Isotope Dilution Method Research Articles

Influence of Vitamin A Status on the Choice of Sampling Time for Application of the Retinol Isotope Dilution Method in Theoretical Children

BackgroundVitamin A status may influence the choice of a blood sampling time for applying the retinol isotope dilution (RID) equation to predict vitamin A total body stores (TBS) in children. ObjectivesWe aimed to identify time(s) after administration of labeled vitamin A that provide accurate estimates of TBS in theoretical children with low or high TBS. MethodsWe postulated 2- to 5-y-old children (12/group) with low (<200 μmol) or high TBS (≥700 μmol) and used compartmental analysis to simulate individual subject values for the RID equation TBS = FaS/SAp (Fa, fraction of dose in stores; S, retinol specific activity in plasma/in stores; SAp, retinol specific activity in plasma). Using individual SAp and group geometric mean FaS values from 1–28 d, we calculated individual and group mean TBS and compared them to assigned values. ResultsMean TBS was accurately predicted for both groups at all times. For individuals, predicted and assigned TBS were closest when the CV% for FaS was low [12–14%; 4–13 d (low), 12–28 d (high)]. The mean percentage error for TBS was <10% from 2–19 d (low) and 7–28 d (high). Predicted TBS was within 25% of assigned TBS for ≥80% of children from 3–23 d (low) and 9–28 d (high). Within groups, RID tended to overestimate lower TBS and underestimate higher TBS. ConclusionsUsing a good estimate for FaS, accurate RID predictions of TBS for individuals will be obtained at many times. If vitamin A status is low, results indicate that early sampling (e.g., 4–13 d) is optimal; if vitamin A status is high, sampling at 12–28 d is indicated. When vitamin A status is unknown, sampling at 14 d is recommended, or a super-subject design can be used to obtain the group mean FaS at various times for RID prediction of TBS in individuals.

Contrasting effects of slurry and mineral fertilizer on N2-fixation in grass-clover mixtures

In dairy systems, grass–clover swards are fertilized with on–farm cattle slurry as a basic dressing and often supplemented with mineral fertilizer throughout the season. Uncertainty remains on consequences for N input from atmospheric N2-fixation by grassland legumes. The aim of this study was to examine the N response of slurry versus mineral N fertilization on clover dynamics, the proportion of N derived from the atmosphere (%Ndfa) and the quantitative N2-fixation (qBNF) using the 15N isotope dilution method. This was done in an on farm setting at two sites in two years with increasing rates of N fertilizer (0–480 kg available N ha-1) either of mineral N only or combined with application of cattle slurry. The leys were at one site composed a 2–species mixture of white clover (Trifolium repens L.) and ryegrass (Lolium perenne L.) and at the other site of a 4–species mixture of white clover, red clover (Trifolium pretense L.), festulolium (Festulolium braunii) and ryegrass. Species dynamics were significantly affected by fertilizer rate in the 2–species sward and by fertilizer rate and type in the 4–species sward. Reduction of the clover proportion in response to fertilization was due to a clover yield decrease (2–species sward) or a grass yield increase (4–species sward). In the 4–species sward, in treatments with mineral N only %Ndfa decreased markedly with N rate, whereas the corresponding slurry + mineral N treatments did not. Thus, apart from fertilization with mineral N only at one site the general picture was a relatively high (>80%) %Ndfa over the season when the N level was below 200–300 kg available N ha-1. The qBNF of the harvested biomass in sward without fertilization reached 193–216 kg N ha-1 in the 2–species sward and 203–286 kg N ha-1 in the 4–species sward. Clover persisted at high fertilization rates, but qBNF was reduced to 16–79 kg N ha-1 and 58–163 kg N ha-1 in 2– and 4–species swards, respectively. Interestingly, in the 4–species sward amended with slurry + mineral N, qBNF was high and independent of fertilization rate. We conclude that response of clover dynamics to N fertilization differed with fertilizer type and level – slurry have lower effect whereas the effect of mineral N only on N2-fixation is less predictable.

