Accurate quantification of selenoproteins in human plasma/serum by isotope dilution ICP-MS : focus on selenoprotein P

Acknowledgements The research leading to these results was funded by the EMRP Joint Research Project “Metrology for metalloproteins” (HLT-05 2012). The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.Peer reviewedPostprin

The Effect of Processing and Seasonallity on the Iodine and Selenium Concentration of Cow's Milk Produced in Northern Ireland (NI): Implications for Population Dietary Intake

Cow’s milk is the most important dietary source of iodine in the UK and Ireland, and also contributes to dietary selenium intakes. The aim of this study was to investigate the effect of season, milk fat class (whole; semi-skimmed; skimmed) and pasteurisation on iodine and selenium concentrations in Northern Ireland (NI) milk, and to estimate the contribution of this milk to consumer iodine and selenium intakes. Milk samples (unpasteurised, whole, semi-skimmed and skimmed) were collected weekly from two large NI creameries between May 2013 and April 2014 and were analysed by inductively coupled plasma-mass spectrometry (ICP-MS). Using milk consumption data from the National Diet and Nutrition Survey (NDNS) Rolling Programme, the contribution of milk (at iodine and selenium concentrations measured in the present study) to UK dietary intakes was estimated. The mean ± standard deviation (SD) iodine concentration of milk was 475.9 ± 63.5 µg/kg and the mean selenium concentration of milk was 17.8 ± 2.7 µg/kg. Season had an important determining effect on the iodine, but not the selenium, content of cow’s milk, where iodine concentrations were highest in milk produced in spring compared to autumn months (534.3 ± 53.7 vs. 433.6 ± 57.8 µg/kg, respectively; p = 0.001). The measured iodine and selenium concentrations of NI milk were higher than those listed in current UK Food Composition Databases (Food Standards Agency (FSA) (2002); FSA (2015)). The dietary modelling analysis confirmed that milk makes an important contribution to iodine and selenium intakes. This contribution may be higher than previously estimated if iodine and selenium (+25.0 and +1.1 µg/day respectively) concentrations measured in the present study were replicable across the UK at the current level of milk consumption. Iodine intakes were theoretically shown to vary by season concurrent with the seasonal variation in NI milk iodine concentrations. Routine monitoring of milk iodine concentrations is required and efforts should be made to understand reasons for fluctuations in milk iodine concentrations, in order to realise the nutritional impact to consumers

Production, characterization and use of isotopically enriched metalloproteins for the analysis of biological samples by species-specific isotope dilution ICP-MS

In this work, the chemical preparation and characterization of an isotopically enriched superoxide dismutase (SOD) is described. Its evaluation as a standard in species-specific isotope dilution analysis by HPLC coupled to ICP-MS is carefully evaluated. The proposed method involved the removal of the enzyme's metal co-factors under various conditions and their replacement with isotopically enriched ⁶⁵Cu and ⁶⁸Zn. SEC-ICP-MS showed that the prepared enriched enzymes had a different metal isotopic abundance compared to the wild-type enzyme. Isotopically enriched and wild-type SOD showed the same migration pattern in 1D-PAGE. An enzyme activity assay provided evidence that incorporated ⁶⁵Cu was bound to the correct SOD-binding motif, since the measured activity correlated directly with the amount of Cu present in the prepared enzyme. The addition of free Cu and Zn or a metal chelator did not result in any exchange or loss of metals from the enzyme at neutral pH. Striking experiments were undertaken to evaluate the use of isotopically enriched SOD in SS-IDMS. The chemical preparation study on SOD was further extended to prepare various other isotopically enriched metalloproteins, including carbonic anhydrase, ceruloplasmin, transferring and haemoglobin. Various enrichment procedures were conducted and their performances then evaluated, using SEC-ICP-MS and protein assays. A procedure for the quantification of SOD in tissue samples using an isotopically enriched SOD spike in combination with 2-dimensional HPLC and SS-IDMS was developed and assessed. The feasibility of employing isotopically enriched protein spikes for the speciation of metalloproteins by utilising gel electrophoresis and LA-ICP-MS was also investigated. Furthermore, the change of the iron speciation of the meat-containing protein myoglobin after various treatments was examined using a combination of SEC-ICP-MS and ESI-MS.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

A species-specific double isotope dilution strategy for the accurate quantification of platinum–GG adducts in lung cells exposed to carboplatin

