Determinants of cellular L-arginine transport

One of the potential causes of hypertension is endothelial dysfunction associated with a decreased production of the vasodilator nitric oxide (NO). Possible factors which may contribute to the reduced NO production include increased reactive oxygen species (eg. superoxides); increased concentrations of homocysteine; or decreased concentrations of L-arginine (cationic amino acids). L-arginine, the precursor of NO, not only increases the bioavailability of NO by increasing its production; but also by reducing the inactivation of NO by superoxides. In patients with hypertension, although fasting plasma L-arginine concentrations are elevated, L-arginine supplementation has been shown to decrease blood pressure. A possible explanation for these data may be that L-arginine uptake into cells is impaired and therefore would not be available for NO production. Indeed, studies have shown that cellular uptake of L-arginine is reduced in lymphocytes from patients with hypertension and individuals genetically predisposed to developing hypertension. However, elucidating the kinetics of L-arginine uptake into endothelial cells is fundamental to determine whether L-arginine uptake is indeed impaired. Previous studies have shown that the uptake of cationic amino acids into endothelial cells is mediated by the high affinity/low rate y+L transporter and the low affinity/high rate y+ transporter. However, data on the kinetics, the relative contribution and physiological importance of the individual transporters in cells expressing more than one transporter, are inconsistent; as most studies determining the uptake of radiolabelled amino acids have assumed Michaelis-Menten kinetics and have calculated constants from Lineweaver-Burk reciprocal plots and Eadie-Hofstee plots. Another approach was therefore required to overcome the limitations and assumptions made in these studies. My first aim was therefore to determine the kinetics of L-arginine uptake into endothelial cells using a general non-linear approach, which allows initial rates of uptake by more than one transporter to be determined and importantly includes the actual concentrations of both the trace radiolabelled and unlabelled amino acid in the model. Furthermore, using this approach no assumptions are made regarding the type of inhibition and the concentrations of inhibitors (or activators) could be included in the model. As the model was additive, the theoretical contribution of uptake by each transporter could be modelled. The present study used raw, rather than transformed data, in non-linear regression analysis to characterize the kinetics of L-arginine uptake into cells. I modelled the initial high affinity/low capacity and low affinity/high capacity uptake of trace L-[3H]arginine by two transporters into ECV304 and umbilical cord vein endothelial cells in the presence of a range of unlabelled L-arginine and modifiers using GraphPad Prism. The contribution of uptake by individual transporters was modelled and showed that leucine inhibited the individual transporters differently and that the inhibition was not necessarily competitive. N-ethylmaleimide inhibited only y+ transport and 2-amino-bicyclo-[2,2,1]-heptane-2-carboxylic acid may be a potential inhibitor of y+L transport. Only the absence of sodium reduced L-arginine uptake by y+L transport and reduced the Km’, whereas reducing sodium decreased L-arginine uptake by y+ transport without affecting the Km’. This non-linear modelling approach allows more than one transporter to be modelled, overcomes many of the assumptions made in reported studies and by using raw, rather than transformed data, avoids the errors inherent in methods deriving constants from the linearization of the uptake processes following Michaelian kinetics. The results of this study therefore provide explanations for discrepancies in the literature and suggest that this modelling approach better characterises the kinetics of amino acid uptake into cells. Having elucidated the kinetics of L-arginine uptake into endothelial cells, I was then equipped to explore possible factors which could impair L-arginine uptake in hypertension. In this regard, although increases in total plasma homocysteine were thought to play a role in hypertension; large prospective clinical trials to reduce total plasma homocysteine by vitaminB6/12/folate supplementation, have failed to show beneficial effects on vascular outcomes. The effects of homocysteine on the vasculature were attributed to the reactive free sulphydryl group; however only a fraction (1.5 – 4%) of total plasma homocysteine is actually present as the free reduced sulphydryl (-SH or thiol) form. In comparison, free oxidized homocysteine, present as the disulphide, homocystine and the mixed disulphide (with cysteine) accounts for 20 – 30% of total plasma homocysteine. In the absence of a clear mechanism by which homocysteine causes vascular disease, one of the other species making up the total homocysteine may be contributing to vascular disease through a different mechanism which may not involve the free sulphydryl group. Earlier studies demonstrated (in isolated nephrons) that the homocysteine disulphide, homocystine, shared the same membrane transporter as L-arginine (the precursor of NO), and competed for uptake with L-arginine. These studies may suggest that increased homocystine concentrations, by inhibiting L-arginine transport, and hence reducing intracellular L-arginine concentrations, may impact on NO production in other cell types. Therefore, the second aim of my study was to determine the effects of homocystine on cellular L-arginine uptake and hence on NO production. The uptake of labelled L-[3H]arginine was measured in confluent, L-arginine depleted HUVEC and ECV304 cells with unlabelled L-arginine, without or with homocystine and modifiers. The kinetic constants were determined in Graphpad Prism using a described non-linear model of uptake for two transporters acting simultaneously. The NO specific fluorescent DAF-2 dye was used to detect NO production by the cells. Elevated physiological concentrations of 2.5μM homocystine significantly inhibited L-arginine uptake by 90% by y+L transport in both HUVEC (p<0.0005) and in ECV304 cells (p<0.05). Homocystine reduced the Kma of y+L transport in HUVEC (<0.0001) affecting uptake in a competitive-like manner. Pre-incubation of the ECV304 cells with L-arginine was able to reverse this inhibition by homocystine. In contrast, homocystine increased uptake by y+ transport in HUVEC (p<0.01). Under the experimental conditions used, effects of homocystine on the rate of NO production could not be shown. By demonstrating that homocystine nearly abolishes L-arginine uptake by y+L transport in both HUVEC and ECV304 cells, these data provide a mechanism as to how homocystine may affect L-arginine concentrations. These data would support studies to determine the association between homocystine concentrations and cardiovascular disease. Lastly, although angiotensin-converting enzyme inhibitors (ACEI’s, as well as angiotensin II receptor antagonists) but not other classes of antihypertensive agents, have been shown to decrease oxidative stress and increase NO availability independent of blood pressure lowering effects, the mechanism is not clear. The ability of ACEI’s to decrease oxidative stress and enhance NO production has been attributed in part to the sulfhydryl groups present in some, but not all, ACEI’s. Hence the mechanisms of the effects of ACEI’s on NO production warrant further investigation, as it is possible that L-arginine transporters may play a role by enhancing L-arginine uptake into cells, and thereby increasing NO production

