Health effects of saturated and trans-fatty acid intake in children and adolescents: Systematic review and meta-analysis

Background Elevated cholesterol has been linked to cardiovascular disease in adults and preclinical markers of atherosclerosis in children, thus reducing saturated (SFA) and trans-fatty acids (TFA) intake from an early age may help to reduce cholesterol and the risk of cardiovascular disease later in life. The aim of this review is to examine the evidence for health effects associated with reducing SFA and TFA intake in free-living children, adolescents and young adults between 2 to 19 years of age. Design Systematic review and meta-analysis of randomised controlled trials (RCTs) and prospective cohort studies. Study selection, assessment, validity, data extraction, and analysis were undertaken as specified by the Cochrane Collaboration and the GRADE working group. Data were pooled using inverse variance models with random effects. Data sources EMBASE; PubMed; Cochrane Central Register of Controlled Trials; LILACS; and WHO Clinical Trial Registry (up to July 2016). Eligibility criteria for selecting trials RCTs involving dietary interventions aiming to reduce SFA or TFA intakes and a control group, and cohort studies reporting the effects of SFA or TFA exposures, on outcomes including blood lipids; measures of growth; blood pressure; insulin resistance; and potential adverse effects. Minimum duration was 13 days for RCTs and one year for cohort studies. Trials of weight loss or confounded by additional medical or lifestyle interventions were excluded. Results Compared with control diets, there was a highly statistically significant effect of reduced SFA intake on total cholesterol (mean difference (MD) -0.16 mmol/l, [95% confidence interval (CI): -0.25 to -0.07]), LDL cholesterol (MD -0.13 mmol/l [95% CI:-0.22 to -0.03]) and diastolic blood pressure (MD -1.45 mmol/l [95% CI:-2.34 to -0.56]). There were no significant effects on any other risk factors and no evidence of adverse effects. Conclusions Advice to reduce saturated fatty acids intake of children results in a significant reduction in total and LDL-cholesterol levels as well as diastolic blood pressure without evidence of adverse effects on growth and development. Dietary guidelines for children and adolescents should continue to recommend diets low in saturated fat.


Introduction
Dietary saturated fatty acids (SFA) and trans-fatty acids (TFA) are strongly correlated with total and low-density lipoprotein (LDL) cholesterol levels in adults [1][2][3][4], both well-established markers of cardiovascular disease (CVD).[5,6] Reduced intakes of SFA have been shown to be are associated with significant reduction in risk of CVD, particularly when replaced by polyunsaturated fatty acids (PUFA) in both randomised trials and cohort studies.[7][8][9][10] Similarly, cohort studies demonstrate that high intakes of TFA are strongly associated with increased likelihood of coronary heart disease and related mortality.[11] Although cardiovascular and coronary heart diseases typically present later in life, atherosclerotic lesions in the aorta and coronary arteries can begin to appear in childhood, [12,13] and are positively associated with dyslipidaemia and other CVD risk factors.[13,14] Elevated total and LDL cholesterol in childhood are, in turn, associated with an increase in CVD risk factors in adulthood [14] including thickening of the carotid artery intima-media, [15][16][17] a marker of subclinical atherosclerosis and predictor of future cardiovascular Dietary saturated fatty acids (SFA) and trans-fatty acids (TFA) are strongly correlated with total and low-density lipoprotein (LDL) cholesterol levels in adults [1][2][3][4], both well-established markers of cardiovascular disease (CVD).[5,6] Reduced intakes of SFA have been shown to be associated with significant reduction in risk of CVD in meta-analyses of both prospective cohort studies and randomised controlled trials (RCTs), with the strongest associations seen when SFA are replaced by polyunsaturated fatty acids (PUFA).[7][8][9][10] Similarly, cohort studies demonstrate that high intakes of TFA are strongly associated with increased likelihood of coronary heart disease and related mortality.[11] Controversially a recent meta-analysis of RCTs incorporating recovered and reanalysed data from two studies conducted fifty years ago [18,19] suggests increased risk of coronary heart disease death and cardiovascular disease when replacing SFA with n-6 PUFA.[18] The limitations of this new analysis have been widely reported.[20,21] The World Health Organization (WHO) is currently updating its guidance on SFA and TFA intake in adults and children.The aim of this systematic review and meta-analysis is to examine the evidence for health effects associated with reducing SFA and TFA intake in children in order to inform and contribute to the development of updated WHO recommendations.

