EFFECTS OF OMEGA-3 FATTY ACIDS ASSOCIATED WITH ANTIOXIDANT VITAMINS IN OVERWEIGHT AND OBESE CHILDREN

Abstract

Introduction

Antioxidants and unsaturated fatty acids have protective effects in obesity.

Aim

We investigated the benefits of Omega-3 fatty acids associated with antioxidant vitamins in obese children. Magnesemia and calcemia were observed in relation with other metabolic parameters, before and after the treatment.

Materials and methods

60 obese children were compared with 35 normal weight children. Each obese child received daily, one pill, containing: 130mg docosahexaenoic acid, 25mg of eicosapentaenoic acid, vitamin A 200µg, vitamin D 1,25µg, vitamin E 2,5mg and vitamin C 30mg for three months. All the participants were instructed not to change their lifestyle.

Results

The serum values for these minerals and for 25(OH) vitamin D were lower in obese children. The obese children had insulin resistance (HOMA-IR) and an imbalance of serum adipocytokines. In obese children, the body mass index was negatively correlated with calcemia (r=-0.34) and serum 25(OH) vitamin D (r=-0.33). The HOMA-IR was negatively correlated with magnesemia (r=-0.34) and serum adiponectin (r=-0.29). The treatment improved the mineral serum level, the insulin sensitivity and the adipocytokines levels.

Conclusion

In obese children, the intake of Omega-3 fatty acids associated with antioxidant vitamins, for three months improved calcemia and magnesemia and increased insulin sensitivity.

INTRODUCTION

Overweight and obesity affect almost 60% of adults and nearly one in three children (29% of boys and 27% of girls) in the WHO European Region (1). Poor eating and sedentary behavior are the main causes of obesity. Also the genetic component is important. FTO rs9939609 polymorphism has been identified as a common gene variant in a Romanian Caucasian small cohort with obese children (2).

Obesity is associated with a high cardiometabolic risk (3) and a good management for obesity should start from childhood.

Nowadays we still need evidence that calcium and vitamin D accelerate weight or fat loss in obesity. The metabolisms of calcium and magnesium are closely related. The intestinal absorption and the renal excretion of these two minerals are interdependent. A high calcium/magnesium intake ratios or a low magnesium intake coupled with a high calcium intake have been associated with increased risk for cancer, prediabetes and cardiovascular diseases. Regarding the intake of these minerals, a Ca/Mg ratio range of 1.7-2.6 (weight to weight) has been proposed as optimum (4).

In girls, it was demonstrated that dietary calcium intake and, to a lesser extent, consumption of dairy products were inversely related to total and abdominal adiposity (5). In healthy male adolescents with different body mass values, the dietary calcium intake was inversely associated with total body and abdominal adiposity values (6).

Magnesium is the fourth most abundant mineral in the human body and it is an important cofactor for different enzymes. As an example, all the enzymes that metabolize vitamin D require magnesium (7). In obesity there is a vicious loop between the metabolic imbalance and the low-grade systemic inflammatory status. A low level of Mg²⁺ in the body, due to unhealthy diets can be one of the culprit. The researchers consider that it is hard to interpret whether Mg²⁺ beneficial effects occur through a direct effect on metabolic pathways or an indirect action on inflammation, or both (8). Mg levels are inversely associated with the degree of obesity It was shown that in the obese children and adolescents Mg level were connected to an unfavorable serum lipid profile and elevated systemic blood pressure compared to healthy controls (9).

Leptin secretion by adipose tissue is in relation with triglycerides storage and its action on adipogenesis is complex. It was demonstrated that in obese children, the level of leptin was higher in children with vitamin D deficiency than in those with normal vitamin D (10). Adiponectin is secreted by fatty tissues in an opposite ratio to body weight and has been shown to have insulin-sensitizing capabilities. It seems that the link between adiponectin and vitamin D is mediated by osteocalcin, an osteoblast-derived protein (11).

The ratio of leptin to adiponectin can be used as an index of insulin resistance and the baseline leptin and adiponectin levels are independent predictors of post-treatment weight gain in children with previous obesity (12).