Illustration of the Importance of Adjustment for within- and between-Person Variability in Dietary Intake Surveys for Assessment of Population Risk of Micronutrient Deficiency/Excess Using an Example Data Set

Nutrition intervention decisions should be evidence based. Single 24-h recalls are often used for measuring dietary intake in large dietary studies. However, this method does not consider the day-to-day variation in populations&rsquo; diets. We illustrate the importance of adjustment of single 24-h recall data to remove within-person variation using the National Cancer Institute method to calculate usual intake when estimating risk of deficiency/excess. We used an example data set comprising a single 24-h recall in a total sample of 1326 1&ndash;&lt;10-year-old children, and two additional recalls in a sub-sample of 11%, for these purposes. Results show that risk of deficiency was materially overestimated by the single unadjusted 24-h recall for vitamins B12, A, D, C and E, while risk of excess was overestimated for vitamin A and zinc, when compared to risks derived from usual intake. Food sources rich in particular micronutrients seemed to result in overestimation of deficiency risk when intra-individual variance is not removed. Our example illustrates that the application of the NCI method in dietary surveys would contribute to the formulation of more appropriate conclusions on risk of deficiency/excess in populations to advise public health nutrition initiatives when compared to those derived from a single unadjusted 24-h recall

Food items consumed by students attending schools in different socio-economic areas in Cape Town, South Africa

Objective: We investigated the food consumption patterns of adolescent students at schools. Our findings are intended to reveal the overall nutritional quality of foods eaten by students at school, including foods brought to school and foods purchased at school. Methods: A questionnaire was completed by 476 students, mostly from grades 7 and 10, from 14 schools in Cape Town, South Africa. The schools were representative of the various ethnic groups and socioeconomic strata of the population. The questionnaire requested information on eating habits at school, foods brought to school and food purchases, and breakfast consumption before school. We also tested whether students knew which foods are healthy and which are less healthy choices. Results: The students were mostly 12 to 16 y of age (mean age 14.5 y). The large majority had breakfast before school (77.8%) and ate at school (79.7%). Food was brought to school by 41% to 56%, whereas 69.3% purchased food at school, mainly at the school store (tuck shop). Predefined “unhealthy” foods brought to school outnumbered “healthy” ones by 2 to 1. Among students who purchased food at school, 70.0% purchased no healthy items, whereas 73.2% purchased two or more unhealthy items. With six foods 84% of students correctly stated whether they were healthy or unhealthy; however, with cola drinks, samoosas (deep-fried pastry with spicy filling), and pies, only 47% to 61% knew that these were less healthy choices. Students’ scores on this question were unrelated to whether they purchased healthy or unhealthy foods. Students who attended schools of high socioeconomic status were twice as likely to bring food to school (64.7% versus 31.0%, P < 0.001), scored higher marks on the quiz of healthy versus unhealthy foods (P < 0.01), but were no more likely to purchase healthy food. Conclusions: The large majority of food eaten by adolescent students in Cape Town is classified as being unhealthy choices. This applies to foods brought to school and food purchases. Consideration needs to be given to policy measures to improve this situation and to improve education of students and their parents

