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.

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.

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.

Effect of calcium supplementation on daily nonheme-iron absorption and long-term iron status.

The short-term effect of calcium supplements (1200 mg Ca/d) on daily nonheme-iron absorption was measured in 14 healthy adult volunteers by using stable isotope extrinsic labeling and fecal monitoring techniques. Mean (+/- SEM) nonheme-iron absorption from a low-calcium (< 320 mg/d), moderately high-iron (15 mg/d) diet was 15.8 +/- 2.1%, but in the presence of calcium (400 mg/meal) as calcium carbonate, absorption fell significantly to 4.7 +/- 1.4% (P < 0.001). The long-term effect of consuming calcium supplements with meals (1200 mg Ca/d) on body iron (functional and storage iron) was investigated in 11 iron-replete adults over a 6-mo period. An unsupplemented control group (n = 13) was also monitored to correct for any seasonal changes in the biochemical measurements. There were no changes in any of the hematologic indexes, including hemoglobin, hematocrit, zinc protoporphyrin, and plasma ferritin resulting from the calcium supplementation. The results clearly show that long-term supplementation with calcium did not reduce plasma ferritin concentrations in iron-replete adults consuming a Western-style diet containing moderate to high amounts of calcium in most meals.

Nonradioisotopic method for measuring iron absorption from a Gambian meal.

Iron absorption from a typical Gambian meal of rice with groundnut sauce was measured by the fecal balance technique in nonanemic adult males with 58Fe as an extrinsic label and neutron activation analysis and compared with ferrous sulphate. Gambian men had a higher efficiency of absorption than UK volunteers but the availability of the food Fe was approximately half that of ferrous sulphate in both groups of subjects.

Bioavailability of iron glycine as a fortificant in infant foods.

The bioavailability of iron glycine added to a vegetable infant weaning food was compared with ferrous sulfate. Stable, isotopically labeled compounds (57Fe or 58Fe) were mixed into the midday meal (1.4 mg added Fe/serving) and fed to 9-mo-old infants on alternate days for 8 d. Bioavailability, expressed as a percentage of the dose consumed, was measured from isotopic enrichment of hemoglobin 14 d after the last test meal. There was no difference between iron glycine and ferrous sulfate (x+/-SEM): 9.0+/-0.7% and 9.9+/-0.8%, respectively. The effect of chelation was examined by measuring iron bioavailability of iron glycine and ferrous sulfate added to a high-phytate (310 mg/100 g) whole-grain cereal weaning food and comparing it with a lower-phytate (147 mg/100 g) vegetable food, as used in the first study. Both iron compounds had lower bioavailability from the high-phytate food, 5.2+/-0.5% for iron glycine and 3.8+/-0.9% for ferrous sulfate, than the lower-phytate food, 9.8+/-1.5% for iron glycine and 9.1+/-1.3% for ferrous sulfate. The results showed no significant difference in bioavailability between the two forms of iron when added to infant weaning foods, suggesting that the glycine complex was fully or partially dissociated in the gastrointestinal tract. It is concluded that chelation does not improve the bioavailability of iron in the presence of dietary inhibitors.

Zinc absorption in infants fed iron-fortified weaning food.

The effect of fortification iron (reduced iron) on zinc absorption from a commercial vegetable-based weaning food was assessed in 11 9-mo-old infants. Each infant was fed a test meal of unfortified or iron-fortified product, labeled extrinsically with 1 mg 67Zn or 70Zn (as citrate), and the next day was fed the second product labeled with the other isotope. A complete fecal collection was carried out for 3-4 d, and the amount of unabsorbed isotope measured by thermal-ionization quadrupole mass spectrometry. Apparent zinc absorption (isotope intake minus fecal excretion, expressed as the % of dose administered) was 31.1 +/- 8.3% (x +/- SD) from the iron-fortified food and 28.6%28.6 +/- 10.5% from the unfortified food. These values were not significantly different, thus iron fortification of the weaning food did not reduce zinc absorption.

