Nicola Langford

Impact of menstrual blood loss and diet on iron deficiency among women in the UK

Women of childbearing age are at risk of Fe deficiency if insufficient dietary Fe is available to replace menstrual and other Fe losses. Haem Fe represents 10-15 % of dietary Fe intake in meat-rich diets but may contribute 40 % of the total absorbed Fe. The aim of the present study was to determine the relative effects of type of diet and menstrual Fe loss on Fe status in women. Ninety healthy premenopausal women were recruited according to their habitual diet: red meat, poultry/fish or lacto-ovo-vegetarian. Intake of Fe was determined by analysing 7 d duplicate diets, and menstrual Fe loss was measured using the alkaline haematin method. A substantial proportion of women (60 % red meat, 40 % lacto-ovo-vegetarian, 20 % poultry/fish) had low Fe stores (serum ferritin <10 microg/l), but the median serum ferritin concentration was significantly lower in the red meat group (6.8 microg/l (interquartile range 3.3, 16.25)) than in the poultry/fish group (17.5 microg/l (interquartile range 11.3, 22.4) (P<0.01). The mean and standard deviation of dietary Fe intake were significantly different between the groups (P=0.025); the red meat group had a significantly lower intake (10.9 (sd 4.3) mg/d) than the lacto-ovo-vegetarians (14.5 (sd 5.5) mg/d), whereas that of the poultry/fish group (12.8 (sd 5.1) mg/d) was not significantly different from the other groups. There was no relationship between total Fe intake and Fe status, but menstrual Fe loss (P=0.001) and dietary group (P=0.040) were significant predictors of Fe status: poultry/fish diets were associated with higher Fe stores than lacto-ovo-vegetarian diets. Identifying individuals with high menstrual losses should be a key component of strategies to prevent Fe deficiency.

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.Conclusions: 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.

Adaptive responses in men fed low- and high-copper diets

The study of Cu metabolism is hampered by a lack of sensitive and specific biomarkers of status and suitable isotopic labels, but limited information suggests that Cu homeostasis is maintained through changes in absorption and endogenous loss. The aim of the present study was to employ stable-isotope techniques to measure Cu absorption and endogenous losses in adult men adapted to low, moderate and high Cu-supplemented diets. Twelve healthy men, aged 20-59 years, were given diets containing 0.7, 1.6 and 6.0 mg Cu/d for 8 weeks, with at least 4 weeks intervening washout periods. After 6 weeks adaptation, apparent and true absorption of Cu were determined by measuring luminal loss and endogenous excretion of Cu following oral administration of 3 mg highly enriched (65)Cu stable-isotope label. Apparent and true absorption (41 and 48 % respectively) on the low-Cu diet were not significantly different from the high-Cu diet (45 and 48 % respectively). Endogenous losses were significantly reduced on the low- (0.45 mg/d; P<0.001) and medium- (0.81 mg/d; P=0.001) compared with the high-Cu diet (2.46 mg/d). No biochemical changes resulting from the dietary intervention were observed. Cu homeostasis was maintained over a wide range of intake and more rapidly at the lower intake, mainly through changes in endogenous excretion.

Use of the stable isotope 106Cd 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.

Absorption of selenium from wheat, garlic, and cod intrinsically labeled with Se-77 and Se-82 stable isotopes.

There is limited information on the absorption of selenium from different foods in humans because of technical difficulties associated with isotopic labeling of dietary selenium. Wheat, garlic, and cod fish were intrinsically labeled with Se-77 or Se-82 stable isotopes. Labeled meals were fed in random order to 14 adults, with a minimum washout period of six weeks between each test meal. Apparent absorption was measured as luminal loss using a fecal monitoring technique over an 8-day period. Plasma appearance of the isotope was measured at 7, 24, and 48 hours post-ingestion. Selenium absorption (+/- SD) was significantly higher (p < 0.001) from wheat (81.0 +/- 3.0%) and garlic (78.4 +/- 13.7%) than from fish (56.1 +/- 4.3%). Lowest plasma concentration was observed after the fish meal at all three time points, with a peak at 24 hours, whereas wheat produced the highest plasma concentration at all three time points and peaked at 7 hours. Selenium absorption from wheat and garlic was higher than from fish, and inter-individual variation was low. Form of selenium and food constituents appear to be key determinants of post-absorptive metabolism.

Changes in the chemical form of selenium observed during the manufacture of a selenium-enriched sourdough bread for use in a human nutrition study

High-performance liquid chromatography interfaced with inductively coupled plasma mass spectrometry, and hydride generation-inductively coupled plasma mass spectrometry were used, respectively, to investigate changes in both the chemical form and the concentration of selenium during its bio-incorporation and bio-accumulation into rye seedlings. A 60-fold increase in the total level of selenium in the seedlings (‘control’ biomass = 0.99 mg kg−1, ‘enriched’ biomass = 55.27 mg kg−1) was accompanied by a change from selenite to several organo-selenium forms, with more than 40% being present as selenomethionine. The seedling biomass was dried, ground and used as an ingredient in the production of a fermented sourdough bread (popular in Poland and many Eastern European countries). The selenium in the resulting bread was also characterized in terms of its speciation, as well as its total selenium content (‘control’ bread = 0.06 mg kg−1, ‘enriched’ bread = 3.56 mg kg−1). The breads were then fed to 24 volunteers as part of a human intervention study designed to establish the efficacy of this mode of selenium supplementation. The human study data subsequently showed the bread was a good source of dietary selenium.

