Effect of a dietary intervention including minimal and unprocessed foods, high in natural saturated fats, on the lipid profile of children, pooled evidence from randomized controlled trials and a cohort study.

AIM: To study the possible effects of a dietary intervention with minimal and unprocessed foods, high in natural saturated fats on the lipid profile and body mass index of children.
METHOD: This study combines three intervention studies; one non-randomized retrospective cohort study and two randomized controlled trials, to a pooled analysis. The intervention group received a dietary intervention of minimal and unprocessed foods for three to six months, consisting of five times per week green vegetables, three times per week beef, daily 200-300 mL whole cow's milk (3.4% fat) and whole dairy butter (80% fat) on each slice of bread. The control group continued their usual dietary habits. Raw data of the three intervention studies where combined into one single dataset for data analysis, using mixed effects analysis of covariance to test the effects of the dietary advice on the main study outcomes, which are measurements of the lipid profile.
RESULTS: In total, 267 children aged 1 to 16 years were followed. 135 children were included in the intervention group and 139 children in the control group. Characteristics (age, gender and follow-up period) were equally distributed between the groups at baseline. In the intervention group HDL-cholesterol increased significantly from 1.22 mmol/L, 95% confidence interval (CI) 1.14-1.32 to 1.42 mmol/L 95% CI 1.30-1.65 (p = 0.007). The increase over time in HDL cholesterol in the intervention group was significantly different compared to the increase in the control group (from 1.26 mmol/L, 95% CI 1.19-1.35, to 1.30 mmol/L, 95% CI 1.26-1.37) (p = 0.04). Due to the increased HDL concentration in the intervention group, the total cholesterol/HDL cholesterol ratio decreased significantly from 3.70 mmol/L, 95% CI 3.38-3.87, to 3.25 mmol/L, 95% CI 2.96-3.31 (p = 0.05).
CONCLUSION: Consumption of minimal and unprocessed foods (high in natural saturated fats) has favourable effects on HDL cholesterol in children. Therefore, this dietary advice can safely be recommended to children.

1. Introduction

Worldwide the prevalence of childhood obesity is increasing dramatically. Childhood obesity often results in obesity in adults with its well-known negative effects on human health, like cardiovascular disease (CVD) [1]. Shockingly, overweight and obesity are linked to more deaths worldwide compared to underweight. According to the World Health Organization (WHO), in 2019, 38.2 million children worldwide under the age of 5 years were overweight or obese [2]. Changes must be made to fight this pandemic. The WHO suggests healthier food choices since these are the most accessible, available and affordable. However, UNICEF investigated so-called healthy food products specially made for children, such as porridge, breakfast cereals and snacks, and concluded that 70% of these ‘healthy’ products are actually unhealthy since they are high sources of energy, trans-fatty acids, sugar and sodium [3]. Many of these products are processed or ultra-processed. Several studies have shown that diets composed of (ultra-)processed food products are associated with negative health effects. Cornwell et al. (2018) obtained information from a cohort study conducted in children (aged 5–12 years) and observed that consumption of ultra-processed foods resulted in lower-quality nutrient profiles of the children [4]. Secondly, Rauber et al. (2015) examined the effects of ultra-processed food consumption in a longitudinal randomized trial, on 345 children’s (aged 3–4 years and 7–8 years) lipid profiles [5]. It was concluded that early ultra-processed food consumption (at the age of 3–4 years) played a role in a negative altered lipid profile of the children later in life (at the age of 7–8 years). Eating more natural food products (unprocessed or minimally processed) at a younger age could contribute to a more positive lipid profile, and therefore to a healthier life.