Iron and zinc absorption in human subjects from a mixed meal of extruded and nonextruded wheat bran and flour.

The effect of extrusion cooking of a bran-flour mixture on iron and zinc retention was measured in normal adults. The stable isotopes 58Fe (1.253 mg) and 67Zn (5.13 mg) were administered with 40 g nonextruded or extruded cereal with milk and isotopic retention was measured from fecal excretion over the next 4-7 d by neutron-activation analysis (Fe) and fast-atom-bombardment mass spectrometry (Zn). 58Fe retention was 15.1 +/- 2.4% (means +/- SEM) with the nonextruded meal and 16.5 +/- 2.7% with the extruded meal. 67Zn retention was 18.9 +/- 1.7% with the nonextruded meal and 18.3 +/- 1.5% with the extruded meal. Extrusion cooking had no effect on 58Fe or 67Zn retention.

Rare earth elements as nonabsorbable fecal markers in studies of iron absorption.

The use of rare earth elements as nonabsorbable fecal markers for studies of iron absorption from sources labeled extrinsically with stable isotopes was evaluated. On 3 successive days 13 healthy fasting adults were given different stable isotopes of iron with samarium, ytterbium, or dysprosium. On day 1, three meals were given with 57Fe (1 mg per meal) plus samarium (0.33 mg per meal); on day 2, identical meals (taken with a calcium supplement to reduce iron bioavailability) were given with equivalent amounts of 58Fe-labeled iron and ytterbium; on day 3, a well-absorbed reference dose of 54Fe (3 mg) was given with 1 mg Dy. A complete fecal collection was carried out for 5-9 d and each stool was analyzed for rare earth elements by inductively coupled plasma-mass spectrometry and iron isotopes by thermal ionization quadrupole mass spectrometry. Mean recovery of rare earth elements was 101%, indicating that they are totally unabsorbed. The excretory pattern of the iron isotopes and the rare earth elements was very similar; the correlation coefficients between samarium and 57Fe, ytterbium and 58Fe, and dysprosium and 54Fe were 0.992, 0.989, and 0.988, respectively (P < 0.001). Iron absorption was calculated as the difference between isotope dose and fecal excretion. Mean (+/-SEM) iron absorption was 16.7 +/- 2.4%, 4.3 +/- 1.6%, and 40.3 +/- 3.1% on days 1-3, respectively. Predicted values estimated from the first 4 d of pooled feces, using the rare earth element recovery data to produce corrected figures for unabsorbed isotope, were in close agreement: 19.1 +/- 2.1%, 4.6 +/- 1.7%, and 40.8 +/- 3.1%, respectively (P < 0.001). With the diet of medium iron bioavailability and with the highly bioavailable reference dose it was possible to predict iron absorption accurately from only one or two stools, provided that they were sufficiently enriched with isotope and a rare earth element.

Oral ferrous sulfate supplements increase the free radical-generating capacity of feces from healthy volunteers.

BACKGROUND: Most dietary iron remains unabsorbed and hence may be available to participate in Fenton-driven free radical generation in conjunction with the colonic microflora, leading to the production of carcinogens or direct damage to colonocytes. OBJECTIVE: Our aims were to measure the proportion of fecal iron available to participate in free radical generation and to determine the effect of an oral supplement of ferrous sulfate on free radical generation. DESIGN: Eighteen healthy volunteers recorded their food intake and collected fecal samples before, during, and after 2 wk of supplementation (19 mg elemental Fe/d). Total, free, and weakly chelated fecal iron were measured and free radical production was determined by using an in vitro assay with dimethyl sulfoxide as a free radical trap. RESULTS: Fecal iron increased significantly during the period of supplementation and returned to baseline within 2 wk. The concentration of weakly bound iron in feces (approximately 1.3% of total fecal iron) increased from 60 micromol/L before to 300 micromol/L during supplementation, and the production of free radicals increased significantly (approximately 40%). Higher-carbohydrate diets were associated with reduced free radical generation. CONCLUSION: Unabsorbed dietary iron may increase free radical production in the colon to a level that could cause mucosal cell damage or increased production of carcinogens.

