In Northern Ghana, 33% of children are stunted due to economic disparities. Dietary fatty acids (FA) are critical for growth, but whether blood FA levels are adequate in Ghanaian children is unknown. The objective of this study was to determine the association between whole blood FAs and growth parameters in Northern Ghanaian children 2–6 years of age. A drop of blood was collected on an antioxidant treated card and analyzed for FA composition. Weight and height were measured and z-scores were calculated. Relationships between FAs and growth parameters were analyzed by Spearman correlations, linear regressions, and factor analysis. Of the 307 children who participated, 29.7% were stunted and 8% were essential FA deficient (triene/tetraene ratio>0.02). Essential FA did not differ between stunted and non-stunted children and was not associated with height-for-age z-score (HAZ) or weight-for-age z-score (WAZ). In hemoglobin adjusted regression models, both HAZ and WAZ were positively associated with arachidonic acid (p0.01), dihomo-gamma-linolenic acid (DGLA, p0.05), docosatetraenoic acid (p0.01) and the ratio of DGLA/linoleic acid (p0.01). These data add to the growing body of evidence indicating n-6 FAs are critical in childhood linear growth. Our findings provide new insights into the health status of an understudied Northern Ghanaian population.
The study was conducted in the northern region of Ghana in the Savelugu-Nanton district [26]. The district covers 2022.6 sq. km with a population density of 68.9 persons per sq. km. The population of Savelugu-Nanton is 139,283 persons with 14,669 households. The average rainfall in the Savelugu-Nanton district is 600 mm. The district is also characterized by high temperatures with an average temperature of 34°C. The district is situated in the Savanna woodland that is capable of sustaining livestock, farming and the cultivation of crops such as rice, groundnuts, yams, cassava, maize, cowpea and sorghum. Over 80% of inhabitants are farmers. The main sources of water in the district are boreholes, rivers and streams, public taps, and pipe borne water. Though the primary source of water for taps and pipe borne water is the same, access to either categorizes households under different income levels, perhaps reflecting differences in hygiene and even nutritional status. Thatch is the main roofing material for housing (50.9%). Illiteracy level is high with 69% of all inhabitants 11 years and above having no education. Some common diseases in the district include malaria, gastroenteritis, upper respiratory tract infection, diarrhea, anemia, and pneumonia. There are three operational community health post zones that deliver health services to the people. The Savelugu-Nanton district was chosen for study as stunting levels are above the national average in the rural communities in this district [27] with overall district stunting level of 38.8% [28]. Additionally, it is one of the few areas in Northern Ghana with road access to rural communities. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects/patients were approved by the Institutional Review Board at Michigan State University (IRB # 16–557) and the Committee on Human Research Publication and Ethics, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana (CHRPE/AP/236/16). The parent or caregiver of the participating child gave consent prior to the child’s participation. A script of the written consent was read and translated in Dagbani to the parents or caregivers of the children. The parents or caregivers thumb printed the consent document to give consent. They were assured that participation was voluntary and confidential, and that their information would remain anonymous. Children (n = 307) between 2 to 6 years of age residing in 5 communities in the Savelugu-Nanton district were recruited for the study. The communities were Janjorikukuo, Pong Tamale, Kparigilanyi, Morglaa and Fazhini. A power analysis was conducted from the results of an earlier study that measured maternal and infant erythrocyte fatty acid intake [29], and the fatty acid variation reported was utilized to run an a-priori sample size calculation for multiple regression based on an estimated medium effect size of 0.5 and significance level p = 0.05. This indicated that 242 participants would yield statistical power of 80% [30]. 