Three previous conducted studies observed the effects of a dietary intervention consisting of minimal and unprocessed food products on children’s lipid profiles [6-8]. This dietary intervention consisted of five times per week green vegetables at dinner, three times per week beef at dinner, daily 200–300 mL whole cow’s milk and whole dairy butter on each slice of bread. There were some concerns about negative effects on the lipid profile of the young children that consumed these products since this dietary intervention was relatively high in saturated fats. However, there is conflicting research regarding the association of natural saturated fat intake and a negative lipid profile. A meta-analysis of prospective cohort studies did not find significant evidence that dietary saturated fat is associated with an increased risk of CVD [9]. Some studies even observed an inverse association. Mozaffarian et al. (2004) observed that a higher natural saturated fat intake led to a more favorable lipid profile with significant higher HDL cholesterol and lower total cholesterol/HDL cholesterol ratio in a randomized trial in postmenopausal women [10]. Secondly, Gillman et al. (1997) examined the association of stroke incidence with intake of fat and type of fat among middle-aged men from the US during 20 years of follow-up and concluded that natural fat, saturated fat, and monounsaturated fat intake were associated with a reduced risk of stroke [11]. However, little research has been done on children.

Another point of discussion concerns the sustainability of this dietary advice. In 2019, the LANCET published the EAT-Lancet sustainable diet for the future. 37 scientists, from various disciplines, set global scientific goals for healthy nutrition and sustainable diets for the future [12]. Table 1 shows the comparison between the EAT-Lancet diet and the diet from the present study. The quantities of the LANCET diet are designed for adults [12]. No separate advice has been issued for younger children since they represent only a small part of the world population according to the authors of the EAT-Lancet committee. However, the dietary advice of the present study, designed for children, does fit within the guidelines of the EAT-Lancet diet. This dietary advice has shown in practice that it has clinical health-promoting effects. This illustrates that a healthy (unprocessed) diet within the global scientific goals for healthy nutrition and sustainable diets for the future, set by the EAT-Lancet committee, is feasible [6-8].

Table 1

Components of the dietary advice of the present study compared to the EAT-Lancet diet.

	Present diet	EAT-Lancet diet
Green vegetables	50–100 gram/day(at least 5x per week)	200–600 grams/day
Beef	50–60 gram/day(3x /week)	0–28 grams/day(equals 0–196 grams/week)
Whole milk	200–300 ml/day	0–500 ml/day
Whole dairy butter	5 grams/slice of bread(equals 10–20 gram/day)	0–12 grams/day

To investigate possible effects of this dietary intervention, a pooled analysis was performed using three previous conducted studies. The aim of the present study was to determine whether this dietary intervention, relatively high in saturated fats, has an influence on the lipid profile or body mass index (BMI) of young children.

2. Material and methods

The present study is a pooled analysis, combining three previous conducted studies all performed at ZGT in the Netherlands (Fig 1). The first study, a non-randomized retrospective cohort study, included children between 1 to 16 years, with at least two measurements of their lipid profile in the period between June 2011 and November 2013 at ZGT (n = 121) [6]. The aim of this retrospective cohort study was to determine whether the developed dietary advice had an influence on risk factors of cardiovascular disease in children. The second study, a randomized controlled trial, included children between 1 and 4 years old (n = 125), referred by their general physician with recurrent upper respiratory tract infections (URTIs), between March 2015 and October 2017, if they had a minimum of 3 URTIs in the last 3 months [8]. The aim of this randomized controlled trial was to evaluated whether the dietary advice can decrease the number and duration of URTIs in children with recurrent URTIs. The third study, a randomized controlled trial, included children aged 1 to 12 years (n = 65) in the period between January 2016 and September 2018, with subclinical hypothyroidism (SH) (Thyroid Stimulating hormone (TSH) > 4.2 mU/L and FT4 within the normal range) [7]. Here the aim was to investigate whether this dietary advice improves or normalizes SH or decreases the presence of Thyroid Stimulating Hormone (TSH) and/or tiredness.

Fig 1

Schematic overview of the data collection of three previous conducted studies used for this pooled analysis.

One retrospective cohort study and two RCTs.