Has Food Security and Nutritional Status Improved in Children 1–<10 Years in Two Provinces of South Africa between 1999 (National Food Consumption Survey) and 2018 (Provincial Dietary Intake Study (PDIS))

The 1999 National Food Consumption Survey in South Africa showed that food insecurity (hunger) was prevalent in households with children aged one to &lt;10 years. A repeat of the survey in two provinces: Gauteng (GTG) and the Western Cape (WC) was undertaken in 2018. Results showed that in all domains (living areas) in GTG, food shortage prevalence decreased between 1999 and 2018, from 55.0% to 29.6% in urban informal areas, from 34.1% to 19.4% in urban formal areas and from 42.1% to 15.6% in rural areas. While the prevalence of food shortage in urban formal areas in the WC remained similar in 2018, prevalence decreased from 81.8% to 35.7% in urban informal areas and from 38.3% to 20.6% in rural areas. Energy and macronutrient intakes improved significantly in GTG between 1999 and 2018 but not in the WC; intakes were significantly higher in the WC at both time points. The only significant change in stunting, wasting, overweight and obesity prevalence was that 7&ndash;&lt;10-year-olds in GTG were significantly more likely to be wasted (BAZ &lt; 2SD) in 2018 than in 1999 (20.2% versus 6.9% respectively). In the WC, 1&ndash;3-year-olds were significantly more likely to be obese in 2018 than in 1999 (8.1% versus 1.7% respectively) and 7&ndash;&lt;10-year-olds were less likely to be stunted (14.5% versus 4.9% respectively). There were significant negative correlations between the hunger score and dietary variables in both provinces in 1999. In GTG in 2018, only the correlation with fat intake remained while there were still several significant correlations in WC in 2018. Changes in top 12 energy contributors reflect a shift to high or moderate energy foods low in nutrients from 1999 to 2018. Nutrient dense (high micronutrients, low energy/g) foods (e.g., fruit) fell off the list in 2018. Logistic regression analyses reflect the importance for food security of having a parent as head of the household and/or caregiver, and parents having grade 12 or higher education and being employed. We conclude that food security nutritional status indicators improved amongst 1&ndash;&lt;10-year-old children especially in GTG between 1999 and 2018. However, the shift to poorer food choices and increase in wasting in older children and overweight in younger children are of concern

Development and validation of a short questionnaire to assess sodium intake

OBJECTIVES: To develop and validate a short food-frequency questionnaire to assess habitual dietary salt intake in South Africans and to allow classification of individuals according to intakes above or below the maximum recommended intake of 6 g salt day-1. DESIGN: Cross-sectional validation study in 324 conveniently sampled men and women. METHODS: Repeated 24-hour urinary Na values and 24-hour dietary recalls were obtained on three occasions. Food items consumed by >5% of the sample and which contributed > or =50 mg Na serving-1 were included in the questionnaire in 42 categories. A scoring system was devised, based on Na content of one index food per category and frequency of consumption. RESULTS: Positive correlations were found between Na content of 35 of the 42 food categories in the questionnaire and total Na intake, calculated from 24-hour recall data. Total Na content of the questionnaire was associated with Na estimations from 24-hour recall data (r = 0.750; P or =2400 mg day-1 equated to a reference cut-off score of 48 and corresponded to mean (standard deviation) urinary Na values of 145 (68) and 176 (99) mmol day-1, respectively (P or =100 and <100 mmol day-1 was 12.4% and 93.9%, respectively. CONCLUSION: A 42-item food-frequency questionnaire has been shown to have content-, construct- and criterion-related validity, as well as internal consistency, with regard to categorising individuals according to their habitual salt intake; however, the devised scoring system needs to show improved sensitivity

Provincial dietary intake study (PDIS) : micronutrient intakes of children in a representative/random sample of 1- to <10-year-old children in two economically active and urbanized provinces in South Africa

