Background
Omega-3 long chain-polyunsaturated fatty acids (LC-PUFAs)—docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA)—are essential for growth, development and general health [
1]. Omega-6 LC-PUFA arachidonic acid (ARA) is essential for brain development [
2]. DHA is especially critical for optimal brain [
2], cognitive [
3] and behavior development. EPA is a precursor of anti-inflammatory eicosanoids (prostaglandins (3 series), leukotrienes (5 series) and thromboxanes (TXA
3)) [
4], and adequate intake of EPA is closely related to positive immunological [
5], inflammatory [
6] and metabolic [
7] outcomes. DPA has been reported to have beneficial effects which include inhibiting platelet aggregation, stimulating endothelial cell migration and regulating gene expression [
8]. Though Arachidonic acid (ARA) is a precursor of pro-inflammatory eicosanoids (prostaglandins (2 series), leukotrienes (4 series) and thromboxanes(TXA
2)) [
4]; it produces some metabolites that are required for systemic homeostasis [
9]. ARA and its metabolite lipoxin A
4 have been shown to function as endogenous anti-diabetic molecules [
10]. ARA together with LC-PUFAs and their anti-inflammatory products: lipoxins, resolvins, protectins and maresins suppress production of pro-inflammatory eicosanoids, limit inflammation, enhance wound healing, resolve inflammation thus restoring normal cellular, tissue and organ function [
10].
Supplementation with omega-3 LC-PUFAs in cardiovascular diseases (CVD) [
11], diabetes mellitus [
12], hypertension [
13], sickle cell anemia [
14], inborn errors of metabolism [
15,
16], non-alcoholic fatty liver disease [
17,
18], attention-defect/hyperactivity disorder [
19] autism [
20] and asthma [
21] has been reported to prevent or alleviate symptoms in children, though other studies have reported conflicting results [
22‐
25].
The above disorders are due to deficiencies in omega-3 fatty acids. Deficiencies in omega-3 fatty acids may result from factors that affect availability of omega-3 LC-PUFAs and influence the metabolism of essential fatty acids (EFA) to LC-PUFAs. These include, imbalances in metabolic pathways [
26], genetics [
27], imbalances in ARA: EPA ratios [
28] and sex hormones [
29]. The imbalances in the metabolic pathway may result from linoleic acid (LA) competing with α-linolenic acid (ALA) for the endogenous conversion of ALA to the long chain derivatives EPA and DHA and also inhibition of incorporation of DHA and EPA into tissues [
26]. Therefore high levels of LA in the diet result in low ALA and low omega-3 LC-PUFA levels. This in turn affects the omega-6: omega-3 (ARA: EPA) ratios which are critical in human health outcomes [
20,
28]. The high levels of LA lead to increased activity of the ARA metabolic pathway [
4], which has deleterious effects such as neurological and neurodevelopmental disorders [
30]. High concentrations of ARA compete with EPA for incorporation into cell membrane phospholipid leading to high ARA: EPA ratios [
28]. The low omega-3 LC-PUFA levels can be due to deficiencies and defects in the Δ6 or Δ5 desaturase enzyme [
31] or mutations in the fatty acid desaturase (FADS) gene [
27]. Protein malnutrition, carnitine and α-tocopherol enzyme deficiency as well as excess oxygen free radical production in chronic diseases also affect LC-PUFA availability [
32]. Oestrogen and testosterone, have been reported to affect EFA metabolism hence availability of long chain metabolites, leading to higher levels in females compared to males [
29]. The conversion of EFA into their long chain metabolites is stimulated by oestrogen and inhibited by testosterone [
29].
Despite the reported benefits of omega-3 LC-PUFAs in children, most studies demonstrating the nutritional importance of omega-3 LC-PUFAs in children have been carried out in developed countries [
2,
3,
11‐
13], [
15‐
17,
19,
21] with a limited number of studies on African children [
14,
33‐
35]. In most of sub-Saharan Africa healthcare facilities, omega-3 LC-PUFA levels are not on clinical laboratories test menus because the laboratories lack the expertise and technology to perform the tests [
36]. In the past the assessment of omega-3 LC-PUFAs has been hindered by difficult methodology [
37] and sample instability [
38,
39]. Analysis has since been revolutionized by the use of minimally invasive dried blood spots (DBS) [
40], which allow the estimation of fatty acid composition of red blood cells and plasma phospholipids that are more reflective of the nutritional status [
41]. To date, only a few studies have developed protocols for testing LC-PUFAs in DBS [
37‐
40,
42], with none being carried out in Africa.
In Southern Africa, a region with high prevalence of childhood infectious diseases [
43], the levels of omega-3 LC-PUFAs in children are unknown, except in South Africa where the positive effects of omega-3 LC-PUFA supplementation on cognitive development were reported in children aged between 6 and 11 years [
33‐
35]. There is paucity of data on omega-3 LC-PUFA levels in the rest of African countries, including Zimbabwe, especially in children whose adequate intake of LC-PUFAs should be ensured for cognitive development and other positive health outcomes [
44].