In the three studies, participants from the intervention group received the same dietary advice, containing minimal and unprocessed food products. This intervention consisted of five times per week green vegetables, three times per week beef, daily 200–300 mL whole cow’s milk (3.4% fat) and whole dairy butter (80% fat) on each slice of bread. The advice was provided with age-specific portion sizes according to the national guidelines. The control group was not informed about the contents of the dietary advice and continued their usual dietary habits. The primary outcome measurements were the measurements of the lipid profile. Standard Deviation (SD) BMI scores of the children were the secondary outcome measurements. Since absolute BMI scores can’t be compared between different age groups in young children, SD scores were determined. BMI was calculated by dividing weight in kilograms by the square of height in meters. The SD BMI score was calculated on the basis of gender, age, height, and weight.

Blood was collected by venapuncture at the start and follow-up of the study. Measurements from the lipid profile are total cholesterol, triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) cholesterol and total cholesterol/HDL ratio. These measurements of the lipid profile were measured using enzymatic colorimetric techniques with the COBAS 6000 (Roche Diagnostics, Almere, The Netherlands). LDL cholesterol was calculated with Friedewald’s formula: LDL = total cholesterol − HDL − (0.45 × TG). SD BMI was calculated based on gender, age, height, and weight. SD scores were used to compare the values with the reference population of Dutch children.

IBM SPSS Statistics 26 (SPSS Inc., Chicago, IL, USA) was used to perform statistical analyses. Raw data of the three intervention studies where combined into one single dataset for analysis. Descriptive statistics were used to obtain mean values with SD, medians, 95% CI and interquartile ranges (IQR). Since this study used results from three previous conducted studies, a mixed effects analysis of covariance (ANCOVA) model was used to test changes in the lipid profile and SD BMI levels between the two groups over time (at intake and at follow-up). Firstly, a one-way analysis of variance (ANOVA) test was performed for every measurement within both the intervention group and control group. Thereafter the ANCOVA test was performed. P-values were considered significant when <0.05.

3. Results

3.1 Baseline characteristics

The non-randomized retrospective cohort study excluded 33 participants. Seven participants had an incomplete lipid profile, 22 participants suffered from a disorder that might influence the lipid profile such as obesity and diabetes mellitus and four participants had other reasons for exclusion. From the first randomized controlled trial, three participants dropped out. Four participants were excluded due to loss of follow-up. Four participants dropped out of the second randomized controlled trial. In total, 267 participants were included in this pooled analysis study. Table 2 shows the descriptive factors age and gender distribution and baseline measurements of the lipid profile of the intervention group and control group per included study of this pooled analysis as well as combined for this pooled analysis. For all dietary components, the compliance was significantly higher in the intervention group. The overall compliance with the dietary advice in the intervention group was 87% compared to 36% in the control group.

Table 2

Baseline characteristics and measurements of the lipid profile of the intervention group and control group of the three intervention study’s and combined for the pooled analysis.

Variable	Intervention group				Control group
	1.Retrospective cohort study n = 48	2.RCT: URTI’s n = 58	3.RCT: SH n = 29	Pooled analysis n = 135	1.Retrospective cohort study n = 40	2.RCT: URTI’s n = 60	3.RCT: SH n = 32	Pooled analysis n = 132
Age in years (median, IQR)	2.6 (1.6–8.0)	2.02 (2.0–2.0)	7.7 (7.4–4.4)	4.2 (3.0–4.0)	4.7 (2.3–9.0)	1.9 (2.0–1.0)	8.1 (7.9–4.8)	4.7 (3.0–5.0)
Male: female (n)	25: 23	37: 21	15: 14	77: 58	24: 16	24: 36	16: 16	67: 72
Total cholesterol (mmol/L) ± SD	4.34 ± 1.01	3.99 ± 0.69	4.23 ± 0.99	4.12 ± 0.84	4.19 ± 0.91	3.91 ± 0.81	4.29 ± 0.77	4.17 ± 0.76
TG (mmol/L) ± SD	1.38 ± 0.74	1.46 ±0.80	1.14 ± 0.78	1.32 ± 0.73	1.35 ± 0.94	1.44 ± 0.87	1.15 ± 0.98	1.36 ± 0.92
HDL cholesterol (mmol/L) ± SD	1.20 ± 0.45	1.11 ± 0.35	1.40 ± 0.46	1.22 ± 0.41	1.22 ± 0.40	1.15 ± 0.36	1.47 ± 0.39	1.26 ± 0.41
LDL cholesterol (mmol/L) ± SD	2.47 ± 0.98	2.22 ± 0.62	2.31 ± 0.97	2.31 ± 0.79	2.32 ± 0.80	2.27 ± 0.67	2.30 ± 0.74	2.32 ± 0.70
Total cholesterol/HDL cholesterol ratio (mmol/L) ± SD	3.96 ± 1.30	3.95 ± 1.29	3.21 ± 1.39	3.70 ± 1.19	3.72 ± 1.26	3.83 ± 1.58	3.19 ± 1.25	3.69 ± 1.43
SD BMI ± SD	-0.26 ± 1.32	-0.13 ± 1.16	0.36 ± 1.60	-0.048 ± 1.37	-0.58 ± 1.21	-0.061 ± 1.26	0.82 ± 1.39	0.023 ± 1.38