Methods
This systematic review and meta-analysis was conducted in accordance with the WHO guideline development process [22] and the methods of the Cochrane Collaboration.[23] As part of the evidence review, results of the meta-analysis were evaluated using the methodology of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) working group.[24] Evidence summaries and GRADE assessments were discussed and reviewed by the WHO Nutrition Guidance Expert Advisory Group (NUGAG) Subgroup on Diet and Health as part of WHO's guideline development process.
The PICO questions (S1 Table ) and priority health outcomes guiding this review were discussed and developed by the NUGAG Subgroup on Diet and Health.A PRISMA checklist was been completed and adhered to (S2 Table ).

Search strategy
Electronic searches were conducted in the following databases without limits on date of publication or publication language: EMBASE (to July 2016); PubMed (to July 2016); Cochrane Central Register of Controlled Trials (to July 2016); LILACS (to July 2016); and WHO Clinical Trial Registry (to July 2016).Search terms can be found in S3 Table .Reference lists of identified papers were hand-searched for additional, relevant articles.

Inclusion criteria
Subjects included male and female children, adolescents and young adults between the ages of 2 to 19 years, from the general population.Studies considered for inclusion were those conducted in healthy individuals as well as in individuals with, or at risk of, hyperlipidaemia (including familial hypercholesterolaemia), hypertension or diabetes (type 1 or 2), or who were overweight or obese.Studies targeting those who were pregnant, acutely ill or with chronic infections such as human immunodeficiency virus (HIV) were excluded.
Randomised controlled trials (RCTs) involving interventions of at least two weeks duration with the primary intention of reducing SFA or TFA intake directly or through reduction in total fat intake, and that reported an outcome of interest were included.Trials where weight loss was the primary outcome were excluded, as were trials involving multifactorial interventions where the effect of SFA or TFA reduction could not be separated from the effect of other changes such as physical activity level.Prospective cohort studies meeting the inclusion criteria and reporting associations between SFA or TFA intake and an outcome of interest were also identified.
Outcomes of interest included total cholesterol, LDL cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, and associated blood lipid ratios; height; body weight, body mass index (BMI) and other measures of adiposity; systolic blood pressure; diastolic blood pressure; insulin resistance and incidence of impaired glucose tolerance, impaired fasting glycaemia or type 2 diabetes; and potential adverse effects.

Study selection
One reviewer (JM) performed the initial screen of all articles identified by the electronic searches.Articles were excluded when the title clearly indicated that it did not meet the inclusion criteria (e.g.non-human studies).Two reviewers (JM, LTM) independently reviewed the abstract and/or full text of the remaining citations.Differences in opinion as to whether particular studies should be included/excluded were resolved by discussion.

