, Saint Quentin Fallavier, France). The carrier gas (nitrogen) flow rate was 15 mL/min at 60 kPa. Samples were injected at 250 °C. The oven temperature remained at 180 °C for 3 min after injection, was increased to 225 °C at a rate of 2 °C/min, and kept constant for 10 min, after which it rose to 235 °C at a rate of 10 °C/min. The temperature was then kept constant at 235 °C until 65 min, ie, until the end of data acquisition. The detector was set at 250 °C. FA methyl esters were identified by using authentic standards (Supelco).

Neuropsychological and behavioral tests
TOVA, an age- and sex-normalized computer-based assessment of inattention, was performed at baseline and after 3 mo (closure of the intervention). This computerized CPT includes a target stimulus and a nontarget stimulus (34). The stimuli are presented for 200 ms at the rate of 30/min. The duration of the test is 22.5 min. Targets are present on 22.5% of trials during the first half of the test and on 77.5% of the trials during the last half.

The variables measured were as follows: 1) errors of omission or failure to respond to the designated target that is interpreted as a measure of inattention, 2) errors of commission or erroneous response to the nontarget that is considered as a measure of impulsivity, 3) response time (in ms) or the latency time required to respond correctly to the target stimulus that is interpreted as a measure of information processing, and 4) response time variability (the SD of response times) that is interpreted as an index of consistency of attention. These results were further compared with normal, same-sex, same-age, and average intelligence quotient groups to be reported as SDs. The number of anticipatory responses (very short latency responses) that represent guessing is interpreted as a measure of impulsivity; this variable, however, is also used to ensure the validity of the results. A high anticipatory response score is interpreted as an invalid result according to the TOVA manual; thus, the total TOVA score could not be calculated. The SD indicates the extent of the disorder, ie, a more negative value points at specific parameter severity, whereas conversely, a more positive deviation from the normal value suggests a better than average performance. The total TOVA score, also referred to as the ADHD index score, includes the response time, d prime or response sensitivity, which is used to interpret the rate of performance change over time and response time variability. This score serves as an indication of the degree of similarity in performance between the assessed children and normative samples (34).

An additional behavior assessment included the Hebrew translation of the parental Abbreviated Conners Rating Scale (40), which is an easy-to-complete 10-item questionnaire graded on a Likert scale of 1–4 (not at all = 0, just a little = 1, pretty much = 2, and very much = 3). These items elicit observations of hyperactive-inattentive behaviors. A Hebrew translation of the 11-item mood or emotional liability subscale of Child Behavior Checklist was also assessed (41) and graded by the parents (0 = not true, 1 = somewhat or sometimes true, and 2 = very true or often true).

Statistical analysis
The statistical analyses were conducted for the subjects who completed the 3-mo intervention and had a minimal compliance of 70% throughout. The results are expressed as means ± SDs. All statistical tests were 2-tailed, and significance was set at a level of 0.05.

Baseline characteristics were compared using an analysis of variance (ANOVA) model for continuous variables and the Kruskal-Wallis test for nonparametric variables, except for randomization and sex, for which the Fisher's exact test was used. An arcsin transformation was made before the statistical analysis for the percentage values of the PL profile in plasma, and their changes were evaluated by using ANOVA followed by post hoc analysis based on Games-Howell comparisons. Baseline values were inserted into the model as covariates if they were found to be statistically different. The assumption of normal distribution and homogeneity of variance for plasma and erythrocyte lipids as well as TOVA variables were unmet. Accordingly, the effect of these interventions was analyzed using the Kruskal-Wallis nonparametric test followed by post hoc testing for differences between means with the use of Gabriel and Games-Howell tests, which adjust {alpha} = 0.05 for multiple comparison. Likewise, the differences within groups were determined by using Wilcoxon's signed-rank test. The differences in the proportion of subjects with total TOVA scores within normative values in the assigned groups at the end of the intervention were tested with a chi-square test followed by comparisons between the treatments by using Fisher's exact test. Spearman's rank-order correlation coefficient was used to evaluate the associations between changes in TOVA variable scores and changes in blood PL FAs over the intervention period. The statistical analyses were carried out by using SPSS statistical software (SPSS Inc, Chicago, IL) version 13.0.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Study subjects
A flow chart of subject participation is shown in Figure 1Go. A total of 102 putative ADHD children were recruited for this study. Eighteen of the applicants were withdrawn from the study because of invalid total TOVA score (34). Another candidate was dropped for unrelated health reasons (Figure 1Go).