QNMR characterization of potential peptide calibrators for isotope dilution LC–MS quantification of a SARS-CoV-2 IgG monoclonal antibody

The year 2020 will be remembered for the most pernicious epidemic of modern history. The SARS-CoV-2 coronavirus has affected millions of people worldwide and has triggered an unprecedented race for the development of diagnostic tests and therapeutical vaccines. These technologies require befitting measurements that underpin reliability and patient safety. The level of blood-borne antibodies against SARS-CoV-2 is correlated initially with the extent of infection and subsequently with the degree of immunity attained. This renders their accurate measurement an important target for immunometric and liquid chromatography–mass spectrometry methods. Suitable reference materials and methods are being developed to ensure the reliability and consistency of such measurements. Solutions of purified monoclonal IgG may prove useful as primary calibrator materials for the development of calibration hierarchies for the measurement of blood-borne antibodies against SARS-CoV-2. Bottom-up approaches can be applied to the quantification of such structurally complex large molecules. These consist of trypsin and Lys C digestion and quantification of the resulting proteotypic peptides as surrogate analytes for an IgG monoclonal antibody. Establishing the metrological traceability of this procedure requires peptides whose purity has been determined accurately, for example with quantitative nuclear magnetic resonance (qNMR). The 1H qNMR method allows for the mass fraction assignment of peptides by accurate quantification of 1H resonance signals specific for both the peptide and for a certified reference material used as internal standard in the peptide solution. The current paper describes the qNMR characterization of five peptides that could be used in a double isotope dilution method for the quantification of SARS-CoV-2 IgG monoclonal antibodies in solution.

Quantitative metabolomics for dynamic metabolic engineering using stable isotope labeled internal standards mixture (SILIS)

The production of chemicals and fuels from renewable resources using engineered microbes is an attractive alternative for current fossil-dependent industries. Metabolic engineering has contributed to pathway engineering for the production of chemicals and fuels by various microorganisms. Recently, dynamic metabolic engineering harnessing synthetic biological tools has become a next-generation strategy in this field. The dynamic regulation of metabolic flux during fermentation optimizes metabolic states according to each fermentation stage such as cell growth phase and compound production phase. However, it is necessary to repeat the evaluation and redesign of the dynamic regulation system to achieve the practical use of engineered microbes. In this study, we performed quantitative metabolome analysis to investigate the effects of dynamic metabolic flux regulation on engineered Escherichia coli for γ-amino butyrate (GABA) fermentation. We prepared a stable isotope-labeled internal standard mixture (SILIS) for the stable isotope dilution method (SIDM), a mass spectrometry-based quantitative metabolome analysis method. We found multiple candidate bottlenecks for GABA production. Some metabolic reactions in the GABA production pathway should be engineered for further improvement in the direct GABA fermentation with dynamic metabolic engineering strategy.

The primary controls on U/Ca and minor element proxies in a cold-water coral cultured under decoupled carbonate chemistry conditions

Culture experiments are uniquely suited for uncovering the fundamental factors controlling skeletal geochemistry because it is possible to explore conditions beyond what is found in the modern ocean and to decouple parameters that co-vary in nature. We cultured juvenile individuals of a cold-water coral (Balanophyllia elegans) in a set of experiments that decoupled carbonate chemistry parameters over a wide range of pH, DIC, and [CO32−] values. Using a multi-element mixed spike isotope dilution method we then analyzed cultured skeletons for U/Ca, which has been proposed as a potential proxy for seawater pH and [CO32−], as well as Sr/Ca, and Mg/Ca.We find that U/Ca and Sr/Ca ratios in cultured B. elegans are most strongly correlated with solution DIC and not pH or [CO32−]. We also confirm previous observations that Metal/Calcium (Me/Ca) ratios follow the same correlated relationships between and among individuals across different experimental conditions. Interpretation of these robust Me/Ca patterns within the framework of a geochemical model of biomineralization allows us to identify two “rules” of skeletal growth for B. elegans. First, changes in seawater exchange rates can explain variability in B. elegans Me/Ca ratios, correlations between these ratios, and sensitivity of Me/Ca to changes in seawater carbonate chemistry. Second, our model best fits our data if we assume that calcifying fluid pH for B. elegans remains constant across widely varying experimental seawater compositions. Our study has implications for the recently developed Sr-U paleothermometer because it refines our understanding of the environmental parameters affecting this proxy. Our model further demonstrates that U/Ca is not a robust indicator of seawater pH or [CO32−]. Instead, U/Ca may record how calcifying fluid [CO32−] responds to changes in the environment or calcification dynamics, which may be useful in evaluating how corals respond to changes like ocean acidification. Measurements of U/Ca and additional Me/Ca ratios in other coral species, evaluated within a similar framework, may elucidate how those species respond to environmental change.