3.2 Lipid profile

The measurements of the lipid profile at follow-up are shown in Table 3. Within the intervention group, HDL-cholesterol increased significantly from 1.22 mmol/L, 95% confidence interval (CI) 1.14–1.32, to 1.42 mmol/L 95% confidence interval (CI) 1.30–1.65 (p = 0.007). This increase over time was significantly different compared to the increase in the control group from 1.26 mmol, 95% CI 1.19–1.35, to 1.30, 95% CI 1.26–1.37 (p = 0.04). Since HDL-cholesterol increased significantly in the intervention group, the total cholesterol/HDL cholesterol ratio was significantly reduced with 0.45 mmol/L, 95% CI 2.96–3.31 (p = 0.00). This reduction was significantly different (p = 0.05) compared to the reduction of 0.25 mmol/L in the control group 95% CI 3.24–3.64 (p = 0.11). TG was significantly reduced in the intervention group with 0.18 mmol/L, 95% CI 0.99–1.26 (p = 0.001), and in the control group with 0.23 mmol/L, 95% CI 0.99–1.23 (p = 0.03). This reduction was similar between the two groups over time (p = 0.83). Total cholesterol levels and LDL cholesterol did not change significantly within the groups. No significant changes occurred in SD BMI in the intervention group. However, SD BMI did increase significantly from -0.0054 to 0.18 in the control group 95% CI 0.086–0.43 (p = 0.01). This change over time was significantly different (p = 0.05) compared to the intervention group, which showed a small reduction from -0.045 to -0.06795% CI -0.42–0.14 (p = 0.87).

Table 3

Changes between the two groups in measurements of the lipid profile and SD BMI at the end (follow-up) of the study period (with 95% CI).

Measurement	Intervention group (n = 135)	Control group (n = 132)	p-value
Total cholesterol (mmol/L) ± SE	4.20 ± 0.062 (4.05–4.33)	4.15 ± 0.064 (4.01–4.29)	0.34
TG (mmol/L) ± SE	1.14 ± 0.055 (0.99–1.26)	1.14 ± 0.059 (0.99–1.23)	0.83
HDL cholesterol (mmol/L) ± SE	1.42 ± 0.067 (1.30–1.65)	1.30 ± 0.034 (1.26–1.37)	0.04*
LDL cholesterol (mmol/L) ± SE	2.36 ± 0.056 (2.19–2.45)	2.35 ± 0.056 (2.24–2.48)	0.86
Total cholesterol/HDL cholesterol ratio (mmol/L) ± SE	3.25 ± 0.077 (2.96–3.31)	3.44 ± 0.091 (3.24–3.64)	0.05*
SD BMI ± SE	-0.067 ± 0.13 (-0.42–0.14)	0.18 ± 0.12 (-0.086–0.43)	0.05*

* indicates a significant p-value when p<0.05. SE (Standard Error).