Data extraction and quality assessment
Two reviewers (JM, LTM) independently extracted data from RCTs using standard data extraction forms, with any discrepancies resolved by discussion.Where necessary, data was extracted directly from graphs using PlotDigitizer Version 2.6.3.Cochrane criteria were used to assess risk of bias for each trial including sequence generation, allocation concealment, blinding of participants, personnel and outcome assessors, incomplete outcome data, selective outcome reporting and other possible sources of bias, which included potential differences in the groups at baseline, evidence of treatment compliance, residual confounding and systematic differences in care between control and intervention groups.[23] Data synthesis and statistical analysis Meta-analysis of trial data was undertaken using the Cochrane Collaboration's Rev Man 5.1 software [25] with generic inverse models and random-effects to account for heterogeneity resulting from differences in trial design, follow-up periods and age of participants.Effects were reported as mean differences for all outcomes except for height and body weight, which were reported as standardized mean differences to account for differences in growth rates through childhood.For trials reporting multiple follow-ups over time, the most recently published or most reliable data was used in the meta-analyses.When cross-over (paired data) studies did not report the mean difference between treatments and its standard error (SE) or other relevant statistics, end of treatment values were analysed as independent samples (a conservative approach).Effect estimates and their standard errors from correctly analysed clusterrandomized trials were included without adjustment in the meta-analyses.For incorrectly analysed cluster ranodmised trials the standard error was inflated to account for unit of analysis errors.The standard error of the effect estimate (ignoring clustering) was mulitplied by the square root of the design effect obtained from a reliable external source.[23] Heterogeneity was assessed by visual inspection of the forest plots, and consideration of the I 2 statistic, where an I 2 statistic of more than 50% indicates a considerable level of inconsistency.Potential causes of heterogeneity were determined by examining characteristics of individual trials and subgroups.When more than one trial was available subgroup analyses were conducted for each outcome by the following pre-specified factors: serum lipid status; age of participants; type of intervention; trial design; duration of intervention; level of SFA or TFA reduction achieved; and the primary nutrient source replacing SFA or TFA.
Sensitivity analyses examined the effects of removing trials at high risk of bias from the analysis.A trial was considered to be at high risk of bias overall if it was graded as inadequate (high) in both the randomisation and at least one other area of bias assessment.Other trials were considered to be at low risk of bias.
Publication bias was not assessed as the number of trials found was not sufficient to conduct funnel plot analyses for any outcome measure.

Trans-fatty acids
No trials were identified meeting the inclusion criteria.The flow of records through screening, exclusion and inclusion of trials is shown in S1 Fig.
Subgroup analysis of the effect of the initial lipid status of the participants found a significantly different effect of SFA reduction on total cholesterol (p = 0.05) between trials involving  Two trials reported achieving SFA intakes of less than 10% of total energy intake (the current WHO population nutrient intake goal [53]) in the intervention group.[35,39] One of these had a high risk of bias, presented data that did not adequately account for baseline differences in lipid levels and reported null effects.[35] The other was a well-designed cluster randomised crossover trial in which the reductions in total (MD -0.29 mmol/l [95% CI: -0.40 to -0.18]) and LDL cholesterol (MD -0.29 mmol/l [95% CI: -0.38 to -0.20]) were significantly greater than the pooled effects for the five trials where SFA intake was greater than 10% of total energy intake in the intervention group (p = 0.03 and p = 0.003 for subgroup differences, respectively) [39]