307 children were enrolled, raising the power to 90%. The exclusion criteria included sick and hospitalized children as well as children who were legally declared intellectually disabled. Data was collected in July 2016. Height of all participants was measured to the nearest 0.1cm with a stadiometer (Seca, USA). Weight was measured using a digital bathroom scale to the nearest 0.1kg (Camry, model number: EB9003, China). All measurements were repeated and averages were reported. The date of birth was recorded from the child’s health card or birth certificate. The sex of the child was also recorded. Blood spots (40ul) were collected on a dried blood spot card (DBS) as previously described by Jumbe et al., 2016 [4, 31]. A sterile single-use lancet was used in puncturing the tip of the middle finger to obtain drops of blood. The first drop of blood was wiped with a sterilized dry pad. The drops of blood were then collected onto the DBS cards. The cards were stored in a dry, cool environment and shipped to the USA for FA analysis at OmegaQuant Analytics, LLC (Sioux Falls, SD). The average time between sample collection and arrival in the US lab was 8 days. Upon arrival in the US lab, the samples were stored at –80°C for 5 days and then analyzed as previously described [29, 32, 33]. Briefly, a punch from the DBS card was combined with the derivatizing reagent [boron trifluoride in methanol (14%), toluene, and methanol (35:30:35 parts)], shaken and heated at 100°C for 45 minutes. Forty parts of both hexane and distilled water were added after the mixture had cooled. After vortexing briefly, the samples were spun to separate layers and an aliquot of the hexane layer that contained the FA methyl esters was extracted. FA analysis was performed as previously described [34–36]. Unless otherwise stated, whole blood FA proportions are expressed as a percent of total identified FAs. Additional drops of blood from the same puncture site were used to assess hemoglobin concentration using a HemoCue photometer (HemoCue 301, Angelholm, Sweden), and malaria status using an antigen-based malaria rapid diagnostic test kit (Standard diagnostic Inc., Korea). Z-scores were calculated for the growth parameters HAZ, WAZ, and WHZ using WHO Anthro v3.2.2 igrowup package for R [37], to calculate z-scores for children < 5 years of age, and WHO Anthro Plus [38] for children ≥ 5. Means and standard deviations were calculated for descriptive analysis. Stunting percentages were calculated based on the WHO standard population and definitions of moderate and severe stunting, wasting, and underweight [39]. FAs were expressed as percent composition of total blood FAs. Mean and standard deviations were calculated for blood FA composition. Total n-3 FA proportions were calculated as ∑ [ALA+ eicosapentaenoic acid (EPA) + docosapentaenoic n-3 (DPA n-3) + docosahexaenoic acid (DHA)]; total n-6 FA proportions were calculated as ∑ [LA + linoelaidic + eicosadienoic (EDA) + dihomo-gamma-linolenic (DGLA) + AA + docosatetraenoic (DTA) + docosapentaenoic n-6 (DPA n-6)]; total n-9 FA proportions were calculated as ∑ [oleic + elaidic + eicosenoic + Mead + nervonic]; total saturated FA proportions were calculated as ∑ [myristic + palmitic + stearic + arachidic + behenic + lignoceric]; total monounsaturated FA (MUFA) proportions were calculated as ∑ [palmitoleic + oleic + palmitelaidic + nervonic + elaidic + eicosenoic]; total polyunsaturated FA (PUFA) proportions were calculated as ∑ [total n-3 + total n-6]. T/T ratio was calculated from the ratio of Mead acid and AA [37]. Product-to-precursor ratios were calculated to estimate PUFA metabolism [40] as follows: EDA/LA to estimate elongase activity, GLA/LA and AA/DGLA to estimate desaturase activity, and DGLA/LA to estimate combined elongase and desaturase activity. All statistical analyses were conducted using software R (R version 3.4.0). Correlations between participant characteristics, anthropometric measurements, and blood FAs were assessed using spearman correlations and graphically displayed using the R package corrplot [41]. Normal probability plots were assessed to verify the validity of regressions. Regression formulas consisted of either the dependent variable HAZ or WAZ, and models were adjusted for each FA and Hb levels (i.e., HAZ = FA + Hb or WAZ = FA + Hb). Hemoglobin was selected as a covariate since it was significantly associated with HAZ and WAZ (p ≤0.01). Regression models were adjusted for Hb and not adjusted for sex as there were few significantly different fatty acids (FAs) between sexes and regression values were unaffected when evaluated with sex adjustment. P-values were considered significant if p≤0.05. Exploratory factor analysis was carried out using the psych package [42]. Briefly, scree plot was used to determine four factors [43]. Palmitelaidic, linoelaidic, and elaidic acids were omitted from the analysis as they were not highly correlated with any other FAs (r<0.3). Varimax rotation was used for orthogonal transformation of the factor loading matrix. FAs correlated with factors r≥0.5 were considered strongly correlated with the factor, regardless of sign. Factor loading scores were generated for each child and used to calculate regressions for each factor. The regressions were HAZ or WAZ = Hb + Factor.