Monitoring of fatty acid levels and results interpretation in individual patients or in populations require availability of reference intervals obtained from apparently healthy individuals [
45]. Fatty acids reference intervals have been established for glycerophospholipids in German children aged 2 and 6 years, for whole blood in apparently healthy European children aged 3–8 years [
46] and in apparently healthy Spanish children who were on a normal diet for their age elsewhere [
15] but these may not be transferable to a different population. However, scanty studies have been done in low income settings particularly in African children and none in Zimbabwe. At present no reference intervals for LC-PUFAs have been established in these settings.
In this study, the levels of omega-3 LC-PUFAs were determined in Zimbabwean children aged between 7 and 9 years using DBS and reference intervals for LC-PUFAs were determined. The LC-PUFAs were compared between groups by gender and by age.
Discussion
To our knowledge, this is the first study to report blood levels of omega-3 and omega-6 LC-PUFAs and to determine LC-PUFA reference intervals in 7–9 year old Zimbabwean children.
The levels for omega-3 LC-PUFAs (EPA, DPAn-3 and DHA) of the children in this study were strikingly low, while those of omega-6 LC-PUFA (ARA) were surprisingly high compared to the determined reference intervals and to the results obtained from a UK study on similar age groups and biomarker [
42] (EPA 0.20 v 0.56, DPAn-3 0.81 v 1.03, DHA 2.15 v 1.9, ARA 10.56 v 8.17). Generally, these children had very low omega-3 PUFAs and very high saturated fats, monounsaturated and omega-6 fatty acids. The essential omega-3 fatty acid, α-linolenic acid, which is the precursor of the omega-3 LC-PUFAs (EPA, DPA and DHA), mainly found in seeds, nuts and some vegetable oils, was also low in the children under study (median level 0.38 %
wt/wt) compared to results obtained from a study on similar age groups and biomarker [
42]. The highest EPA value obtained in this study of 0.55 %
wt/wt was lower than the mean values obtained from a study on similar age groups and biomarker [
42]. Results of the present study also demonstrated the lowest EPA value of 0.06 %
wt/wt reported in apparently healthy children compared to results obtained from a study on similar age groups and biomarker [
42]. This might be a reflection of the different geographical backgrounds, diet and genetic make-up of the children in the different studies.
The low EPA and high ARA levels are of health concern because they lead to very high ARA: EPA ratios, which are pro-inflammatory, and to very high total omega-6 PUFA: total omega-3 PUFA ratios [
30]. The high ratios observed in this study reflect possible imbalances in the dietary intake of omega-6 and omega-3 rich foods. The imbalances could be as a result of contemporary changes in human nutrition caused by increased consumption of diets rich in saturated fats (rich in beef), monounsaturated and omega-6 fatty acids including the use of cooking oils, vegetable oils and bread spreads rich in omega-6 PUFAs, accompanied by a decreased intake of omega-3 PUFA-rich foods [
50]. Deficiencies in DHA exposes children between the ages of 7 and 9 to impaired brain development during the 7–9 year old “Brain Spurt” [
44], possibly leading to compromised intellectual development, academic performance, low verbal learning ability, memory and learning difficulties [
33,
34].
The LC-PUFA levels of all parameters except DHA were lower in the present study compared to the expected values from the University of Stirling Aquaculture laboratory [
39] that used the same method of analysis and sample type (DBS) as the present study (EPA 0.20 v 0.91, DPA 0.81 v 2.47, DHA 2.15 v 2.47, ARA 10.56 v 13.88). The median EPA level in the present study was similar to that obtained by Mohammed et al. on pregnant Zimbabwean black women [
36], indicating a general view of the dietary intake of foods poor in omega-3 LC-PUFAs and α-linolenic acid in the population.
Our findings of no differences by gender in median LC-PUFAs levels were in agreement with those of Glaser et al. on a paediatric population [
45]. However, another study on a paediatric population reported a more pronounced low omega-3 and omega-6 LC-PUFA status in girls than boys [
42], while another study reported slightly higher omega-6 ARA in boys than in girls [
46]. Yet another study, reported that sex hormones (testosterone and oestrogen) influence the enzymatic synthesis of LC-PUFAs, leading to gender related differences in LC-PUFA status with higher levels occurring in adult females [
29]. The reason for the lack of gender differences in LC-PUFA levels observed in this study was perhaps due to the younger age of the participants.
The observed differences in median EPA and ARA: EPA ratio across and between the children’s age groups is probably due to differences in dietary content. The 7 year old children had lower EPA and higher ARA values leading to high ARA: EPA ratio. The low EPA values observed in the children understudy are however constrained by the lower sample size in this particular age group; hence the results should be interpreted with caution. These findings were similar to UK study on similar age groups and biomarker [
42] and also similar to the study with European children though with no age dependence for ARA [
46]. An Italian study with differences in fatty acids by age groups concluded that the differences resulted either from lower intakes or the rates of utilization and resulting physiological requirements which are higher in younger age groups compared to older age groups [
51].
