Selenium concentration and speciation in biofortified flour and bread: Retention of selenium during grain biofortification, processing and production of Se-enriched food.

The retention and speciation of selenium in flour and bread was determined following experimental applications of selenium fertilisers to a high-yielding UK wheat crop. Flour and bread were produced using standard commercial practices. Total selenium was measured using inductively coupled plasma-mass spectrometry (ICP-MS) and the profile of selenium species in the flour and bread were determined using high performance liquid chromatography (HPLC) ICP-MS. The selenium concentration of flour ranged from 30ng/g in white flour and 35ng/g in wholemeal flour from untreated plots up to >1800ng/g in white and >2200ng/g in wholemeal flour processed from grain treated with selenium (as selenate) at the highest application rate of 100g/ha. The relationship between the amount of selenium applied to the crop and the amount of selenium in flour and bread was approximately linear, indicating minimal loss of Se during grain processing and bread production. On average, application of selenium at 10g/ha increased total selenium in white and wholemeal bread by 155 and 185ng/g, respectively, equivalent to 6.4 and 7.1µg selenium per average slice of white and wholemeal bread, respectively. Selenomethionine accounted for 65-87% of total extractable selenium species in Se-enriched flour and bread; selenocysteine, Se-methylselenocysteine selenite and selenate were also detected. Controlled agronomic biofortification of wheat crops for flour and bread production could provide an appropriate strategy to increase the intake of bioavailable selenium.

Prioritizing micronutrients for the purpose of reviewing their requirements: a protocol developed by EURRECA.

BACKGROUND: The EURRECA (EURopean micronutrient RECommendations Aligned) Network of Excellence (http://www.eurreca.org) is working towards the development of aligned recommendations. A protocol was required to assign resources to those micronutrients for which recommendations are most in need of alignment. METHODS: Three important 'a priori' criteria were the basis for ranking micronutrients: (A) the amount of new scientific evidence, particularly from randomized controlled trials; (B) the public health relevance of micronutrients; (C) variations in current micronutrient recommendations. A total of 28 micronutrients were included in the protocol, which was initially undertaken centrally by one person for each of the different population groups defined in EURRECA: infants, children and adolescents, adults, elderly, pregnant and lactating women, and low income and immigrant populations. The results were then reviewed and refined by EURRECA's population group experts. The rankings of the different population groups were combined to give an overall average ranking of micronutrients. RESULTS: The 10 highest ranked micronutrients were vitamin D, iron, folate, vitamin B12, zinc, calcium, vitamin C, selenium, iodine and copper. CONCLUSIONS: Micronutrient recommendations should be regularly updated to reflect new scientific nutrition and public health evidence. The strategy of priority setting described in this paper will be a helpful procedure for policy makers and scientific advisory bodies.

Reticulated platelets interfere with flow cytometric reticulocyte counts.

As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis.

Estimation of iron absorption in humans using compartmental modelling.

OBJECTIVE: To evaluate whether a compartmental model could estimate iron absorption as accurately as the well-validated technique of plasma area under the curve using labelled test meals. DESIGN: The study is a randomised cross-sectional intervention. SETTING: The study was carried out at the Human Nutrition Unit at the Institute of Food Research, Norwich, UK. SUBJECTS: A total of nine female volunteers, aged 33+/-8 y. INTERVENTIONS: Volunteers were given an oral dose (approximately 5 mg) of Fe-57 as iron sulphate in an orange juice test drink and simultaneously infused Fe-58 (approximately 200 microg) as iron citrate over 90 min. Multiple blood samples were taken for the following 6 h. The samples were analysed by mass spectrometry and iron absorption was estimated using a mathematical model based on the appearance of Fe isotopes in plasma and the area under the curve technique. RESULTS: The geometric mean (-1 s.d., +1 s.d.) absorption of the model estimate is 16% (9, 31) and the area under curve estimate is 18% (8, 29). CONCLUSIONS: Results indicate that a compartmental model can be used to estimate labelled iron absorption although it is unlikely that this new method will be used in favour of an existing one. Further studies are now needed with unlabelled iron to assess whether the technique could have application in the assessment of total (haem+nonhaem) iron absorption from food.