4. Discussion

This pooled analysis aimed to get more information about the effects of a dietary intervention with minimal and unprocessed foods that are relatively high in natural saturated fats, on the lipid profile and SD BMI of young children. There were some concerns about negative effects on the lipid profile regarding the saturated fats. However, consumption of whole dairy products, green vegetables and beef did not result in a negative lipid profile. It even showed a significant increase in the beneficial HDL cholesterol in the children who consumed the dietary advice. This is favourable since higher HDL cholesterol levels can reduce the risk of developing obesity and CVD in later life [9,10].

A previously conducted meta-analysis of randomized control trials in adults supports this finding since it was observed that consumption of natural saturated fatty acids increased HDL cholesterol even more compared to consumption of unsaturated fatty acids [13]. Secondly, a large meta-analysis of prospective cohort studies that investigated the relationship between consumption of fat from whole cow’s milk and adiposity in children (aged 1–18) concluded that a higher intake of fat from whole cow’s milk was associated with lower adiposity in the children [14]. Furthermore, it had a favourable effect on the lipid profile since TG was reduced and HDL cholesterol was increased. Similar results were observed by Engel et al. (2018) in a 3-week randomized crossover study where whole milk consumption was compared to skimmed milk consumption [15]. Whole milk consumption increased HDL cholesterol significantly compared to skimmed milk. Still, consuming a diet with a high saturated fat percentage is not accepted by many health organizations like the national Heart Foundation of Australia [16]. Besides saturated fats, whole dairy products also contain natural trans-fats, trans-palmitoleic acid, which have been associated with lower LDL cholesterol and TG levels, and higher HDL cholesterol in several large cohort studies [17-19]. However, the mechanism remains unclear. One of the possible mechanisms, by which whole cow’s milk does not cause unfavourable lipid profiles, is the presence of calcium. Whole dairy products contain a high amount of calcium, of which several studies observed a positive association with body fat modulation [20-22]. These studies have shown that calcium potentially inhibits fat absorption as faecal fat excretion was increased. A second potential mechanism of saturated fats from whole dairy products is that it reduces chronic inflammation and oxidative stress [23]. Fats from whole dairy products, especially butter, are sources of butyric acid which has anti-inflammatory properties [24]. Butyric acid downregulated the NFκB-mediated inflammatory pathways resulting in decreased chronic inflammation in the gastrointestinal tract, leading to beneficial effects on the lipid profile, body weight and metabolic health [25,26]. A third possible mechanism that could be involved in lowering measurements of the lipid profile are sphingolipids present in whole dairy products. Ohlsson et al. observed reduced cholesterol absorption in healthy adult men who consumed whole dairy products with sphingolipids [27]. However, they remained inconclusive about the exact mechanism behind this.

The significant increase in HDL cholesterol contributes to the significant decrease in total cholesterol/HDL cholesterol ratio in the intervention group since total cholesterol remained similar after consumption of the diet. The total cholesterol/HDL cholesterol ratio is used as a predictor of developing CVD in later life [1]. Contrary to expectations, this predictor was also slightly decreased in the control group, indicating that not only this dietary advice decreases the risk of developing CVD in later life, but other factors can also influence this measurement. It is known that some commonly consumed food products have lipid-lowering properties. For example, thylakoids (the photosynthetic parts of chloroplasts) and phytochemicals in green vegetables have been shown to lower blood lipids [28]. An intervention study observed significantly lower free fatty acids in serum when healthy normal-weight individuals consumed a high-fat meal with the addition of thylakoids [29]. Secondly, a study with rats treated with thylakoids and phytochemicals from spinach showed a 62.3% reduction in TG [30]. This is in line with results from the present study since the TG were reduced in both groups. However, as expected the compliance to the green vegetables was higher in the intervention group (82%) compared to the control group (57%). Still, this resulted in a similar decrease in TG in both groups, showing green vegetables is not solely responsible for lowering the TG.