Important contributors for the low serum vitamin D3 concentrations in obesity are: an increased uptake of vitamin D by the adipose tissue (13), less time spent in the sun and less 25(OH) vitamin D provided by the liver (14).

Vitamin D and long-chain n-3 polyunsaturated fatty acids (PUFA) provide benefits for the prevention of osteoporosis and cardiovascular events, and both are involved also, in controlling inflammatory and immunity pathways (15, 16).

There are only few studies about supplements of both Vitamin D and Omega-3 fatty acids. The results of a large randomized controlled trial of Vitamin D 2000 UI/day and marine Omega-3 fatty acid 1g/day supplements (Vital trial) demonstrated that these supplements may have additive and possibly synergistic antioxidant, anticancer and cardioprotective effects (17). Also, in a recent systematic review and meta-analysis a significant inverse relationship between childhood obesity and serum antioxidant levels has been shown (18).

All of the nonbone activities of vitamin D are in concert with vitamin A (9-cis retinoic acid) (19). Vitamin C is useful in combination with other antioxidants, can support calcium absorption and magnesium absorption and maximize the antioxidant effect of vitamin E (20).

This research aimed to examine the benefits of Omega-3 fatty acids associated with antioxidant vitamins in obese children. Magnesemia and calcemia were observed in relation with other metabolic parameters, before and after the treatment.

MATERIALS AND METHODS

The participants for this study, 65 obese and 35 normal weight children were recruited from the Emergency Hospital “Grigore Alexandrescu”, from Bucharest, Romania. The control and the obese groups were balanced according to sex and age. Safety exclusions criteria were: renal failure, endocrine diseases, hereditary diseases, cancer or systemic inflammation. The children involved in the study have followed the correct rickets prophylaxis only until 2-3 years old due to socio-economic reasons.

Each obese child received daily, one pill (323mg), containing: 130 mg docosahexaenoic acid (DHA), 25 mg of eicosapentaenoic acid (EPA), vitamin A 200 µg, vitamin D 1,25 µg (50IU), vitamin E 2,5 mg and vitamin C 30 mg for three months. All the participants were instructed not to change their lifestyle (dietary and drinking habits, physical activity) during the whole study and not to take any additional medication including vitamin supplements. In the first two weeks a placebo was given both to obese children and control subjects. At the end of this time, blood samples were collected. In the next three months the dietary supplement was administered once daily after lunch, only to obese children. A washout period was not considered necessary. The general practitioner was involved in monitoring the treatment by maintaining contact with the parents by phone. Five obese subjects gave up taking part of the study, and the final number of the participants was 60. The study protocol was approved by the Ethical Commission of the Emergency Hospital “Grigore Alexandrescu”, from Bucharest and a written informed consent was obtained from each parent.

Clinical Characteristics

Anthropometric measurements: body mass index (BMI) and waist circumference (WC) were determined. BMI was calculated as body weight (kg) divided by square height (m²). Overweight is defined as a BMI at or above the 85^th percentile and lower than the 95^th percentile. Children above the 95^th percentile are considered obese. The obese group (n=60) was formed by overweight and obese children.

Biochemical measurements

Blood samples were taken after an overnight fast. Standard enzymatic methods were used for measuring the levels of serum total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDLc). Total analytical variability, expressed as coefficient of variation (CV) was 2%, 1.9% and 0.9% respectively. Metabolic markers: fasting serum glucose and creatinine were measured by colorimetric methods. Enzymes activities for alanine aminotransferase (ALT) and aspartat aminotransferase (AST) were performed by standard methods using an automatic analyzer BS 400 Mindray and kits.

Total serum calcium was measured by titration with a standardized solution of ethylenediaminetetraacetic acid and free ionized calcium was measured directly using ion selective electrodes. Magnesium was measured by colorimetric methodology by using xylidyl blue as colorimetric agent.