Copper absorption from foods labelled intrinsically and extrinsically with Cu-65 stable isotope.

OBJECTIVE: To determine copper absorption from copper containing foods labelled either intrinsically or extrinsically with a highly enriched Cu-65 stable isotope label. DESIGN: A longitudinal cross-over study. SETTING: The study was conducted at the Institute of Food Research, Human Nutrition Unit, Norwich, UK. SUBJECTS: Subjects were recruited locally via advertisements placed around the Norwich Research Park. A total of 10 volunteers (nine female, one male) took part in the study, but not all volunteers completed each of the test meals. INTERVENTIONS: A highly enriched Cu-65 stable isotope label was administered to volunteers in the form of a reference dose or in breakfast test meals consisting of red wine, soya beans, mushrooms or sunflower seeds. Faecal monitoring and mass spectrometry techniques were used to estimate the relative quantities of copper absorbed from the different test meals. RESULTS: True copper absorption from the reference dose (54%) was similar to extrinsically labelled red wine (49%) and intrinsically labelled sunflower seeds (52%), but significantly higher than extrinsically labelled mushrooms (35%), intrinsically (29%) and extrinsically (15%) labelled soya beans and extrinsically labelled sunflower seed (32%) test meals. CONCLUSIONS: The use of Cu-65 extrinsic labels in copper absorption studies requires validation according to the food being examined; intrinsic and extrinsic labelling produced significantly different results for sunflower seeds.

Iron contents of Malawian foods when prepared in iron cooking pots.

The aim of this study was to determine the iron content of Malawian foods prepared in iron pots and to examine the effects of continuous cooking time and added oil on the iron content of the food prepared. Foods prepared, which included a staple (Nsima), relish vegetables, and beans, had an increased iron content when prepared in an iron compared to a glass pot. For these three foods, iron content per gram increased by 3.15 micrograms, 35.8 micrograms, and 147.32 micrograms, respectively. Continuous use of the iron pot for cooking could have a positive effect on the amount of iron added to the food, as the three foods' iron content increased by a further 2.9 micrograms iron/g, 7.6 micrograms iron/g and 20.1 micrograms iron/g, respectively. This effect needs more study. Food pH was significantly negatively correlated with food iron content. The use of oil reduced the amount of iron added to stir-fried vegetables by 52.37 micrograms/g and increased iron added to Nsima (1.2 micrograms/g). Malawian foods increased their iron content when cooked in iron pots. This method of food preparation potentially provides a low-cost sustainable means of improving the iron intakes of families and communities where this traditional method of food preparation is acceptable.

Validity and reproducibility of a computerised tool for assessing the iron, calcium and vitamin C intake of Belgian women.

OBJECTIVE: To determine the relative validity of a newly developed iron intake assessment tool, designed specifically to assess iron, calcium and vitamin C intake. DESIGN: Estimates of iron, calcium and vitamin C intake from a computerised iron intake assessment tool compared with those from 11-day estimated dietary records. SETTING: Region of Ghent (N= +/- 225,000), a city in Flanders, the Dutch-speaking part of Belgium. SUBJECTS: In all, 50 women aged 18-39 y, participating in a large-scale epidemiological study on iron intake and iron status. MAIN RESULTS: Mean dietary iron intake from the 11-day food record, the unadjusted dietary iron intake assessment tool and the adjusted tool was, respectively, 10.5 +/- 2.7, 10.4 +/- 4.3 and 9.6 +/- 2.9 mg. For the different nutrients, the correlation coefficients vary from 0.45 to 0.60 for adjusted intake. The mean difference of iron intake by the two methods (0.8 +/- 2.9 mg) did not differ significantly from zero. The new method correctly classified 38% (iron), 38% (calcium) and 58% (vitamin C) of the subjects to the correct tertile. The correlation coefficients ranged from 0.48 for adjusted vitamin C intake to 0.73 for adjusted calcium intake between two administrations. CONCLUSION: The newly developed instrument can be used to assess mean group intakes of iron, calcium and vitamin C in women consuming a Western diet. However, since the ranking capability of the new tool is rather weak, further refinement of the tool is required to produce a robust method for assessing iron, calcium and vitamin C intakes of individuals. SPONSORSHIP: This research was supported by the Belgian National Fund for Scientific Research/Flemish Division (Fund No G.0152.01).