Quantity of phenibut in dietary supplements before and after FDA warnings

Introduction Phenibut is used to treat anxiety, insomnia, alcohol withdrawal and other conditions in Russia. The drug, however, has abuse potential and may cause lethargy, delirium, psychosis and coma. In the United States (US), the US Food and Drug Administration (FDA) has never approved the use of phenibut as a prescription medication, but the drug is available over-the-counter in dietary supplements. More than 80 cases of coma and death have been associated with phenibut consumption and withdrawal, and the FDA recently warned that the drug is not permitted in over-the-counter supplements. We designed our study to determine the presence and quantity of phenibut in over-the-counter supplements before and after the FDA warnings. Methods Phenibut products were included if they (a) listed phenibut or a synonym as an ingredient on the label, (b) were labeled as a dietary supplement, and (c) were available for sale both before and after the FDA warning. Supplements were analyzed by liquid chromatography time-of-flight mass spectrometry; quantification was performed by isotope dilution method. Results Four brands of dietary supplements labeled as containing phenibut met the inclusion criteria. Prior to the FDA warnings, two of the four brands contained phenibut, at dosages of 484 mg and 487 mg per serving. After the FDA warning, all four products contained phenibut, ranging in dosages from 21 mg to 1,164 mg per serving. Phenibut was first detected only after the FDA warnings in two brands, and the quantity of phenibut increased in three of four products after the FDA warnings. Quantities detected per dose were as much as 450% greater than a typical 250 mg pharmaceutical tablet manufactured in Russia. Conclusion Following FDA issuing an advisory that phenibut is not permitted in dietary supplements, the quantity of phenibut increased in 3 of 4 brands of over-the-counter phenibut supplements.

A Dilute-and-Shoot UHPLC–MS/MS Isotope Dilution Method for Simultaneous Determination and Confirmation of 11 Mycotoxins in Dried Distiller’s Grains with Solubles

Natural contamination with mycotoxins in dried distiller's grains with solubles (DDGS) as a mainstream animal feed ingredient poses a risk to animal health. A regulatory method was needed for the agency to simultaneously detect 11 mycotoxins of high regulatory priority in DDGS. A DDGS sample (10 g) was extracted twice with acetonitrile-water under mildly acidic condition. Two aliquots from the combined crude extract were taken and processed separately: (1) diluted 400-fold with solvent for analysis of deoxynivalenol and fumonisins B1 and B2; and (2) with the pH adjusted to 7.5, and then diluted 15.7-fold for analysis of aflatoxins B1, B2, G1, and G2, ochratoxin A, zearalenone, and T-2 and HT-2 toxins. Uniformly labeled 13C-isotopologues of these mycotoxins were added as internal standards to the diluted extracts for quantitative analysis by ultra-high-performance LC-tandem MS. The linear quantitation ranges (µg/kg) were: aflatoxins B1, B2, G1, and G2, 1.57-105; zearalenone, 16.3-1090; T-2 toxin, 3.14-208; HT-2 toxin, 48.2-3220; ochratoxin A, 0.47-31.4; deoxynivalenol, 240-16000; fumonisin B1 and B2, 320-21200. Accuracies for these analytes at each of three fortification levels ranged from 70.7 to 100%, with corresponding RSDs between 1.4 and 10.5%. True recoveries were all higher than 83%. This method was successfully validated to meet the agency's performance guidelines for regulatory methods. This method is easy, quick, and robust to simultaneously quantify and confirm the presence of 11 regulated mycotoxins in DDGS.