Adiponectin was measured by the immunoenzymatic ELISA method, kit catalog nr ABIN509829, Mediagnost, Reutlingen, Germany, in Synevo lab and leptin was measured by the radioimmunologic method, by using the kit Human-leptin-RIA-sensitive LEP-R40, Mediagnost, Reutlingen, Germany, in Synevo lab. The homeostatic model assessment (HOMA) is a method used to quantify insulin resistance and beta-cell function. The HOMA-IR value was calculated according to Matthews formula, HOMA-IR=Glycaemia x Insulin/405. Glucose and insulin were measured during fasting and insulin was given in µU/mL. The method used for vitamin D determination was the immunochemical method with electrochemiluminescence detection. The reference range of the total level of 25(OH) vitamin D is 30-100 ng/mL. Levels of 20-29 ng/mL define vitamin D insufficiency, and levels below 20 ng/mL, the deficiency of vitamin D. The level of this vitamin was measured only at the beginning of the study and it wasn’t done after the treatment, because of high costs.

RESULTS

The serum biological values in the obese versus the control children are shown in the Table 1. It can be noticed that: the anthropometric parameters, the blood pressure values, the lipid profile, glycaemia, the transaminases, the albumin, the adipocytokines (leptin and adiponectin), the minerals (calcium and magnesium), the HOMA-IR and the 25(OH) vitamin D were modified significantly between the groups. After the treatment the differences between the groups were reduced and the values for the parameters were improved.

Table 1.

Serum biological values in obese and control children

	Control group (n=35)	Obese group (n=60)	Obese children after the treatment	Control versus obese group. p value	Obese group before and after the treatment p value
Average age (years)	11.6± 3.4	11.8±3.5	-	Ns	-
Gender (F/M)	17/18	35/25	-	Ns	-
SBP(mmHg)	115.8±9.5	133.2±12.9	119.2±12.2	<0.001	<0.01
DBP (mmHg)	53.8±8.8	69.1±8.4	58.5±5.2	<0.001	<0.01
BMI (kg/m2)	18.05±4.7	28.3±2.8	27.3±2.9	<0.001	<0.06
WC (cm)	61.1±13.7	93.1±11.6	85±8.9	<0.001	<0.01
HOMA-IR %	1.89±0.62	4.52±1.81	2.96±1.01	<0.0001	<0.01
Cholesterol (mg/dL)	121.2±24.9	157.8±32.9	122.2±22.7	<0.04	<0.03
Triglycerides (mg/dL)	50.9±19.3	106.6 ±54.3	82.2±29.5	<0.0004	<0.003
HDL-C (mg/dL)	64.3±8.6	43.5±12.1	60.4±12.1	<0.02	<0.05
LDL-C (mg/dL)	59.6±15.7	97.7±25.17	81.1±21.2	<0.01	<0.05
25(OH) vitamin D ng/dL	24.9±9.7	20.3±7.5	Not measured	<0.01	Not calculated
ALT (U/L)	13.6±4.8	28.0±8.9	19.9±7.4	<0.001	<0.01
AST (U/L)	16.8±3.7	28.1±8.913.5	22.8±7.1	<0.01	<0.03
Glycaemia (mg/dL)	69.8 ±5.1	87.9±12.3	75.6±8.6	<0.01	<0.05
Albumin (g/dL)	4.34±0.43	3.91±0.46	4.21±0.40	Ns	<0.01
Leptin(ng/mL)	14.8±7. 20	20.39±6.87	15.53±4.30	<0.001	<0.01
Adiponectin (ng/mL)	8.8±3.7	5.32±1.83	10.56±1.93	<0.02	<0.01
Albumin/Globulins	1.4±0.2	1.30±0.21	1.48±0.22	<0.01	<0.001
Total Calcemia (mg/dL)	9.9±0.54	8.87±0.57	9.59±0.50	<0.01	<0.03
Ionic calcemia (mg/dL)	4.91±0.34	3.82±0.24	4.06±0.14	<0.01	<0.05
Magnesemia (mg/dL)	2.4±0.6	2±0.13	2.12±0.14	<0.01	<0.02
Creatinine (mg/dL)	0.65±0.1	0.71±0.12	0.63±0.10	<0.001	<0.01

Legend: WC=waist circumference. BMI =body mass index DBP=diastolic blood pressure. SBP=systolic blood pressure.