Bioavailability of selenium from fish, yeast and selenate: a comparative study in humans using stable isotopes.

OBJECTIVE: To measure the bioavailability of selenium from cooked and raw fish in humans by estimating and comparing apparent absorption and retention of selenium in biosynthetically labelled fish with labelled selenate and biosynthetically labelled selenium in brewers yeast. DESIGN: The intervention study was a parallel, randomised, reference substance controlled design carried out at two different centres in Europe. SETTING: The human study was carried out at the Institute of Food Research, Norwich, UK and at TNO Nutrition and Food Research, Zeist, The Netherlands. SUBJECTS: In all, 35 male volunteers aged 18-50 y were recruited; 17 subjects were studied in Norwich (UK) and 18 in Zeist (Netherlands). All of the recruited subjects completed the study. INTERVENTIONS: Biosynthetically labelled trout fish (processed by two different methods), biosynthetically labelled brewers yeast and isotopically labelled selenate were used to estimate selenium apparent absorption and retention by quantitative analysis of stable isotope labels recovered in faeces and urine. Subjects consumed the labelled foods in four meals over two consecutive days and absorption was measured by the luminal disappearance method over 10 days. Urinary clearance of isotopic labels was measured over 7 days to enable retention to be calculated. RESULTS: Apparent absorption of selenium from fish was similar to selenate and there was no difference between the two processing methods used. However, retention of fish selenium was significantly higher than selenate (P<0.001). Apparent absorption and retention of yeast selenium was significantly different (P<0.001) from both fish selenium and selenate. CONCLUSION: Fish selenium is a highly bioavailable source of dietary selenium. Cooking did not affect selenium apparent absorption or retention from fish. Selenium from yeast is less bioavailable.

Use of stable isotopes to assess the bioavailability of trace elements: a review.

The bioavailability (utilization) of trace elements is very variable, depending on the characteristics of the element itself, composition of the diet, gastrointestinal conditions and other physiological factors. To derive dietary recommendations and make use of emerging information on diet-gene interactions relating to nutritional requirements for trace elements, methods for assessing bioavailability are required. The trace elements of particular interest include iron, zinc, selenium and copper because of problems of deficiency, purported links with chronic diseases, and the growing market in fortified foods and supplements. The absorption and subsequent metabolism of trace elements can be monitored using stable isotope tracers, but there are difficulties associated with this approach. Although radioisotopes offer many advantages over stable isotopes, their use is restricted due to safety and ethical issues. The difficulties and limitations of methodologies associated with labelling of trace elements in foods with stable isotopes, design of human studies to generate results that predict bioavailability, and detection and quantification of stable isotopes in biological samples are discussed.

Chronic exposure to high levels of dietary iron fortification increases lipid peroxidation in the mucosa of the rat large intestine.

There is increasing evidence that excess dietary iron may be a risk factor for colorectal cancer. However, the majority of animal studies looking at possible mechanism have used unrealistically high concentrations of iron. The current study was designed to test whether chronic exposure to high levels of iron fortification affects the free radical generating capacity of the lumenal contents, mucosal lipid peroxidation and crypt cell proliferation. Rats were fed diets containing either 29 mg/kg or 102 mg/kg of elemental iron for 6 mo. The free radical generating capacity of lumenal contents was assessed using an in vitro assay. Crypt cell proliferation rate was measured in tissues taken from the cecum and colon, with the remaining tissue being used for the assessment of lipid peroxidation. Chronic feeding of iron did not increase crypt cell proliferation rate in either the colon or cecum, but it was associated with an increase in free radical generating capacity in the colon and increased lipid peroxidation, particularly in the cecum. These results may be relevant to epidemiological evidence showing that dietary iron is associated with the risk of proximal colon cancer in humans.