Exploration of apatite (U[sbnd]Th)/He geochronological analysis of volcanic units in fossil-bearing strata of the Homa Peninsula, southwestern Kenya

Many geological contexts lack suitable material for commonly applied absolute dating methods. Even if some regions contain potentially datable materials, they may be inappropriate for dating by existing geochronological techniques. Paleoanthropological localities on the Homa Peninsula, southwestern Kenya, have yielded important paleoanthropological material. These localities are, however, poorly constrained in time, and few radioisotopic ages have been published, because sediments derived from the Homa Mountain carbonatite complex commonly do not contain zircons for UPb dating or feldspars for 40Ar/39Ar dating. Additionally, apatite grains that are present have failed to produce precise isotope dilution-thermal ionization mass spectrometer (ID-TIMS) results due to their high common lead content. Apatite (UTh)/He dating using single-crystal laser fusion He determinations and the UTh isotope dilution method was applied to three formations with well-constrained ages to test the suitability of this technique for dating sediments on the peninsula. Two of the three Kanam Formation samples provide dates close to the approximately 6 million year age (Ma) provided by 40Ar/39Ar dates. The Abundu Formation was dated to 2.39 ± 0.29 Ma overlapping with the age of the Rawi Formation (C2An.1n Subchron, 3.05–2.59 Ma) to which it has been correlated. The age for the Kanjera South Member 1.54 ± 0.85 Ma overlaps with the expected 2 Ma age, though it has a large uncertainty. The apatite raw (UTh)/He ages are somewhat younger than the expected dates, but the technique shows promise.

Probing the Hidden Role of Mitochondrial DNA Damage and Dysfunction in the Etiology of Aristolochic Acid Nephropathy.

Aristolochic acid nephropathy (AAN) is a unique type of progressive renal interstitial fibrotic disease caused by prolonged exposure to aristolochic acids (AAs) through AA-containing herbal medicines or AA-tainted food. Despite decades of research and affecting millions of people around the world, the pathophysiology of AAN remains incompletely understood. In this study, we tested the potential causative role of mitochondrial dysfunction in AAN development. Our findings revealed AA exposure induces an exposure concentration and duration dependent lowering of adenosine triphosphate in both cultured human kidney and liver cells, highlighting an AA exposure effect on mitochondrial energy production in the kidney and liver, which both are highly metabolically active and energy-demanding organs. Analysis with liquid chromatography-tandem mass spectrometry coupled with stable isotope dilution method detected high levels of mutagenic 8-oxo-2'-deoxyguanosine and 7-(deoxyadenosine-N6-yl)-aristolactam adduct on mitochondrial DNA isolated from AA-treated cells, unmasking a potentially important causative, but previously unknown role of mitochondrial DNA mutation in the pathophysiology of AAN development.

Selective and sensitive UHPLC-ESI-Orbitrap MS method to quantify protein oxidation markers

A targeted UHPLC-MS/MS isotopic dilution method has been developed for the simultaneous quantification of 18 different free and protein-bound aromatic amino acid oxidation products in food and biological matrices. All analytes, including critical isomeric pairs of Tyr, o-Tyr, m-Tyr, and dioxyindolylalanine diastereomers were chromatographically resolved to obtain high selectivity, without the need for derivatizing or ion pairing agents. The results of method validation showed adequate retention time reproducibility [0.1–0.6% coefficient of variation (CV) for over 224 injections], accuracy (within ±1–20% of the nominal concentration), and precision (1–17% CV) for all target analytes. The lower limit of quantification was calculated in different matrices using both the Hubaux-Vos approach and accuracy and precision data showing values in the range of 0.2–15 ng/mL. Use of stable isotope-labelled internal standards compensated errors due to matrix effects and artefactual degradation of analytes. Both acid and enzymatic hydrolyses were tested to obtain the best possible results for the quantification of protein oxidation products, demonstrating the stability of target analytes under hydrolytic conditions. The method was successfully applied to quantify target analytes in serum, tissue, milk, infant formula, pork liver pâté, chicken meat and fish. The method was also applied to assess the role of Fenton's reagent in oxidizing Trp, Phe and Tyr residues in different proteins, with results showing o-Tyr, dioxyindolylalanine diastereomers, kynurenine, dityrosine being the main oxidation products. The Fenton chemistry favored the formation of o-Tyr over m-Tyr from Phe with 2–36 folds higher yields. 3-Nitrotyrosine, a marker of protein nitration, was also detected in samples treated with Fenton's reagent.

