The present study shows that the combination of plant sterol esters (1300 mg), fish oil (1000 mg EPA+DHA) and B vitamins impacts on LDL-cholesterol and homocysteine levels in hypercholesterolemic children and adolescents. To our knowledge, this is the first report of the use of the combination of these natural compounds for early dietary management of potential CVD risk.
Elevated LDL-cholesterol level is a well-established independent risk factor for CVD. Reductions in the LDL-cholesterol levels by 8.7% in the group of 15 adolescents aged over 17 years and by 9.5% in the group of 10 children aged 10 to 16 years were observed. The results are in line with previous studies on the use of plant sterols and stanols in children and adolescents with hypercholesterolemia with the significant LDL-cholesterol reduction observed in range of 9 to 15% with daily intake ranging from 1.2 to 3 g of plant sterol or stanols [5–7, 20, 21]. It has been proposed that the mechanism of such cholesterol lowering involves competition between intestinal plant sterols/stanols and intestinal cholesterol for absorption in mixed micelles and upregulation of the enterocyte ATP-binding cassette transport proteins .
The atherogenic index (Total cholesterol to HDL-cholesterol ratio) improved during the study with no significant change in HDL-cholesterol, apolipoprotein A-1 and hsCRP.
Measurement of LDL subfractions may provide additional assessment of CVD risk. There is growing evidence that small dense LDL subfractions may be more atherogenic than larger less dense LDL subfractions due to their lower binding affinity for the LDL receptor, impaired clearance from the circulation, susceptibility to oxidative modification and easier penetration into the subendothelial space of the arterial wall, leading more readily to the formation of cholesterol deposits and inflammation . In the present study, the combined effect of plant sterol esters, n-3 PUFA and B vitamins resulted in less atherogenic profile. The levels of the atherogenic lipoprotein VLDL, IDL-1 and IDL-2 subfractions decreased significantly in both age groups. However, only trace levels of the LDL-3 subfraction were found in 15 participants (2 in the age group 10-16 years) with no LDL-4 to LDL-7 subfractions detected. In addition, significant reductions in the predominant large dense LDL-2 subfraction was observed in both age groups without any change in non-atherogenic subfractions IDL-3 and LDL-1. In contrast to our results, Engler et al.  reported significantly increased LDL-1 subfraction by 91% and decreased small dense LDL-3 by 48% after supplementation with DHA (1.2 g/day) for 6 weeks in children with either initial LDL-cholesterol levels above 130 mg/dl (familial hypercholesterolemia) or LDL-cholesterol levels above 130 mg/dl or TAG >150 mg/dl or both (familial combined hyperlipidemia). To our knowledge there are no reports on the administration of plant sterols/stanols linked with LDL subfraction analysis in children and adolescents. In hypercholesterolemic adults, following plant sterol and stanols supplementation in various forms and doses, the results are inconsistent with no significant changes in LDL particle size  or a significant reduction in all LDL subfractions  or small LDL subfraction .
Accumulating evidence suggests that elevated plasma TAG may pose another significant independent risk for CVD . The n-3 PUFA lowering effect on plasma TAG levels is well established in adults, but no significant changes in TAG levels in children and adolescents with dyslipidemia were seen after daily supplementation with 1.2 g of DHA [10, 11]. In our study, the TAG levels decreased in both age groups, but a significant reduction was observed in the group of children aged 10 to 16 (baseline mean TAG level 1.1 mmol/l). Recently, a pooled analysis of 12 randomised controlled trials showed that plant sterols alone modestly lower TAG levels (by 6% or 0.12 mmol/l) in hypercholesterolaemic adults depending on baseline concentration . Moreover, Micallef et al  showed a 22.6% reduction in overall cardiovascular risk of hyperlipidemic adults consuming the combination of 2 g/day plant sterols and 1.4 g/day omega 3 PUFA compared to 15.1% and 15.3% CVD risk reduction in the plant sterols and fish oil groups alone.
A reduction in lipid levels affects plasma levels of fat soluble vitamins [30, 31]. In the present study, the concentrations of vitamins and provitamins were unchanged after standardisation for LDL-cholesterol levels, except for a reduction in α- tocopherol levels.
Elevated homocysteine concentration is an independent risk factor for ischemic stroke and thromboembolism in children [32, 33] and is strongly influenced by folate and B vitamins status . The levels of total homocysteine seen in the present study were within the normal ranges for both age groups agreeing with previous studies on healthy or hypercholesterolemic children [13, 34–36]. However, we noted a significant reduction in total homocysteine by 3.25 μmol/l after 16 weeks intervention, similar to significant reduction by 3.99 μmol/l in children with familial hypercholesterolemia supplemented daily with 5 mg folate for 3 months . A meta-analysis of observational studies in adults suggested that lowering total homocysteine levels by 3 μmol/l would reduce the risk of ischemic heart disease by 11% and stroke by 19% . Results for the metabolites of the homocysteine metabolic pathway, methionine and cysteine, indicate that the intervention may have had an effect on the degradation of homocysteine through the transsulfuration pathway to cysteine rather than on re-methylation of homocysteine to methionine. Cysteine may be utilised in protein synthesis or as a precursor in the synthesis of glutathione.
The pre-emulsification of fish oil significantly improves absorption of EPA and DHA . The addition of fish oil (590 mg EPA+410 mg DHA/day) into the emulsified intervention resulted in increased levels in plasma EPA by 220% and DHA by 130% and significant reduction of the n-6 PUFAs; AA and DGLA. Engler et al  reported increases in the DHA concentration and reduction in the n-6 PUFA in the plasma phospholipids of hyperlipidemic children after algal DHA oil supplementation (1.2 g/day). Reducing the n-6:n-3 fatty acid ratio in the diet may contribute to cardioprotective health in children and adolescents.
Our pilot study has limitations; this was an investigational uncontrolled study and the observed within-group differences may be subject to confounding factors. Also the small number of participants included in this study means that the study was underpowered to detect all statistical significant differences which could explain the observed trends for some results especially in the younger group (10-16 years old) with only 10 participants.