The final 83 subjects were randomly assigned into 3 groups; 23 dropped out because of a dislike of the preparation taste (n = 10), noncompliance with the dietary regime (n = 6), adverse effects (n = 3 for gastrointestinal-related discomfort and vomiting; n = 1 for rash), reassignment to methylphenidate (n = 2), and reoccurrence of tics (n = 1). Most of these dropouts occurred within the first 2-wk after enrollment. Both the PL-n–3 and FO treatments were generally well tolerated by the participants, and there were no differences between the attrition rates of the different intervention groups (PL-n–3, 11/29; FO, 7/28; and placebo, 5/26; P = 0.1585). Biochemical and neuropsychological endpoints were unavailable for the dropouts, so that any change from baseline values could not be calculated for these subjects.

No significant differences were detected between the baseline characteristics of the 60 schoolchildren in the PL-n–3, FO, and placebo groups who completed the intervention (Table 2Go). The scores for the parental assessment of child behavior ratings at baseline suggested that these children had symptoms of inattention.


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  		TABLE 2. Baseline characteristics of the 60 children who completed the interventions

 
Plasma phospholipid profiles
The composition of serum PLs are shown in Table 3Go. The baseline PL profile analysis showed that the plasma phosphatidylcholine (PC) and phosphatidylethanolamine (PE) concentrations of the PL-n–3 group were statistically different from those of the FO group (P = 0.044 and 0.032, respectively) but not from the placebo group (P = 0.066 and 0.150, respectively). The tested interventions did not affect plasma PL concentration ({approx}32–42 mg/L; Table 3Go). Likewise, after the 3-mo consumption of lipid preparations, no differences were noted in plasma lysophosphatidylcholine, sphingomyelin, PC, phosphatidylinositol, and PE concentrations (P = 0.448, 0.278, 0.083, 0.432, and 0.317, respectively). Plasma PS concentrations were below the detection level for all groups, including PL-n–3–fed children. These results are consistent with previous observations (42), which suggest that within a few hours of dietary ingestion, PS is converted to other PLs via its lyso form and the subsequent decarboxylation in the mucosal cells.


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  		TABLE 3. Composition of phospholipids in the plasma of children with impaired visual sustained attention performance at baseline and changes after 3 mo of intervention1

 
Changes in the plasma FA profiles as a result of dietary interventions were found primarily in the PL rather than in the triacylglycerol or cholesterol ester fractions (data not shown). Plasma and erythrocyte PL FA compositions at baseline and the changes after 3 mo in the 3 intervention groups are listed in Table 4Go. There were no differences between the children's FA profiles in plasma and erythrocyte PLs at baseline.


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  		TABLE 4. Fatty acid composition in plasma and erythrocyte phospholipids in children with abnormal total Test of Variables of Attention (TOVA) scores at baseline and the subsequent changes after 3 mo of treatment1

 
Saturated fatty acids
Of the saturated fatty acids (SFAs) in plasma PLs, no differences across diets were detected (Table 4Go). In erythrocyte PLs, long-chain SFA, arachidonic acid (20:0), behenic acid (22:0), and lignoceric acid (24:0) concentrations, however, decreased significantly in the PL-n–3–fed children compared with the FO group (P = 0.033, 0.021, and 0.005, respectively) and, to a lesser extent, the placebo group (P = 0.114, 0.046, and 0.045, respectively). These alterations did not affect (P = 0.162) the proportion of total SFAs (data not shown).

cis Monounsaturated fatty acids
The largest difference between the n–3 dietary matrices and placebo was observed in the n–9 rather than in the n–7 FAs. There were no differences across diets in the plasma and erythrocyte eicosenoic acid (20:1n–9) and erucic acid (22:1n–9) concentrations (Table 4Go). In the erythrocyte PLs, an increase ({approx}230%) in nervonic acid (24:1n–9) concentrations was detected in response to PL-n–3 administration compared with the placebo (P = 0.029) but not the FO (P = 0.389) treatment. There was a tendency toward a reduction in total monounsaturated fatty acid (MUFA) concentrations in plasma PLs (P = 0.063), but the dietary matrices did not significantly affect (P = 0.140) total MUFAs in the erythrocyte fraction (data not shown).