The diet used in this intervention consisted of minimal and unprocessed food products, such as green vegetables and whole dairy products. A previous study conducted by Lee et al. (2014) investigated the association between added sugars in (ultra)processed foods and HDL cholesterol in young females in a 10-year follow-up (from 9 years to 19 years of age) cohort study [31]. They observed an increased HDL cholesterol in individuals who consumed less than 10% energy from added sugars. Secondly, previous studies have demonstrated that consumption of (ultra)processed foods in early life contribute to a negative lipid profile in young children [5,32]. The intervention group from this pooled analysis study consumed whole dairy products instead of ultra-processed artificially flavoured dairy products. This could have contributed to the increase in HDL cholesterol [14]. Data of the dietary intake before they started with the dietary advice were incomplete and therefore not shown.

Besides the lipid profile, consumption of whole dairy products, green vegetables and beef did not lead to a change in SD BMI levels in the young children. In contrast, the SD BMI of the children that continued their usual dietary habits increased significantly. This could also be explained by the type of food these children consumed, as they continued their usual dietary habits. UNICEF concluded that 70% of the products made for young children in the Dutch supermarket, such as breakfast cereals and snacks, are unhealthy [3]. These products are consumed by a lot of children in the Netherlands since parents simply do not know that these are unhealthy. According to previous studies, the consumption of (ultra)processed foods leads to poor diet quality [4,33]. Costa et al. (2019) concluded that early (ultra)processed food consumption was associated with increased abdominal obesity in children [34]. Consumption of these products results in negative health outcomes later in life, such as obesity and CVD [3].

It is becoming clear that a healthy and natural diet is necessary to nurture human health and to support environmental sustainability [12]. For children with developing and growing bodies, an adequate intake of macro- and micronutrients is important and required. Food from animal sources can be a valuable source since they contain a high nutrient density [35].

The strength of this pooled analysis is that it combines the results of intervention studies and not of cross-sectional studies. This is not/seldomly done in children of this age group. The number of investigated children is increased compared to each single study and therefore, conclusions can be drawn. Some limitations must be considered as well. The laboratory could not make a distinction in the LDL cholesterol subclasses (large, intermediate or small dense). Previous research has suggested that the LDL subclass pattern and size of the LDL particles are important in the development of CVD [36]. Since this distinction could not be made, LDL cholesterol was categorized as one group and not further analysed. The dietary habits of the children before they started with the dietary advice were incomplete. Suggestions for follow-up studies could be the inclusion of additional information on the composition of the diet (macronutrient composition, fatty acid composition, fiber, plant sterols/stanols) as well as beverages intake. Certainly, children from the investigated age group consume many beverages and notably the caloric ones can influence the lipid profile as well. Secondly, the level of physical activity should be reported in future studies to strengthen the results.

The findings from this pooled analysis can further support the available scientific evidence for nutritional guidelines worldwide. An easily accessible, simple and low-cost diet can lead to beneficial outcomes for young children.

5. Conclusion

This pooled analysis, including minimal and unprocessed foods, shows that consumption of whole dairy products, green vegetables and beef had no negative effects on the lipid profile, but increased the prognostic favourable HDL cholesterol significantly. Secondly, this diet had no adverse effect on the SD BMI levels of the children. Therefore, this dietary advice of minimal and unprocessed foods, conform guidelines of the EAT-Lancet sustainable diet for the future, can safely be recommended for children.

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23 Apr 2021

PONE-D-21-01126

Effect of a dietary intervention including minimal and unprocessed foods, high in natural saturated fats, on the lipid profile of children

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Reviewer #1: Specific comments

1) Additional information on the composition of the diet (specific foods, macronutrient composition, fatty acid composition, fiber, plant sterols/stanols) that control subjects consumed as well as the level of physical activity would be useful, not only for the present report but for comparison with previous and future studies.

2) Additional information on the foods (macronutrient composition, fatty acid composition, fiber, plant sterols/stanols) consumed by subjects in the intervention group besides the 4 foods listed in Table 1 as well as the level of physical activity would also strengthen the manuscript.

3) The manuscript indicates that normality was checked by the Shapiro-Wilk test, but was normality found? If not, what action was taken?