The study also determined DBS LC-PUFA reference intervals for the apparently healthy 7–9 year old Zimbabwean children. However, these DBS LC-PUFA reference intervals cannot be generalised to the rest of the population since the LC-PUFA results were from children from a select group born to a cohort residing in a peri-urban setting, which did not include rural and urban children. The determined LC-PUFA reference intervals were not comparable to those of three other studies which determined LC-PUFA reference intervals perhaps due to methodological differences [
15,
45,
46].
Our results showed generally low values across the omega-3 LC-PUFA range. The levels of these LC-PUFAs could be improved by identifying and encouraging the intake of locally available omega-3 LC-PUFA rich foods. Supplementation with EPA and DHA omega-3 fish oils and algae based oils to balance ARA levels is recommended in the children since low omega-3 LC-PUFA levels are recognized confounders of general health. Limited intake of ARA-rich foods is also recommended if the desirable total omega-6 PUFA: total omega-3 PUFA ratio of 1–4:1 [
6] is to be achieved. There is need for a public awareness campaign on food sources rich in omega-3 LC-PUFAs and the benefits of omega-3 LC-PUFAs throughout life. We recommend further studies on children under the age of 5 years and inclusion of children from rural and urban Zimbabwe to ascertain their omega-3 LC-PUFA levels. Results from such studies could be used as the basis for establishing reference intervals that can be generalized to the whole Zimbabwean paediatric population, as well as the basis for food fortification and the implementation of omega-3 LC-PUFA supplementation policies.
The study has a number of limitations. Firstly no dietary intake assessment was done during specimen collection to ascertain the practices that could explain the low omega-3 LC-PUFA levels, hence, the causes of low omega-3 LC-PUFA levels are assumption based. Secondly, the determined reference intervals are limited to the children born to the specified cohort as a limited age group was used for this study. The study population was also restricted to children in a peri-urban setting that may not be truly reflective of the Zimbabwean population. The determined DBS reference intervals could also not be compared to those from other populations because of analytical method differences [
15,
45,
46]. Lastly, the three age groups were unequal and this could distort the distribution of omega-3 LC-PUFAs findings by age.
Abbreviations
% wt/wt, % weight to weight; ALA, α-linolenic acid; ARA, arachidonic acid; BHT, butylated hydroxytoluene; CI, confidence intervals; CVD, cardiovascular diseases; DBS, dried blood spot; DGLA, dihomo-gamma-linolenic acid; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; EFA, essential fatty acids; EPA, eicosapentaenoic acid; FADS, fatty acid desaturase; FAME, fatty acid methyl ester; GLC, gas liquid chromatography; IQR, inter-quartile ranges; LA, linoleic acid; LC-PUFA, long chain polyunsaturated fatty acids; PMTCT, prevention of mother to child transmission; SPE, solid phase extraction; TXA2, thromboxanes; TXA3, thromboxanes
Acknowledgements
This study was funded by the Letten Foundation (Oslo, Norway). We would like to thank the Letten Research Center for access to samples, the participants, their parents and legal guardians without whom this study would not have been possible. We would like to express our gratitude to Professor Bell of the Institute of Aquaculture University of Stirling Scotland UK, for training the first author in the analysis of the fatty acids, James Dick, Elizabeth MacKinlay and Irene Younger for hands on training and analysis of the samples.
Grace Mashavave (gracemashavave@yahoo.com)
MSc Clinical Biochemistry (UZ), Department of Chemical Pathology, University of Zimbabwe College of Health Sciences.
Patience Kuona (patiekuona@gmail.com)
MMED Paediatrics (UZ), Department of Paediatrics and Child Health, University of Zimbabwe College of Health Sciences.
Willard Tinago (wtinago@gmail.com)
PhD, Medical Statistics (University College Dublin, Ireland), Department of Community Medicine, University of Zimbabwe College of Health Sciences.
Babill Stray-Pedersen (babill.stray-pedersen@medisin.uio.no)
PhD, Division of Women and Children, Rikshospitalet, Oslo University Hospital and Institute of Clinical Medicine, University of Oslo, Norway.
Marshall Munjoma (marshall@uz-ucsf.co.zw)
PhD, Epidemiology and Diagnosis of Sexually Transmitted Infections (University of Oslo, Norway), Department of Obstetrics and Gynaecology, University of Zimbabwe College of Health Sciences.
Cuthbert Musarurwa (curtbertm@yahoo.com)
MSc Clinical Biochemistry (UZ), MSc Clinical Epidemiology (UZ), Department of Chemical Pathology, University of Zimbabwe College of Health Sciences.