n–6 Polyunsaturated fatty acids
In both plasma and erythrocyte PLs, LA concentrations significantly increased (17 ± 23% and 22 ± 17%) from baseline after consumption of PL-n–3 compared with FO (1 ± 14% and 6 ± 17%; P = 0.029 and 0.008, respectively), and, to a lesser degree, compared with placebo (5 ± 12% and 10 ± 21%; P = 0.221 and 0.046, respectively) (Table 4Go). In contrast, a significant decrease in the AA and adrenic acid (22:4n–6) content in the PL-n–3–and FO-fed groups compared with the placebo group was detected in the plasma but not in the erythrocyte PLs.

n–3 Polyunsaturated fatty acids
The response of n–3 LC-PUFAs in plasma PLs to the diets mirrored that of the n–6 LC-PUFAs, although the magnitude of the effect was more pronounced. In plasma PLs, EPA, docosapentaenoic acid (DPA; 22:5n–3), and DHA concentrations increased significantly at the end of the interventions in both the PL-n–3 and FO groups compared with the placebo group (Table 4Go). The combined effects of the PL-n–3 and FO supplements on the n–3 and n–6 FA concentrations in plasma PLs resulted in a distinct increase ({approx}190–220%) in the ratio of EPA to AA (change from baseline values of 0.06 ± 0.05 and 0.04 ± 0.11, respectively) as compared with placebo (–0.01 ± 0.04; P < 0.0001). In erythrocyte PLs, the PL-n–3 and, to a lesser extent, the FO supplements significantly increased DPA concentrations compared with the placebo (P = 0.022 and 0.133, respectively). Erythrocyte EPA and DHA concentrations were not altered by the dietary treatments at the end of the feeding interventions (Table 4Go).

Computerized test of attention
Changes in sustained attention and impulsivity before and after the interventions, as measured by the TOVA, are summarized in Table 5Go. No significant differences were detected between the baseline variables of attention in this objective analysis. The baseline total TOVA scores indicate that these children were markedly different (total mean: –5.1 SD) from those of age- and sex-matched normative values (≥ –1.8 SD). The total TOVA score was significantly affected by the treatments to a different extents: PL-n–3 > FO > placebo (P < 0.001). Accordingly, the ratio of subjects with total TOVA scores within the normative range at the end of the treatment was significantly different between the interventions (Figure 2Go). The proportion of subjects with normative scores in the PL-n–3 group (11/18) was statistically different from that of the placebo group (3/21; Figure 2Go), but not from the FO group (7/21; P = 0.120). Furthermore, a treatment effect was obtained in all TOVA-adjusted variables, except for the errors of omission (Table 5Go). The PL-n–3 and, to a limited extent, the FO treatment significantly improved the executive functioning in errors of commission, response time, and response time variability as compared with placebo intervention (PL-n–3: P = 0.050, 0.031, and 0.003, respectively; FO: 0.126, 0.673, and 0.028, respectively).


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  		TABLE 5. Mean changes from baseline in Test of Variables of Attention (TOVA) scores of the children presenting abnormal executive functioning after the 3 mo of dietary interventions1

 

Figure 2
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  		FIGURE 2.. Proportions of children presenting normal ({square}, ≤ –1.8 SD) or abnormal ({blacksquare}, > –1.8 SD) total Test of Variables of Attention (TOVA) scores after the consumption of phospholipids enriched with n–3 fatty acids (PL-n–3; n = 18), fish oil (n = 21), or placebo (n = 21) for 3 mo. Chi-square analysis followed by comparison between the treatments with Fisher's exact test showed a significant effect of diet (P = 0.015) and a significant difference between placebo and PL-n–3 treatments (P = 0.004).

 
These alterations in the TOVA variables were shown to correlate with the FA profile changes in both the plasma and erythrocyte PLs (Table 6Go). Across the study interventions, there was a significant positive correlation with the changes in total TOVA scores only with the changes in plasma DHA concentrations (Table 6Go). In the PL-n–3 group, significant correlations were negatively associated between the changes in total TOVA scores and the changes in plasma PL EPA concentrations, EPA/AA ratios (–0.515; P < 0.05), and erythrocyte PL LA concentrations and positively associated with changes in erythrocyte PL lignoceric acid and DHA (0.560; P < 0.05) concentrations. Similarly, significant correlations were found for changes in adjusted TOVA variables and other biochemical alterations, such as plasma PL DHA, AA (data not shown), and erythrocyte PL behenic acid (data not shown), mostly in children who were assigned to the PL-n–3 treatment.