(S6 and S7 Figs).
There were no significant associations observed for HDL cholesterol, triglycerides or apolipoproteins A1 or B (Table 3).
Anthropometric measures.There were no significant associations observed between reduced SFA intake and weight, height, body mass index (BMI) or waist circumference (Table 3).
Blood pressure.Three trials reported blood pressure outcomes [29,35,47] but only two reported data in sufficient detail for the meta-analysis.[29,47] A statistically significant decrease in diastolic blood pressure associated with reduced SFA intake was observed (MD -1.45 mm Hg [95% CI: -2.34 to -0.56]) (Fig 4).There were no significant effects of SFA reduction on systolic blood pressure (Fig 5).In addition, one of these trials separately reported on risk of high blood pressure and found that reduced SFA intake reduced the risk of high blood pressure in adolescents (relative risk [RR] 0.83 [95% CI: 0.70 to 0.99]).[54] Insulin sensitivity.One trial reported improvements in insulin sensitivity as measured by the homeostasis model of insulin resistance (HOMA-IR) at 9 years of age [46] (p = 0.02 for intervention effect) and between 15-20 years [49] (p trend = 0.005 for intervention effect).At age 19 the MD was -0.14 [95%CI: -0.28, 0.01] (Table 3).Multivariate analyses suggest that the improvements were largely due to reduced SFA saturated fat intakes, but increased dietary fibre intakes may have also have been a factor.Adverse effects.In addition to no observed adverse effects on anthropometric measures, there was no evidence of adverse effects of reducing SFA intake in children on micronutrient intakes, cognitive development or sexual maturation in the small number of trials reporting these outcomes.
Two trials assessed micronutrient intakes.[28,31,42] One noted that self-reported intakes of some micronutrients were lower in the reduced SFA group, however no adverse effects  were observed in serum indicators of nutritional status and there was no significant difference in adjusted serum ferritin levels (MD -2.1 μg/L [95%CI: -4.9 to 0.8]; p = 0.08) between groups.This effect was reduced to 1.5 μg/L (p = 0.12) when an outlier was removed.[28,31] Similarly, serum ferritin levels in 3-4 year old children participating in a second trial were not different between groups (MD -2.6 μg/L [95%CI: -8.2 to 2.9]; p > 0.05), nor were there differences observed in intakes or other indicators of iron and zinc status.[42] Two trials assessed the effect of SFA reduction on cognitive development.There were no significant differences in the relative risk of failing tests of speech and language skills, gross motor functioning plus perception, or in visual motor skills for 5 year old children in the intervention compared with the control group.[44] In addition, there was no difference in parental perceptions of their children's behavior at 3 years between the intervention and control groups.[43] Similarly, children in another trial exhibited no signs of adverse effects of SFA reduction in terms of academic functioning, psychological symptoms or family functioning.[30] In one trial that assessed sexual maturation, reduced saturated fat intake had no effect on Tanner stages of puberty in males or females at any point during the trial.[31,55,56] Cohort studies.Three prospective cohort studies were identified that reported on associations between SFA intake at baseline, or changes in SFA intake during follow-up, and relevant health outcomes in children.The Bogalusa Heart Study compared total and LDL cholesterol concentrations in children who had maintained consistent SFA intakes during 6.5 years of follow-up.[57] Total and LDL cholesterol concentrations of the children in the highest vs lowest tertiles of SFA intake were compared but no significant association was found.Magarey et al. examined the association between SFA intake (measured periodically over the 15 years) and measures of adiposity, and found a statistically significant association between energy adjusted SFA intake and BMI standard deviation score after adjustment for baseline fatness, sex and parental BMI.[52] The Avon Longitudinal Study of Pregnancy and Childhood reported significant associations from reverse stepwise multiple regression models examining the effect of nutrient intakes (assessed at 18 months of age) on cholesterol concentrations at 31 months of age.[50] There was a significant association between SFA intake and total cholesterol in boys and on the total:HDL cholesterol ratio in girls.Data from the cohort studies was not included in the meta-analysis or GRADE assessment.

Quality of the body of evidence
Some degree of bias was detected for most trials (Fig 6) but only one trial was assessed as being at high risk of bias over a range of criteria.Three trials had a high risk of bias for possible differences in care between intervention and control group [28][29][30][31][32][33][41][42][43][44][45][46][47][48][49], one of which also had a high risk of bias for selective reporting.[32,33] One trial had high risk of bias in terms of randomisation [37,38].Another raised further questions relating to allocation concealment and other areas of potential bias, suggesting high risk of bias overall [35].Sensitivity analyses examining the influence of this trial showed that its exclusion slightly strengthened the associations between SFA reduction and total and LDL cholesterol but did not change our interpretation of the overall findings.
Assessment of the quality of the results for outcomes included in the main analysis can be found in GRADE evidence profile 1 (S6 Table ).All outcomes for children were rated as "high" with the exception of triglyceride, apolipoprotein A1, systolic blood pressure and insulin resistance which were rated as "moderate".Similar assessments were made for threshold and replacement analyses (GRADE evidence profiles 2-5, S6 Table ).