4) The manuscript indicates that “All of these external factors did not affect the lipid profile of the children significantly (data not shown)”. The results of these statistical tests should be included in the manuscript whether statistically significant or not.

5) The type and amounts of beverages, notably the caloric ones, consumed by the intervention and control subjects should be documented in the manuscript if possible, and discussed.

Reviewer #2: Title page: the short title 'a pooled analysis' is insufficiently descriptive of this study; please suggest a more informative title. I would also suggest that the main title include the description that this is a pooled analysis e.g. by adding " - pooled evidence from randomised controlled trials and a cohort study".

Abstract: The methods need a bit more detail e.g. one sentence to explain how you conducted the pooled analysis, since this is a key feature of this study. The results in the abstract also don't seem to make sense: for example, you say BMI increased from 0.023 to 0.15 - these are impossibly low values of BMI, unless you meant that these were the changes in BMI (but even then the sentence would still not make sense). Please recheck these. Lastly, the appropriate way to report results is to report the effect/difference, its 95% confidence intervals and the p-value for the hypothesis test of 'no-difference'.

Methods: the current methods are not sufficiently detailed. The study setting, participants, interventions and outcomes are reasonably well reported, but the statistical methods are glossed over. You could add a bit more description, perhaps one or two sentences each, of the underlying studies, e.g. what their objectives were. For the methods, there are several things you need to add to improve the reporting. First, you need to report how you summarised the participant characteristics. This is typically reporting counts and proportions for categorical variables and means or medians with standard deviations or IQRs, respectively, for continuous ones. This should preferably be done separately for each data source (e.g. see Table 1 in http://www.thelancet.com/journals/langlo/article/PIIS2214-109X(17)30484-9/fulltext). Secondly, for this analysis of a pooled dataset you can't simply resort to t-tests, there are statistical issues relating to within-study clustering of the contributing studies to deal with. You need an appropriate method to pool estimates from studies, and this could be done either through (1) analysing the datasets independently (e.g. analysis of covariance/baseline-adjusted models for the continuous outcomes) and then combining the study specific estimates (separately for each outcome) using an appropriate method such as inverse variance weighting to pool them, or (2) combining all the studies into a single dataset and then using a mixed effects ANCOVA/baseline adjusted model on the combined dataset for each outcome, with random effects across studies. Method (1) would be further enhanced by including a forest plot of the study-specific effects and the pooled effect similar to what would be done in a meta-analysis. Comparison of baseline characteristics between treatment groups is unnecessary, especially for randomised trials where baseline imbalance is not expected anyway.

Results: see comment above about comparing baseline characteristics between groups. Furthermore, in table 2 you have also compared them in terms of dietary compliance, which is a feature of the intervention which is necessarily expected to be different - please remove the p-value column from this table. In Table 3 you should not even be including the baseline values in the comparison; as suggested above, you should use the baseline values only for adjustment in the ANCOVA model i.e. a model of the follow-up values with treatment group as the main explanatory variable and including the baseline value of the outcome as a covariate. Table 3 should then report the follow-up mean and standard error (not SD unlike the baseline table) for each arm, followed by the effect (i.e. the coefficient for treatment in the ANCOVA model described above) with its 95% confidence intervals and p-value. You can have additional columns for effect/95%CI/p-value if you report both unadjusted and adjusted effects.

I am unable to assess the manuscript further given the shortcomings of the approach to analysis.

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Reviewer #1: No

Reviewer #2: No

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4 Jul 2021

Dear reviewers,

I would like to thank you for your effort and time you've put into my manuscript. Your input definitely helped to improve the manuscript. I hope you feel the same!

Submitted filename: Rebuttal Letter.docx

Click here for additional data file.

27 Jul 2021

PONE-D-21-01126R1

Effect of a dietary intervention including minimal and unprocessed foods, high in natural saturated fats, on the lipid profile of children, pooled evidence from randomized controlled trials and a cohort study.