High bioavailability of calcium in fortified Horlicks.

OBJECTIVE: To compare the bioavailability of calcium carbonate-fortified Horlicks with calcium naturally present in milk. DESIGN: Randomised crossover within-subject comparison using a double label stable isotope technique. SETTING: Institute of Food Research, Human Nutrition Unit. PARTICIPANTS: Sixteen pre-menopausal women aged 23-40 y, habituated to a high-calcium diet (mean 1240 mg/day). RESULTS: Mean true fractional calcium absorption was 38.8% (s.d.+/-14.5) from Horlicks and 21.2% (s.d.+/-4.6) from milk. Significantly more calcium was absorbed from a serving of Horlicks than from the same quantity of calcium present in 420 g semi-skimmed milk (P<0.001). CONCLUSIONS: Fortified Horlicks is a highly bioavailable source of calcium. A single serving, containing at least 500 mg calcium, provides half the reference nutrient intake for the population sub-group with the highest requirement (adolescent boys) and more than half for all others. SPONSORSHIP: SmithKline Beecham funded this research project.

Iron absorption from a breakfast cereal: effects of EDTA compounds and ascorbic acid.

Sodium iron ethylenediaminetetracetic acid (NaFeEDTA) has been recommended for food fortification programmes to improve iron status but its performance in commercial products has not been evaluated. The effect of EDTA on iron absorption from fortified cornflakes, given as part of a typical Western breakfast, was determined in a double-blind randomised study with 20 non-anaemic female volunteers, using experimentally prepared iron compounds, enriched with 58Fe, and faecal monitoring. Five meals were compared: hydrogen reduced iron, hydrogen reduced iron plus Na2EDTA (molar ratio EDTA:Fe 1:2), hydrogen reduced iron plus NaFe(III)EDTA at two different molar ratios (EDTA:total Fe 1:3 and 1:2), and hydrogen reduced iron plus 15 mg ascorbic acid (ascorbic acid:Fe 1.3:1). The iron and EDTA compounds were accurately weighed into gelatine capsules and taken with unfortified cornflakes, semi-skimmed milk and tea on two consecutive days; the iron dose per meal was 3.75 mg. Iron absorption from all five test meals was measured in each volunteer with a minimum wash-out period of 2 weeks between tests. Geometric mean iron absorption (%) from the 5 tests was 14.1, 17.6, 20.6, 24.4 and 17.5 respectively (equivalent to 0.5-0.9 mg absorbed iron). There was a significantly higher iron absorption from the mixture of reduced iron and NaFe(III)EDTA (EDTA:Fe 1:2) than from reduced iron alone (p = 0.014). It is not known whether the higher absorption was from reduced iron or NaFeEDTA or both. Absorption was not increased significantly with NaFe(III)EDTA (EDTA:Fe 1:3), Na2EDTA (EDTA:Fe 1:2) or ascorbic acid (15 mg).

Iron.

The most useful and appropriate methods for assessing the bioavailability of (nonheme) iron supplements are described. When the supplement can be labeled isotopically, the best method for measuring bioavailability is hemoglobin incorporation, followed by fecal monitoring. Caco-2 cell in vitro systems can be used for rapid screening to predict potential availability for absorption. If the compound cannot be labeled, then the plasma appearance/disappearance of oral iron given together with an intravenous dose of iron isotope can be used to quantify absorption. With oral doses in excess of 25 mg, the 4- to 6-h plasma concentration can provide a qualitative assessment of bioavailability. Approaches for normalizing results to minimize intraindividual and interindividual variability in efficiency of iron absorption are discussed.

Selective extraction of blood plasma exchangeable copper for isotope studies of dietary copper absorption.