CITATION: Senekal M, et al. 2020. Provincial Dietary Intake Study (PDIS): Micronutrient Intakes of Children in a Representative/Random Sample of 1- to <10-Year-Old Children in Two Economically Active and Urbanized Provinces in South Africa. International Journal of Environmental Research and Public Health, 17(16). doi:10.3390/ijerph17165924The original publication is available at https://www.mdpi.com/journal/ijerphIn 1999, the National Food Consumption Survey found serious risk of dietary deficiency for a number of micronutrients in 1- to 9-year-old children in South Africa. To address these shortfalls, fortification with vitamin A, thiamine, riboflavin, niacin, vitamin B6, folic acid, iron and zinc of maize meal and bread flour was made mandatory in 2003. The aim of this study was to examine micronutrient intakes of 1- to <10-year-old children after nearly 20 years of fortification in two of the most urbanized and economically active provinces, Gauteng (GTG) and the Western Cape (WC). A multistage stratified cluster random sampling design and methodology was used. Households were visited by fieldworkers who interviewed caregivers and obtained dietary intake data by means of a multiple-pass 24-h recall. Two additional 24-h recalls were completed among a nested sample of 146 participants to adjust the single 24-h recall data of the total sample using the National Cancer Institute Method. Results show that median intake of all the fortification nutrients were above the estimated average requirement (EAR), with the only concern being folate in the WC. Between a quarter and a third of children in the WC, where maize porridge intake was significantly lower than in GTG, had a folate intake below the EAR. Nutrients that are not included in the fortification mix that remain a serious concern are calcium and vitamin D, with intake of dairy and vitamin D sources being very limited in both provinces. The improvement in micronutrient intakes of children is encouraging, however the outstanding nutrient deficiency risks need attention.https://www.mdpi.com/1660-4601/17/16/5924Publishers versio

Provincial Dietary Intake Study (PDIS) : energy and macronutrient intakes of children in a representative/random sample of 1–<10-year-old children in two economically active and urbanized provinces in South Africa

CITATION: Steyn, N. P. et al. 2020. Provincial Dietary Intake Study (PDIS): Energy and Macronutrient Intakes of Children in a Representative/Random Sample of 1–<10-Year-Old Children in Two Economically Active and Urbanized Provinces in South Africa. International Journal of Environmental Research and Public Health, 17(5). doi:10.3390/ijerph17051717The original publication is available at https://www.mdpi.com/journal/ijerphThe double burden of malnutrition is still prevalent in South Africa, hence the importance of a dietary survey to identify risks of under- and over-nutrition. A multistage stratified cluster random sampling design was applied in two economically active provinces, Gauteng (GTG) (N = 733) and Western Cape (WC) (N = 593). Field workers completed questionnaires, and a 24 h recall with children taking part aged 1–<10-years (N = 1326). Important findings were that 71% and 74%, respectively, of 3–<6-year-olds and 6–<10-year-olds had an energy intake below the estimated energy requirement (EER), while 66% 1–<3-year-olds had intakes above the EER. The percentage of children with a total fat intake below recommended levels decreased as age increased ((51%, 40% and 5%) respectively, for the three age groups). Similarly, the percentage of those who had a total fat intake above the recommendation increased with increasing age (4%, 11% and 26%, respectively, for the three age groups). Saturated fat intake above 10%E was highest in the youngest and oldest children (33% and 32%, respectively). The percentage of children with a free sugars intake above 10%E was 47%, 48% and 52% respectively, and 98–99% had a fibre intake that was less than recommended. Overall, the diet was not healthy, with the main food items being very refined, and the diet being high in salty snacks and sugary items, and low in fruit, vegetables and legumes.https://www.mdpi.com/1660-4601/17/5/1717/htmPublishers versio

Mean ± Standard Deviation Intake Values for 1–<10-Year-Old South African Children for Application in the Assessment of the Inflammatory Potential of Their Diets Using the DII® Method: Developmental Research

This study aimed to develop a set of mean &plusmn; standard deviation (SD) intake values for South African (SA) children for 36 of the 45 food parameters included in the original Dietary Inflammatory Index (DII&reg;) tool. The SA food composition database contains 30 of the 45 food parameters included in the original DII&reg;, and a supplementary database was developed for six of the food parameters not included in the SA database. The SA child mean &plusmn; SD intake of macronutrients, micronutrients and select flavonoids was calculated by age in years, using eight data sets from dietary surveys conducted in SA in the last three decades. A total sample of 5412 children was included in the calculation of the mean &plusmn; SD. The current study sample was determined to be representative of 1&ndash;&lt;10-year-old children in SA, and the plausibility of the mean intake values was confirmed by being in line with age-appropriate recommendations. Furthermore, an increase in energy, macronutrient, and most micronutrient intakes with increase in age was evident. The generated mean &plusmn; SD values for SA children can be used for calculation of the inflammatory potential of the dietary intake of SA children in the age range of 1&ndash;&lt;10-year-old children