PLOS ONE

Dear Dr. Hendriksen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

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Massimiliano Ruscica, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Abstract

- SD BMI is introduced suddenly in the abstract without explanation; this should be described. Is it the standardised body mass index? If so, say so and also explain how the standardisation was done.

- the methods should summarise how the pooled analysis was conducted; there is nothing in the current abstract that gives the reader any sense of what this pooled analysis constituted: for example, did you combine all data into a single dataset and then conduct regressions looking at the associations between exposures and outcomes in this combined/pooled dataset? If so, a sentence saying exactly this should be included in the methods after the sentence that currently ends on line 52.

- the statements from the second half of line 46 to the end of line 47 pertaining to the numbers included in the analysis are results and should be moved to the results section of the abstract.

- I appreciate that the authors have included effect estimates, confidence intervals and p-values in the results. However there is a problem with the results as presented. The point estimate of the difference between intervention and control group of 0.20 would be expected to lie within the confidence intervals; so a confidence interval of 1.30 to 1.65 is not consistent with this point estimate. Additionally, the correct order to cite the numerical results is: estimate 95%CI p-value, not estimate p-value 95%CI as currently done on line 55-56. Lastly, the result at the end of line 56-57 should also include an estimate of the magnitude of increase with 95%CI and p-value. I do wonder though what the relevance is of reporting changes over time in the control group - it is the difference in difference over time between the intervention and control groups which would be important to report here.

Methods

- more clarity is needed on what SD BMI means, especially how the standardisation was done to obtain this outcome measure.

- more clarity is needed on how the 'pooling' was done here: did you pool raw data from the studies into a single dataset then analyse them, or did you pool the estimates from the three studies to obtain pooled estimates?

- in line 129 you say participants were divided into control group and intervention group without describing what those groups received. This statement as it serves no particular purpose (and you say nothing about the same for the other two studies), and you should probably remove it from this section, as you later describe the interventions from line 141.

- I think you should remove all statements about tests for normality; they are not useful, as they are extremely sensitive to very slight deviations from normality. In any case, normality of outcomes in not a requirement for regression analyses or ANOVA/ANCOVA (multivariate normality or normality of residuals is what is important). Furthermore, the statements about outcome measures in line 156 to 158 are results and would not be included in the methods even if they were relevant. Please remove these and the tests for normality.

Results

- effects or differences should be reported in this format: effect or difference, 95% confidence interval, p-value.

- as noted in the abstract, it is not possible for the increase of 0.20 mmol/L to have a confidence interval that does not straddle/include 0.20; the lower and upper 95%CI of 1.30 to 1.65 do not include 0.20 so either the point estimate or the confidence interval is incorrect.

- it would be more meaningful to present the difference in pre-treatment and post-treatment values of the outcomes compared between the intervention and controls groups i.e. difference-in-difference analysis. To do this, table 1 should include the baseline means and SDs of each of the outcomes in each group. Table 2 should then include the mean and SE (not SD) of each outcome in each arm in the follow-up period, along with the baseline-adjusted differences between the groups with 95%CI and p-values. You may also further adjust for other relevant factors if desired.

Please consult a statistician to advice on these and other issues with the analysis and presentation of results.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

29 Sep 2021

We would like to thank the reviewer for the time and valuable comments on the manuscript. All comments were carefully studied and answered. The comments were valuable for the improvements of this manuscript.

Submitted filename: Rebuttal Letter.docx

Click here for additional data file.

3 Dec 2021

Effect of a dietary intervention including minimal and unprocessed foods, high in natural saturated fats, on the lipid profile of children, pooled evidence from randomized controlled trials and a cohort study.

PONE-D-21-01126R2

Dear Dr. Hendriksen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Massimiliano Ruscica, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

14 Dec 2021

PONE-D-21-01126R2

Effect of a dietary intervention including minimal and unprocessed foods, high in natural saturated fats, on the lipid profile of children, pooled evidence from randomized controlled trials and a cohort study.

Dear Dr. Hendriksen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Massimiliano Ruscica

Academic Editor

PLOS ONE