Measuring mineral absorption by fecal monitoring is labor-intensive and relies on good volunteer compliance. Blood indicators of absorption could be advantageous and we have developed a method for selective extraction-of recently absorbed (exchangeable) copper based on dialysis of plasma with histidine and subsequent copper extraction using Chelex resin. The potential for measuring copper absorption by transient enrichment of exchangeable copper with the stable isotope 65Cu from an ingested tracer, was also investigated. This method was compared with that of the fecal monitoring technique in a human volunteer, who consumed a 6 mg dose of 65Cu with inhibitors of copper absorption. Holmium was used as a non-absorbable rare-earth marker of unabsorbed tracer excretion, allowing estimation of re-secreted 65Cu (44 microg d(-1)), and hence calculation of true tracer absorption, which was only 10.8%. Monitoring plasma tracer kinetics showed potential for estimation of copper absorption without the need for fecal copper analysis.

Copper supplementation in humans does not affect the susceptibility of low density lipoprotein to in vitro induced oxidation (FOODCUE project).

The oxidative modification of low-density lipoprotein cholesterol (LDL) has been implicated in the pathogenesis of atherosclerosis. Copper (Cu) is essential for antioxidant enzymes in vivo and animal studies show that Cu deficiency is accompanied by increased atherogenesis and LDL susceptibility to oxidation. Nevertheless, Cu has been proposed as a pro-oxidant in vivo and is routinely used to induce lipid peroxidation in vitro. Given the dual role of Cu as an in vivo antioxidant and an in vitro pro-oxidant, a multicenter European study (FOODCUE) was instigated to provide data on the biological effects of increased dietary Cu. Four centers, Northern Ireland (coordinator), England, Denmark, and France, using different experimental protocols, examined the effect of Cu supplementation (3 or 6 mg/d) on top of normal Cu dietary intakes or Cu-controlled diets (0.7/1.6/6.0 mg/d), on Cu-mediated and peroxynitrite-initiated LDL oxidation in apparently healthy volunteers. Each center coordinated its own supplementation regimen and all samples were subsequently transported to Northern Ireland where lipid peroxidation analysis was completed. The results from all centers showed that dietary Cu supplementation had no effect on Cu- or peroxynitrite-induced LDL susceptibility to oxidation. These data show that high intakes (up to 6 mg Cu) for extended periods do not promote LDL susceptibility to in vitro-induced oxidation.

Use of the stable isotope (106)Cd for studying dietary cadmium absorption in humans.

Hydroponically grown wheat was intrinsically labelled with the stable isotope 106cadmium (106Cd) and the flour made into a porridge. The abundance of the isotope in the porridge was approximately 30 times the natural abundance, but the total level of Cd in the porridge was 0.03 mg/kg fresh weight, which was the same as expected in a normal diet. Cadmium measurements were made using inductively coupled plasma-mass spectrometry (ICP-MS). The porridge was eaten at breakfast by adult and infant volunteers. Bulked faecal collections were analysed for unabsorbed Cd. Initial results suggest that the apparent absorption of Cd may be higher than 5% as commonly quoted, but longer faecal collection times may be necessary to confirm this.

Lack of effect of dietary chromium supplementation on glucose tolerance, plasma insulin and lipoprotein levels in patients with type 2 diabetes.

Chromium is essential for the regulation of insulin action, thereby influencing carbohydrate and lipid metabolism. An uncontrolled pilot study was designed to measure the habitual daily intake of chromium in a group of healthy individuals with type 2 diabetes and to monitor the effect of daily supplementation with high chromium yeast on glucose tolerance, plasma insulin and lipoproteins. Twelve free-living adults with type 2 diabetes underwent a glucose tolerance test (GTT) on recruitment, at 4 weeks (after a 7-d duplicate diet collection) and at 12 weeks (following 8 weeks daily supplementation with 100 micrograms of chromium). Urine samples were collected on the day before and the day of each GTT. Blood samples were taken at half hourly intervals for 3 hours during the GTT and the plasma glucose, cholesterol, triglyceride, HDL, LDL and insulin concentration measured. The chromium content of diets and urine samples was determined. Fasting glucose concentrations and glucose area under the curve profiles did not alter significantly post supplementation with the chromium rich yeast. No significant changes in insulin and lipoprotein concentrations were observed. The results of this study do not support the hypothesis that individuals with type 2 diabetes benefit from yeast-based chromium supplements (100 micrograms/day).