Development of a high-resolution ICP-MS method, suitable for the measurement of iron and iron isotope ratios in acid digests of faecal samples from a human nutrition study

High-resolution ICP-MS has been used to determine iron concentrations and isotope ratios in acid digests of faecal material from a human nutritional study (designed to investigate the absorption of iron from different types of diet). A resolution setting of 4000 was employed to separate the analyte isotopes from argon-based spectral interferences. Accurate correction of chromium and nickel isobaric overlaps required a correction for mass bias between the monitor isotopes 53Cr+ and 60Ni+, and the interfering isotopes 54Cr+and 58Ni+. Control faecal samples collected from each volunteer, and known to contain natural isotopic abundance iron, were used to calculate the mass bias correction factor, which was then applied to the remaining samples. The method was validated through the measurement of 190 separate acid digests of the certified reference material NIST 1577b (Bovine Liver), measured over a seven month period. The mean iron concentration of all 190 digests was 182 ± 16 µg g−1 compared to the certified value of 184 ± 15 µg g−1. Good agreement with the published natural 57Fe/58Fe isotope ratio was also observed, with a typical ‘in-batch’ precision of 1% RSD for the three replicates of NIST 1577b that were routinely analysed as part of each sample digestion batch. No significant difference was found between isotope ratios measured by ICP-MS (acid digested faecal samples) and by TIMS (fully cleaned up solutions originating from the same set of faecal samples). The preparation and analysis of samples by the ICP-MS method allowed a single analyst to digest, dilute and analyse 120 samples in duplicate in a period of 10 working days.

Comparison of three different instrumental approaches to the determination of iron and zinc isotope ratios in clinical samples

Stable isotopes can be used to investigate the absorption of nutritionally important elements in humans. Although thermal ionisation mass spectrometry (TIMS) is the benchmark technique for accuracy and precision in isotope ratio measurement, the lengthy sample preparation procedures and analysis times required can present significant disadvantages. Inductively coupled plasma mass spectrometry (ICP-MS) is now a well-established technique for isotopic determinations and can be used following only a simple sample preparation (microwave-assisted digestion followed by a dilution step). The advantage of this is that it can provide comparable data to that from TIMS, but in a significantly shorter analysis time. This study assesses the accuracy and precision of two ICP-MS instruments for the determination of Fe and Zn isotope ratios in faecal samples by comparison with TIMS data. Using standard statistical techniques, no significant differences were found between data obtained from TIMS, double-focussing ICP-MS or a single-focussing ICP-MS equipped with a collision cell.

An ICP-MS methodology using a combined high-resolution/multi-collector detector system for the measurement of total zinc and zinc isotope ratios in faecal samples from a human nutrition study

Inductively coupled plasma (ICP), using a novel combination of high-resolution and multi-collector mass spectrometric detectors, was used for the determination of zinc totals and zinc isotope ratios in nitric acid digests of freeze dried faecal material. The samples were collected as part of a human study investigating the absorption of iron, copper and zinc from three different diet types. A solution of 70Zn was administered to each volunteer and its absorption monitored by measuring the changes to the natural isotopic ratio as the non-absorbed dose appeared in the faeces. 70Zn was measured, at a resolution of 2350, on an electron multiplier in the axial channel of the multi-collector array, whilst 67Zn, 68Zn, 69Ga and 71Ga were simultaneously measured at low resolution (400), using Faraday collectors. Internal and external standardisation procedures were compared for the correction of instrumental drift, but the effectiveness of internal standardisation was limited due to the use of the two different types of detector (electron multiplier and Faraday). Mass bias was corrected for, using the isotope ratio measured in the first faecal sample from each volunteer on the study (t = 0), known to contain only zinc of natural isotopic abundance. The method was validated through the measurement of 190 separate digests of the reference material NIST 1577b, analysed over a seven month period. The mean of all 190 replicates was 125 ± 14 µg g−1, compared to the certified value of 127 ± 16 µg g−1. Zinc isotope ratios measured in the reference material digests showed excellent agreement with published natural isotopic abundance data. Within-sample precision was approximately 0.1 % RSD for 67Zn/70Zn, 68Zn/70Zn and 67Zn/68Zn, and was well inside the requirements of this human study. Good agreement was also found between the isotope ratios measured by TIMS analysis of fully cleaned up samples and by ICP-MS analysis of acid digested solutions of the same set of faecal samples. The preparation and analysis of samples by the ICP-MS method developed allowed a single analyst to digest, dilute and analyse 120 samples in duplicate in a period of 10 working days.