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.

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.

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.

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.

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).

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.

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.

Increases in the concentrations of available iron in response to dietary iron supplementation are associated with changes in crypt cell proliferation in rat large intestine.

High concentrations of iron in the diet have been shown to increase chemically induced colorectal tumors in rats. It is therefore important to understand the influence of dietary iron on the concentration of unabsorbed iron in the large intestine and its distribution between soluble and insoluble pools in the luminal compartment. We sought to investigate this issue and to establish whether iron modifies mucosal cell proliferation, which is thought to influence initiation and progression through the adenoma carcinoma sequence. In the first experiment, four groups of seven rats were fed diets at two concentrations of iron, 29 and 102 mg/kg, with or without the addition of 2.5 g phytic acid/kg. The concentrations of iron in the contents of the large bowel extractable with water ("free iron") or a buffered EDTA solution ("exchangeable iron") were determined. The concentration of freely soluble iron increased approximately 100% with iron supplementation in both the cecum and the colon, and there was an approximately five- to sixfold increase in exchangeable iron at both sites (P < 0. 05). In a second experiment with identical feeding conditions, there was a significantly greater number of cell divisions per crypt in the colon of the high iron group and a significantly greater number of cell divisions in the upper part of the crypt in the cecum. The concentrations of free and exchangeable iron observed in colonic contents in this study are consistent with those reported by others to increase free radical production in fecal material. Further studies are required to determine whether the small changes in crypt cytokinetics are a consequence of oxidative mucosal damage.

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.

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.

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.

The validity of extrinsic stable isotopic labeling for mineral absorption studies in rats.

The use of extrinsic stable and radioisotopic labels (Fe, Zn, Cu and Se) was compared with the use of intrinsic labels by measuring label retention in rats. Saccharomyces cerevisiae (Hansen strain CBS 1171) was prepared intrinsically enriched with a stable isotope of iron, zinc, copper or selenium, and unenriched freeze-dried yeast was extrinsically labeled with the appropriate stable and/or radioisotope. Male Wistar rats, weighing 80-100 g and fed a purified diet, were given a test meal of one of the above labeled yeasts. Isotopic retention was determined by fecal monitoring. Retention of the stable isotopes was determined by thermal ionization quadruple mass spectrometry (TIQMS) and retention of the radioisotopes by counting feces in a whole-body counter. The results indicated that the behavior of the labels differed among the minerals, with copper as the only one in which the intrinsic and extrinsic stable isotopes were comparably retained. With zinc, retention of the extrinsic radiolabel and intrinsic label was similar, but retention of the extrinsic stable isotope label was higher. With iron, the intrinsic label had a significantly lower retention than the two extrinsic labels; with selenium, retention of all three labels was different, but these differences were not of a sufficient magnitude to conclude that extrinsic stable isotopic labelling is not valid. These results demonstrate that an extrinsic stable isotope label can be used for copper, selenium and inorganic iron, but that such a label is not valid for studies on zinc.

Effects of double-blind controlled calcium supplementation on calcium absorption in Chinese children measured with stable isotopes (42Ca and 44Ca).

A double-blind controlled Ca supplementation trial was conducted for 6 months in thirty-four 7-year-old Chinese children from Hongkong and Jiangmen, China. The children were randomly allocated to the study group (n 17) or control group (n 17), and a CaCO3 tablet (300 mg Ca) or a placebo tablet was taken daily. True fractional Ca absorption (TFCA) was evaluated before and after the trial using stable isotopes: 8 mg 44Ca mixed in 100 g chocolate milk was given after an intravenous injection of 0.75 mg 42Ca. There was no significant difference in baseline TFCA between the study group (60.6 (SD 11.4)%) and the controls (58.2 (SD 9.0)%; P = 0.55). Serum 25-hydroxycholecalciferol levels were comparable between the two groups (P = 0.71). After 6 months, TFCA of the study group (55.6 (SD 12.7)%) was significantly lower than that of the controls (64.3 (SD 10.7)%; P = 0.015). By comparing the individual changes in TFCA after the trial between the two groups there was a non-significant reduction in TFCA (5.03 (SD 12.4)%; P = 0.11, Wilcoxon signed-rank test) in the study group (60.6-55.6%), whereas a significant increase in TFCA (6.17 (SD 7.7)%; P = 0.004, Wilcoxon signed-rank test) was observed in the controls (58.2-64.3%). The differential in TFCA between the two groups after 6 months was significantly different (P = 0.001), and remained significant after adjustment for baseline dietary intakes, weight and height by multiple-regression analysis (P = 0.003). If the mechanism of TFCA from chocolate milk in response to the treatment effects is similar to that from the total diet, then our results suggest that children with adequate vitamin D status can adapt to a change in Ca intake by adjusting the efficiency of TFCA. In corollary, children on habitually-low Ca diets have a higher TFCA than the counterparts with higher Ca diets.

Riboflavin deficiency and iron absorption in adult Gambian men.

Iron absorption from 3.38 mg 58Fe was measured in riboflavin-deficient Gambian men with haemoglobin (Hb) less than 11.5 g/dl before and after oral riboflavin therapy, and the results compared with a group not receiving riboflavin. Riboflavin status (as determined by erythrocyte glutathione reductase activation coefficient) and Hb increased in teh riboflavin-supplemented but not placebo group. Plasma ferritin levels were low and did not change in either group. There was very wide variation in percentage iron absorption between individuals and also within single individuals on two separate occasions but no measurable change with riboflavin supplementation. The results of the study indicate that the efficiency of iron utilization is impaired in riboflavin deficiency, but that iron absorption is unaffected.

Apparent zinc absorption by rats from foods labelled intrinsically and extrinsically with 67Zn.

A variety of foods (peas (Pisum sativum), chicken meat, eggs, goat's milk, human milk) enriched with the stable isotope 67Zn were prepared by means of intrinsic- and extrinsic-labelling procedures. They were fed to rats and apparent absorption of 67Zn determined from faecal excretion measurements using thermal ionization mass spectrometry. There were significant differences in the absorption of the extrinsic and intrinsic label which differed in magnitude between the foods tested. The extrinsic 67Zn was less well absorbed in peas, chicken meat, eggs, and human milk than intrinsic 67Zn, but in goat's milk the extrinsic 67Zn was better absorbed than the intrinsic label. These results demonstrate that extrinsically-added stable Zn isotopes do not fully exchange with endogenous Zn in many foods, and illustrate the need for caution when using extrinsic labels for Zn bioavailability studies.

Intrinsic labelling of different foods with stable isotope of zinc (67Zn) for use in bioavailability studies.

Intrinsically-labelled foods are required to validate extrinsic-labelling techniques used to study the bioavailability of trace elements. Wheat (Triticum aestivum), peas (Pisum sativum), goat's milk, human milk, eggs and chicken meat were selected for intrinsic-labelling studies with 67Zn. Peas were grown hydroponically in enriched nutrient solution and wheat was grown in sand and watered with enriched nutrient solution. Some of the wheat plants were also given stem injections of 67Zn solution. Eggs and chicken meat were prepared by administering 67Zn intravenously to chickens, and human milk was collected after an oral dose of 67Zn in a cola drink. All the foods investigated were sufficiently enriched with 67Zn for Zn absorption studies except wheat prepared by the sand and water-culture method.