Platinum–DNA adducts, and in particular Pt–GG, have been identified as the major cytotoxic species during chemotherapy treatment with platinum containing drugs. This paper reports for the first time a strategy based on the use of double species-specific isotope dilution analysis (IDA) for the quantification of carboplatin–GG adducts formed by exposing human lung carcinoma cells to carboplatin. The main challenge posed by the use of carboplatin in this pre-clinical application, in comparison with most previously reported studies using cisplatin, includes the relatively low reactivity of this drug, thus demanding for improved limits of detection to be achieved in order to perform accurate quantification of the adducts at low ng Pt per mg DNA levels with relatively small uncertainty in micro-volumes of the biological sample. This was alleviated by developing micro-flow HPLC reversed phased methodology coupled to sector field ICP-MS (R = 300), showing a limit of detection of 0.2 ng Pt per mg DNA. To perform IDA, carboplatin–GG calibrants and spikes (194Pt-enriched GG adducts) were synthesized in house and characterised for Pt mass fraction, Pt distribution and structural composition. In order to assess the accuracy of the developed procedure, in the absence of certified reference materials, a reference sample prepared by incubation of calf thymus DNA with carboplatin and characterised in house (e.g. for its total P and Pt contents and Pt–GG concentration) was analysed in parallel. The use of this sample as a quality control of the cleavage of DNA and recovery of Pt adducts from real samples makes the described strategy particularly novel. Moreover, spike recovery experiments on the cell samples with the reference carboplatin–DNA sample were undertaken. The validated methodology was applied to cultured human lung carcinoma cells exposed to carboplatin; Pt–GG adducts were found to be at a level of 5.54 ng Pt per mg DNA with a relatively expanded combined uncertainty (k = 2) of approximately 20%. The major contributing factors to the overall measurement uncertainty were the mass fraction of Pt in the natural carboplatin–GG standard, the measured isotope ratio precision of sample and calibration blends and the blend to blend variation. The SI traceable methodology presented here will be invaluable for the provision of reference values to clinical measurements and cancer clinical trials

Cow's milk consumption increases iodine status in women of childbearing age in a randomized controlled trial

Background: Recent evidence has highlighted the prevalence of mild-to-moderate iodine deficiency in women of childbearing age and pregnant women, with important public health ramifications owing to the role of iodine, required for thyroid hormone production, in neurodevelopment. Cow’s milk contributes the greatest amount to iodine intakes in several countries. Objective: The objective of this study was to investigate the effect of increased cow’s milk consumption on iodine status, thyroid hormone concentrations and selenium status. Methods: A 12 week, randomized-controlled trial was conducted in 78 low-moderate milk consuming (<250ml/d) healthy women (18-45 years). The intervention group were asked to consume 3L of semi49 skimmed milk per week, while the control group continued their usual milk consumption (baseline median (IQR): 140 (40-240) mL/d). At baseline, week 6 and week 12 participants provided a spot51 urine sample [urinary iodine concentration (UIC); creatinine] and a fasting blood sample [thyroid hormone concentrations; serum total selenium; selenoprotein P]. This study was registered at ClinicalTrials.gov Study (Ref: NCT02767167). Results: At baseline, the median (IQR) UIC of all participants was 78.5 (39.1-126.1)μg/L. Changes in the median UIC from baseline to week 6 (35.4 vs. 0.6 μg/L; P=0.014) and week 12 (51.6 vs. -3.8 μg/L; P=0.045) were significantly greater in the intervention group compared with the control group. However, despite being higher within the intervention group at weeks 6 and 12, the change in the iodine:creatinine ratio from baseline was not significantly different between groups at either week 6 (P=0.637) or 12 (P=0.178). There were no significant differences in thyroid hormone concentrations or selenium status between groups at any time point. Conclusions: The present study has demonstrated that the consumption of additional cow’s milk can significantly increase UIC in women of childbearing age. These results suggest that cow’s milk is a potentially important dietary source of iodine in this population group

Cow Milk Consumption Increases Iodine Status in Women of Childbearing Age in a Randomized Controlled Trial

Background: Recent evidence has highlighted the prevalence of mild-to-moderate iodine deficiency in women of childbearing age and pregnant women, with important public health ramifications owing to the role of iodine, required for thyroid hormone production, in neurodevelopment. Cow’s milk contributes the greatest amount to iodine intakes in several countries. Objective: The objective of this study was to investigate the effect of increased cow’s milk consumption on iodine status, thyroid hormone concentrations and selenium status. Methods: A 12 week, randomized-controlled trial was conducted in 78 low-moderate milk consuming (<250ml/d) healthy women (18-45 years). The intervention group were asked to consume 3L of semi49 skimmed milk per week, while the control group continued their usual milk consumption (baseline median (IQR): 140 (40-240) mL/d). At baseline, week 6 and week 12 participants provided a spot51 urine sample [urinary iodine concentration (UIC); creatinine] and a fasting blood sample [thyroid hormone concentrations; serum total selenium; selenoprotein P]. This study was registered at ClinicalTrials.gov Study (Ref: NCT02767167). Results: At baseline, the median (IQR) UIC of all participants was 78.5 (39.1-126.1)μg/L. Changes in the median UIC from baseline to week 6 (35.4 vs. 0.6 μg/L; P=0.014) and week 12 (51.6 vs. -3.8 μg/L; P=0.045) were significantly greater in the intervention group compared with the control group. However, despite being higher within the intervention group at weeks 6 and 12, the change in the iodine:creatinine ratio from baseline was not significantly different between groups at either week 6 (P=0.637) or 12 (P=0.178). There were no significant differences in thyroid hormone concentrations or selenium status between groups at any time point. Conclusions: The present study has demonstrated that the consumption of additional cow’s milk can significantly increase UIC in women of childbearing age. These results suggest that cow’s milk is a potentially important dietary source of iodine in this population group