Discussion
This systematic review and meta-analysis showed that reduction of SFA intake in children and adolescents between 2 to 19 years of age was associated with statistically significant reductions in total and LDL cholesterol and diastolic blood pressure.These effects were consistent across all of the trials included in the meta-analyses.The effects on cholesterol were greatest among those in which SFA was replaced primarily with PUFA or MUFA, and when the intervention group achieved a reduction in SFA to below 10% of total energy intake.

Findings in Context
The very low incidence of cardiovascular events in children makes an examination of the direct effects of saturated fat intake on CVD impractical.Total and LDL cholesterol can readily be measured in children, however, and both are well established risk factors for coronary heart disease in adults; a meta-analysis of prospective cohort studies involving over 900,000 adults found a linear association between blood cholesterol concentrations and coronary mortality [6], while clinical trials show that cholesterol-lowering with statin therapy reduces coronary heart disease risk.[51] Furthermore, elevated LDL cholesterol in children has been found to be associated with preclinical signs of atherosclerosis.[12] There is strong evidence from controlled clinical trials in adults that replacing dietary SFA with MUFA, PUFA or carbohydrate reduces total and LDL cholesterol concentrations.[1,3] In addition there is reasonably strong evidence from randomised trials in adults that replacing SFA with PUFA from plant oils, but not refined carbohydrates, reduces the incidence of cardiovascular events and mortality.[8][9][10] However recent meta-analyses of randomised trials examining the effect of replacing saturated fat with plant oils rich in linoleic acid (n-6 PUFA), including recovered data from Sydney Diet Heart Study (SDHS) and the Minnesota Coronary Survey (MCS), both conducted over 50 years ago, indicates no benefit with respect to coronary heart disease mortality despite reduction in total cholesterol.[18,19] These new findings should be viewed with caution given that in the SDHS and MCS intakes of PUFA intervention groups exceeded current recommendations, very high rates of loss to follow-up occurred, there was evidence of selective outcome reporting and neither study was powered to show an effect of n-6 PUFA on mortality.[20,21] We believe our findings in children are compatible with the totality of evidence in adults indicating beneficial effects in terms of lower coronary heart disease risks when replacing dietary SFA with PUFA-rich unsaturated fatty acids.[36,39] Questions have been raised about the appropriateness of recommending low-fat diets to children over concerns that excessive restriction of dietary fat could result in inadequate nutrient intakes-particularly of iron-which could negatively impact normal growth and development.[58,59] In the analysis there was no evidence of adverse effects of reducing SFA intake on anthropometric measures of growth, cognitive development or micronutrient intake, including iron.A primary focus of both the Dietary Intervention Study in Children (DISC) [28] and Special Turku Coronary Risk Factor Intervention Project (STRIP) [41] was to assess the safety of a reduced SFA diet in children in terms of growth and development, and authors of both trials concluded that a diet low in SFA was safe for children.
That no relevant trials of TFA intake in children were found may be because most epidemiological studies on TFA intake have assessed cardiovascular outcomes which generally only present in later life.Several trials assessing the effects of conjugated linoleic acid (CLA) supplementation on weight loss in children were identified but none met the inclusion criteria.[58,[60][61][62][63]