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  		TABLE 6. Spearman's rank-order correlations between changes in Test of Variables of Attention (TOVA) scores and changes in plasma and erythrocyte phospholipid fatty acid concentrations1

 
The parental Abbreviated Conners Rating Scale questionnaire failed to show a significant effect between the treatments. Rather, it showed a significant change from baseline in all treatment groups (PL-n–3: –5.00 ± 8.32; FO: –3.20 ± 6.05; and placebo: –2.35 ± 3.73). Minimal and insignificant changes from baseline values were noted by the parents in the subscale of Child Behavior Checklist after all 3 dietary regimens (PL-n–3: –1.47 ± 5.10; FO: –0.53 ± 3.10; and placebo: –0.78 ± 3.52).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our main finding was that consumption of EPA and DHA in similar amounts, but in different carriers, to children with deviated attention performance for 3 mo resulted in different outcomes: in the incorporation in blood FAs, in executive functioning as assessed by computerized CPT, and in correlations between these effects. To the best of our knowledge, the randomized controlled trial presented herein is the first short-term intervention study with n–3 LC-PUFAs to show a correlation between these biochemical and cognitive function outcomes.

In the current study, supplementation with PL-n–3 and FO to children increased EPA, DPA, and DHA concentrations but decreased AA and adrenic acid concentrations in plasma PLs, but not in the triacylglycerol and cholesterol ester fractions. These findings agree with published observations (43), which showed that {approx}60% of EPA and DHA from digested FO was selectively incorporated into the PL fraction of plasma. Furthermore, it was shown previously (24, 44, 45) that n–3 LC-PUFA interventions in children increased the n–3 LC-PUFA and reduced the n–6 LC-PUFA concentrations in plasma PLs. These metabolic alterations could plausibly be associated with the balance in intake of n–3 and n–6 LC-PUFAs and the possible negative feedback mechanism on desaturase enzymes, such as {Delta}6 (46) and {Delta}5 (47) desaturase, which catalyze the conversion of both n–3 and n–6 from precursors to DHA and adrenic acid, respectively.

In erythrocytes, our findings suggest that PL-n–3 and FO had a limited effect on n–3 LC-PUFA concentrations, whereas the n–6 LC-PUFA concentrations were unchanged. These observations are inconsistent with previous work (29). The counterintuitive findings could be explained by the low n–3 LC-PUFA dose intervention, the different effect, and the kinetics of dietary EPA and DHA and/or the participants' metabolism. In the aforementioned study, 5-fold more DHA (480 mg/d) was provided to the putative ADHD children (Table 1Go), and n–6 LC-PUFA oils were added as well. Furthermore, the daily consumption of 1 or 0.2 g DHA by hyperlipidemic adults receiving statin therapy resulted in different kinetics of DHA in the plasma and erythrocytes (48). A new equilibrium was reached in the plasma within 1 mo, whereas in the erythrocytes it was observed only after 4–6 mo of supplementation. Moreover, consumption of the low-dose supplementation in that study was associated with a slightly slower incorporation rate for both of these blood compartments. Additionally, dietary EPA and DHA were suggested to affect FA metabolism differently (49). EPA or DHA supplementation may result in a distinct increase in the administered FAs, albeit via different kinetics, and with a minimal elevation of the counter fatty acid in the plasma PL fraction (48). Specifically, EPA and DHA interventions have shown that EPA accumulated faster than did DHA in plasma (50, 51), leukocytes (52), and erythrocytes (53, 54). Finally, early reports have associated ADHD children with symptoms of EFA deficiency and reduced blood concentrations of n–3 and n–6 LC-PUFAs, with no obvious lack of dietary intake when compared with control children (55). The underlying mechanism for this deficiency in ADHD subjects is still unclear. However, an increased breakdown of FAs via oxidation and/or a decrease in EFA metabolism could play, at least in part, a role in this pathophysiology. The latter was recently supported by preliminary findings associating a single nucleotide polymorphism in the EFA desaturase gene FADS2 with ADHD (56). Our finding of an effect of PL-n–3 consumption on erythrocyte FA profile alterations, such as an increase in DPA rather than in DHA concentrations and a decrease in >C20 SFA and in LA concentrations, might be associated with the impaired FA metabolism of these children.