Iron deficiency in infancy; easy to prevent--or is it?

Despite major advances in our knowledge of iron nutrition in infancy, iron deficiency remains a significant problem in the UK. The precise size of the problem is unknown, but there appears to be a higher risk amongst certain ethnic groups. Nutritional iron deficiency appears after the age of 6 months and is undoubtedly linked to inappropriate feeding practice. High bioavailability of iron in weaning foods is of paramount importance in preventing iron deficiency, whether using family foods or commercially available products, and further information about different foods and diets is needed. The long-term detrimental effect on mental development makes prevention of iron deficiency a high priority public health objective for the 1990s.

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.

Factors affecting iron stores in infants 4-18 months of age.

OBJECTIVES: To determine the effects of dietary, physiological or environmental factors on body iron levels in infants aged 4-18 months. DESIGN: The daily iron intake of the infants was measured from a diet history obtained by interview using a standardised question sheet, previously validated against weighed intake (minimum 3 days) in an independent sample of 8 and 18 month old infants. Capillary blood samples were analyzed for haemoglobin, mean cell volume, haematocrit, zinc protoporphyrin and plasma ferritin concentration. Ferritin values were log-transformed prior to analysis to give a better approximation to the normal distribution and forward stepwise multiple linear regression was carried out using SPSS. SETTING: The city of Norwich, UK and some of its suburbs. SUBJECTS: One hundred and eighty-one healthy infants in age groups 4, 8, 12 and 18 months. RESULTS: Main determinants of iron stores in the 4 month old infants were birth weight (+ve (P < 0.001)) and body weight (-ve (P < 0.005)). In the 8 month old infants intake of cow's milk (-ve (P < 0.05)), belonging to a smoking household (-ve (P < 0.05)) and quantity of commercial babyfood consumed (+ve (P < 0.05)) were significant. In this age group there was a gender effect (girls > boys (P < 0.01)) and the gender effect remained at 12 months (girls > boys (P < 0.05)), but at 18 months only non-haem iron intake was a significant factor (-ve (P < 0.05)). CONCLUSIONS: At 4 months of age birth weight and body weight exert the greatest influence on iron stores, whereas by 8 months components of the weaning diet have an effect (commercial babyfood (+ve), cow's milk (-ve)); there is also a gender effect (girls > boys), possibly reflecting the different growth rate between boys and girls. At 12 and 18 months the only significant factors are gender (girls > boys) and non-haem iron intake (-ve) respectively.

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 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.

Zinc metabolism in non-insulin dependent diabetes mellitus.

Low plasma zinc concentrations and increased zinc excretion have been reported in patients with non-insulin dependent diabetes mellitus (NIDDM). This study was designed to compare zinc metabolism in ten NIDDM patients with ten healthy controls, using stable isotopes of zinc. Zinc absorption from a standard breakfast labelled extrinsically with 3 mg 67Zn was measured using a fecal monitoring technique and thermal ionization quadrupole mass spectrometry. One hour after the test meal each volunteer was given 0.5 mg 70Zn (as citrate) intravenously and blood samples taken at intervals for up to 6 days. Isotopic enrichment was measured and the data entered into a simple kinetic model to estimate the size and rate of turnover of exchangeable pools of zinc. Urinary zinc excretion was higher in the male diabetics than controls (p < 0.05), but not in females. Since the efficiency of absorption and endogenous losses of zinc were very variable between individuals, and there was only a limited amount of data, it was not possible to draw conclusion about the effect of NIDDM on zinc absorption and excretion. However, the fact that there were no differences in exchangeable zinc pool sizes suggests that zinc metabolism is not altered in NIDDM.

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 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.