The BMI was negatively correlated with ionic calcemia (r =-0.34, p<0.05) and also with 25(OH) vitamin D level (r =-0.33, p<0.05). The serum ionic calcium level was also negatively correlated with another anthropometric parameter, the waist circumference (r =-0.36, p<0.05).

The insulin resistance index, HOMA-IR was negatively correlated with magnesemia (r= -0.34, p<0.05, Fig. 3) and with adiponectin (r=-0,29, p<0.05). Magnesemia was negatively correlated, at the limit of statistical significance with glycaemia (r=-0.24, p<0.05) and with the level of beta1-globulins, as a marker of inflammation (r=-0.23, p<0.05).

Figure 3.

Correlation between HOMA-IR and magnesemia (r= -0.29, p<0.05).

DISCUSSION

In our study, the obese children had lower calcemia and magnesemia in comparison with the control. Although during the treatment period, the children have not received minerals, didn’t change their lifestyle, the intake of supplements with antioxidant vitamins improved the serum level for calcium and magnesium (Table 1). Maybe this result is a consequence of increased absorption of these minerals under the action of the low doses of vitamin D and of vitamin C from the supplement.

In the obese children group, the ionic serum calcium level was negatively correlated with the body mass index (Fig. 1) and also with the waist circumference. The level of 25(OH) vitamin D was negatively correlated with the BMI values (Fig. 2). In contrast with calcium, serum magnesium has not had any significant statistically correlation with the anthropometric parameters.

Figure 1.

Correlation between BMI and ionic calcemia (r = -0.34, p<0.05).

Figure 2.

Correlation between BMI and 25(OH)vitamin D (r=-0.33, p<0.05).

It was demonstrated that the high waist circumference and the high BMI present in obesity are inversely associated with the serum vitamin D level. Increased serum vitamin D and higher dairy calcium intake are related to greater diet-induced weight loss (21).

In a recent meta-analysis of epidemiologic studies it has been demonstrated that there is a negative relationship between blood vitamin D levels and the risk of abdominal obesity in children and adolescents. Each 10 ng/ml increase in serum vitamin D levels could be related to a 7% decreased odds of abdominal obesity. Waist circumference percentiles might better identify those with abdominal obesity than waist to height ratio and this should be important for clinical practice (22).

Also, waist circumference is the most effective anthropometric measurement for predicting the development of insulin resistance in obese adolescents and children. As vitamin D levels increase, anthropometric measurements are more stable and do not increase (23).

For vitamin D supplementation, the dosage and duration of cholecalciferol administration has not been established (24).

The parathyroid hormone (PTH) serum level is the link between calcemia, serum vitamin D level and anthropometric markers. Reinehr T et al. demonstrated that the weight status was positively correlated with the PTH levels and negatively with 25(OH) vitamin D serum level (25). Low calcium intake increases serum calcitriol levels in a direct manner or mediated by PTH. In adipose tissue, the calcitriol is sequestrated and influences the activity of many enzymes. It inhibits the expression of hormone-sensitive lipase and stimulates the fatty acid synthase, promoting lipogenesis (26), it inhibits the expression of uncoupling protein-2 (involved in thermo genesis and body weight control) (27) and in adipocytes, it stimulates the expression of 11b-hydroxysteroid dehydrogenase 1, which catalyzes the conversion of cortisone to cortisol (involved mainly in fat deposition, especially in the abdominal region) (28). Other studies have shown that dietary calcium and calcium supplements may increase the fecal excretion of fat by forming insoluble complexes in the intestine (29).

It should be emphasized that there is a vicious cycle between body fat and vitamin D deficiency. This liposoluble vitamin is sequestered and stored in adipocytes and its bioavailability is reduced. The PTH levels will be increased and it will promote lipogenesis which will worsen vitamin D deficiency (26).