Strengths and limitations
A strength of this review is the quality and consistency of the data, particularly that relating to total and LDL cholesterol.Results of the meta-analysis are also highly consistent with what has been observed in trials in adults.The data covers children between 2 to 19 years of age and consistently showed that advice given to parents and to older children was effective in reducing SFA intakes.Data from the STRIP trial [41][42][43][44][45][46][47][48][49] reporting effects across the course of childhood through to adulthood illustrates this consistency (S8 Fig) .Adolescents in the DISC trial similarly demonstrated seemingly consistent levels of compliance over three years of follow-up [28][29][30][31].
These findings are subject to the limitations inherent to most dietary intervention studies, notably difficulties in obtaining reliable dietary intake data, maintaining the blind among participants and personnel, and variation in the nature and quality of the interventions, all of which may bias the results.It is generally accepted that it becomes increasingly difficult for participants to maintain adherence to dietary interventions over time although this may be more of an issue in trials involving adults with previously established eating habits.Since children are typically reliant on adults to provide nourishment, they may be less prone to stray from the intervention diet, provided caregivers comply with the intervention.The possibility that the effects may be partially explained by factors other than saturated fat intakes such as differences in consumption of fruit and vegetables or physical activity levels between control and intervention groups cannot be ruled out.Although the primary objective of the STRIP trial was fat modification [41], some reports of follow-up conducted later in later childhood and adolescence indicate that the intervention group received additional advice regarding intake of fruit, vegetables and dietary fibre, smoking and physical activity.[54] Thus we cannot be entirely certain the effects observed were primarily due to SFA reduction.However, the significant effects on total and LDL cholesterol remain even when the STRIP data is excluded from the analyses (data not shown).Given the careful consideration of the potential sources of bias described above in assessing the quality of the evidence, and the observation that the greatest effects on cholesterol levels were seen in the most controlled trials, our confidence in the results of the meta-analysis is high.

Conclusion
Advice to reduce saturated fat intake of children results in a significant reduction in total and LDL-cholesterol levels as well as diastolic blood pressure without adverse effects.Results of this review suggest that the greatest effect on cholesterol occurred when SFA was replaced with PUFA or a mixture of PUFA/MUFA, although benefit was observed even when replacement was unclear.Results further suggest that the greatest reduction in cholesterol occurred when SFA intake was less than 10% of total energy intake.Elevated cholesterol has been linked to CVD in adults and preclinical markers of atherosclerosis in children, thus reducing saturated fat intake from an early age may help to reduce the risk of CVD later in life.CVD is a major contributor to the substantial global economic burden of NCDs.Projected increases in NCDs over the next two decades could cost up to 75% of global gross domestic product (GDP) as a result of healthcare costs and lost productivity due to death or disability.[64] Interventions targeting reduction in saturated fat intakes amongst children and adolescents could translate into major cost savings by reducing risk of CVD.Interventions should focus on reducing intakes of highly processed fried and nutrient-poor fast foods and snacks, processed meats and fatty meats.High-fibre fruits and vegetables, nuts and seeds, lean meats and reduced fat dairy foods should be the core components of children's diets.

Table 2 . Dietary intake data for included trials. Study ID Achieved SFA intake Replacement nutrient a
Int, intervention group; Cont, control group; %EI, % of total daily energy intake; SFA, saturated fatty acids; PUFA, polyunsaturated fatty acids; TFA, trans fatty acids; MUFA, monounsaturated fatty acids a Based on dietary intake data b As assessed at three month follow-up of a one year trial (data not sufficient to calculate for later timepoints).c p < 0.05 d The fat content of the food supplements provided 26.2% of total fat intake to the participants, 3.9% of which was saturated fatty acids in the intervention group and 17.4% of which was saturated fatty acids in the control group.Total SFA intake was not reported, however, estimates based on other dietary information provided about the trial indicate that the whole milk (control) provided approximately 6.5% of total energy intake as SFA and the milk preparation (interventions) provided approximately 1.4% of total energy intake as SFA.As the milk/milk preparation provided 26.2% of total fat intake, it is very likely that those consuming whole milk were consuming >10% of total energy intake as SFA.It is less clear if those consuming the milk preparation achieved an SFA intake of <10% of total energy intake.e Food supplement provided to intervention group contained 70% MUFA, 15% PUFA, 15% SFA.Food supplement provided to control group contained 30% MUFA, 3% PUFA, 67% SFA.f Actual SFA intake varied throughout 19 years of follow-up but was consistently lower in intervention groups https://doi.org/10.1371/journal.pone.0186672.t002

Table 3 . Summary of effect estimates for randomised trials that compared usual diets with reduced saturated fat diets in children. Outcome Trials Participants a (n) Effect Estimate (95%CI) p value I 2
a subjects in crossover trials counted once only https://doi.org/10.1371/journal.pone.0186672.t003