PL-n–3 consumption induced an increase of the total TOVA score of 94% or 3.35 SD (from –4.53 to –1.18 SD), from baseline. Changes in this score that exceeded 80% or 2.5 SD were previously reported for stimulants (57, 58). Introducing similar amounts of EPA+DHA via FO also had significant effect of 1.72 SD or 37% (from –5.69 SD to –3.97 SD), although to a limited extent (Figure 2Go). Our findings differ from previous reports in which DHA (345 mg/d) (24) or EPA+DHA (100 + 514 mg/d) (25) supplementation in ADHD children for 2–4 mo was shown to result in elevated plasma PL DHA concentrations and in no effect on the CPT scores. Likewise, in young patients with recurrent self-harm, the 12-wk administration of EPA+DHA (1220 + 908 mg/d) failed to affect impulsivity as assessed by computerized CPT (9). One possibility for resolving this discrepancy might be the use of different n–3 LC-PUFA carriers and/or food matrices. Earlier reports (see review in reference 59) suggest that PS affects memory, learning, and behavior. These observations were mainly associated with the stimulation of the cholinergic system. Few studies have suggested that the cholinergic system may also be involved in the pathophysiology of ADHD (60). Interestingly, nicotinic stimulation was shown to affect executive functioning, specifically sustained attention, and working memory in CPT. A second possibility could be the different n–3 LC-PUFA proportions used in the interventions. Recent reports favored EPA rather than DHA (EPA/DHA ratios {approx}3; range: 1.2–6.8) for improving behavior in children with neurodegenerative or neurodevelopmental disorders (26, 28, 44, 45, 61, 62). Our n–3 LC-PUFA–containing interventions were characterized by a high EPA/DHA ratio ({approx}1.6). Moreover, the noted effectiveness was attained while providing a considerably modest daily dose of EPA+DHA (250 mg compared with {approx}1200 mg/d; range: 345–4300 mg/d). Taken together, our findings suggest that providing preparations with a high EPA/DHA ratio could affect visual sustained attention performance, even at subgram amounts, in pediatric populations.

In the PL-n–3 group, plasma and erythrocyte PL FA alterations were correlated with changes in the TOVA indexes. Elevated concentrations of n–3 LC-PUFAs in blood membranes were previously shown to correlate with modest improvement in behavioral assessments in children (29, 44, 45, 63). Similarly, infants consuming n–3 LC-PUFAs showed a lower visual evoked potential latency that was correlated with an increase in DHA in erythrocyte lipids (44, 64). Interestingly, in our study the highest Spearman's rho value was calculated for the changes in erythrocyte LA concentrations after PL-n–3 intervention (Table 6Go). This finding could, at least in part, be associated with the induced cellular fatty acid metabolism alterations observed after n–3 LC-PUFA consumption. This may be associated with the difference in n–3 LC-PUFA carriers, because it was previously suggested that PL-n–3 is a better vehicle for n–3 LC-PUFA accretion (65, 66).

In conclusion, our findings indicate that providing n–3 LC-PUFAs esterified to PLs or triacylglycerols for 3 mo to children with impaired visual sustained attention performance results in different FA profiles in blood fractions and affects their executive functions differently. Moreover, the blood lipid alterations were correlated with the changes in the CPT scores, mainly in the PL-n–3 group. A better understanding of the physiology of n–3 LC-PUFAs and the role of their carriers throughout the metabolic process in children with n–3 LC-PUFA deficiency could place the results of the current investigation in clearer context. In the meantime, these observations could assist in planning future trials of the role of n–3 LC-PUFA carriers in psychiatric as well as in healthy pediatric populations.


   ACKNOWLEDGMENTS
 
We thank Doron Comaneshter (Statistics Services Unit, Tel Aviv Sourasky Medical Center) for help with the statistical analyses and Esther Eshkol for editorial assistance.

The authors' responsibilities were as follows—NV (Principal Investigator): designed the trial; NV, DP, GB, GZ, and JB: interpreted the data and wrote the manuscript; YZ-A: coordinated the study, responsible for patient recruitment and management, and assisted with the blood work; JB and GB: performed the laboratory analyses; and NK: performed the TOVA analyses and interpreted the corresponding data. All authors critically revised the manuscript and approved the final report. NV is a consultant at Enzymotec LTD and DP is the Director of Clinical Studies at Enzymotec LTD. The other authors had no personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication September 9, 2007. Accepted for publication November 30, 2007.




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