Both low calcium intake and PTH may stimulate renin release and consequently angiotensin II and aldosterone synthesis and the blood pressure will be increased (30).

In our study, after the treatment, the calcemia was improved and the waist circumference and the blood pressure were reduced although the children were not on a specific diet (Table 1). After the treatment, the serum levels of the minerals (ionic calcium, total calcium and magnesium) were increased in 93.33% patients. The best increase was observed for total calcium, but we noticed, also that serum albumin, the transporter for calcium, increased significantly after the intake of the supplement (Table 1). We emphasize that magnesium is the second most important intracellular cation after potassium and serum magnesium does not reflect intracellular magnesium. However, hypomagnesemia can be proved to be one of the important predictors of obesity and usually is secondary to decreased dietary magnesium intake (31, 32). Clinical researchers have demonstrated an inverse association between dietary magnesium and insulin resistance (33). In our study, magnesemia was inversely associated with HOMA-IR (Fig. 3) and after the treatment, HOMA-IR was reduced significantly (Table 1) and magnesemia was improved (Table 1). Magnesemia was negatively correlated, at the limit of statistical significance with glycemia and with the level of beta1-globulins, as a marker of inflammation.

In a recent study, the researchers underlined that favorable effects of vitamin D are not only related directly to insulin signaling, but also indirectly “with reduction of oxidative stress, sub-inflammation and epigenetic regulation of gene expression as well as Renin-Angiotensin-Aldosterone-System” (34). Many studies have demonstrated that vitamin D supplementation reduses insulin resistance (35, 36).

In our study, in the obese children, the imbalance between serum adiponectin (low level) and leptin (high level) was present (Table 1). We have not found any significant correlation between serum 25(OH) vitamin D level and adipokines, but the serum adiponectin was negatively correlated with the insulin resistance index, HOMA-IR.

We underline that after the treatment the serum concentrations of these adipocytokines and the HOMA-IR level were improved (Table 1).

In the literature there are articles which have demonstrated that each component from the supplement used in our paper could influence the insulin resistance. Omega-3 fatty acids in combination either with vitamin D (37) or with vitamin E (38) are effective in reducing insulin resistance. Vitamin C and E are antioxidants, act as scavengers in aqueous and respectively in lipid cellular medium, are effective in prevention of non-enzymatic glycosylation of proteins and reduce insulin resistance (39).

The strength of our research study is the fact that a supplement (Omega-3 fatty acids, associated with antioxidant vitamins) reduces waist circumference and insulin resistance in obese children. We emphasize that the children were not on diet and a good compliance of the treatment was obtained due to parents and general practitioner involvement.

This study has some limitations because of the relative low number of children recruited, lack of vitamin D determination at the end of the study and because it is not a placebo controlled, double blind study. Also, it would have been interesting to specify what the intake was prior to the start of the study by introducing a diet evaluation questionnaire. Magnesium deficiency was demonstrated in a high calorie food rich in carbohydrates and fats and this diet provokes the accumulation of excess visceral fat (40). As an example, in the questionnaire we could ask about the carbonated drinks that contain a lot of phosphorus which interfere with magnesium absorption. Nowadays, compensation of magnesium deficiency in children through the use of organic magnesium salts is open (40).

In conclusion, in obese children, the intake of Omega-3 fatty acids associated with antioxidant vitamins, for three months, improved calcemia and magnesemia and increased insulin sensitivity. The antioxidant vitamins: A, D, E and C, even if in low doses have additive and possibly synergistic effects on mineral metabolism. The waist circumference, a surrogate marker of insulin resistance and of visceral fat was reduced after the treatment, even if the children haven’t lost weight. The improved magnesemia and the intake of Omega-3 fatty acids increased the insulin sensitivity and reduced the adipocytokines imbalance.

The answer if calcium and vitamin D accelerate weight or fat loss in obesity will be given by the large-scale prospective cohort studies and randomised trials. Our study underlines that antioxidant vitamins and Omega-3 fatty acids reciprocally potentiated even at low doses.

Conflict of interest

The authors declare that they have no conflict of interest.