Quantitation of γ-Glutamylcysteine-Formaldehyde Conjugate in Formaldehyde- and Oxidative Stress-Exposed Cells by Liquid Chromatography-Tandem Mass Spectrometry.

Humans are constantly exposed to formaldehyde (FA) of both exogenous and endogenous sources, and FA exposure is associated with the development of many human diseases, including cancers. Marker molecules that can provide information on exposure history and amounts will assist disease risk assessment and early interventions. To develop marker signatures of FA exposure, we explored in this study the conjugation reaction of FA with γ-glutamylcysteine (GGC), one of the precursors to glutathione biosynthesis, under physiologically relevant conditions. The results showed that the reaction produced a stable metabolite of FA, (S)-1-((((R)-2-amino-2-carboxyethyl)thio)methyl)-5-oxopyrrolidine-2-carboxylic acid (COCA). Using liquid chromatography-tandem mass spectrometry coupled to a stable isotope-dilution method, we then quantitated for the first time the formation of this novel metabolite in FA- and Fe2+-EDTA-exposed human cells. The results revealed the exposure time- and concentration-dependent formation of COCA in FA- or Fe2+-EDTA-exposed cells, suggesting that COCA may serve as a biomarker of FA and oxidative stress exposure. Furthermore, the study sheds light on a previously unknown protective role of GGC against FA and oxidative stress.

Absolute Quantitation of Tryptophan-Containing Peptides and Amyloid β-Peptide Fragments by Coulometric Mass Spectrometry.

Isotope-labeled internal standards are routinely used for mass spectrometry (MS)-based absolute quantitation. However, syntheses of isotope-labeled peptides are time-consuming and costly. To tackle this issue, we recently developed a coulometric mass spectrometric (CMS) approach for absolute quantitation without the use of standards, based on the electrochemical oxidation of cysteine or tyrosine-containing peptides followed by mass spectrometric measurement of the oxidation yield. To further expand the utility of this method, herein we present the CMS method for absolute quantitation of peptides based on tryptophan electrochemical oxidation. Several tryptophan-containing peptides, such as WGG, WQPPRARI, WAGGDASGE, RTRPLWVRME, and KVPRNQDWL, were successfully quantified with a quantification error ranging from -4.5 to +4.3%. Furthermore, this quantitation approach is also applicable to protein, in which protein can be digested and a surrogate peptide can be selected for quantification to reflect the amount of the parent protein, as exemplified by CMS analysis of peptide GITWK from cytochrome c. The CMS result agreed well with the traditional isotope dilution method, with only a small difference of 3.5%. In addition, CMS was used to successfully quantify amyloid beta (Aβ) peptide fragments (up to 28 amino acid residues) based on tyrosine oxidation. The validity of the CMS method for peptide and protein absolute quantitation without using isotope-labeled peptide standards would greatly facilitate proteomics research.

230-OR: Exercise Training Improved Antilipolytic Sensitivity to Insulin in Obese Adults with Low Sensitivity to Insulin before Training

Low sensitivity to the antilipolytic effects of insulin in adipose tissue has been linked with impaired insulin-mediated glucose uptake in skeletal muscle. The aims of this study were: 1) to compare adipose tissue morphology in obese adults with high- (HIGH) vs. low-sensitivity (LOW) to the antilipolytic effects of insulin, and 2) compare the effects of exercise training on adipose tissue from HIGH vs. LOW subjects. Using isotopic dilution methods before and during a hyperinsulinemic-euglycemic clamp, we measured the antilipolytic response to insulin in a total of 36 obese adults - and we identified sub-cohorts of 13 LOW (64±7% lipolytic suppression by insulin; BMI: 34±4kg/m2) and 13 HIGH (82±3% lipolytic suppression by insulin; BMI:33±3kg/m2). Abdominal subcutaneous adipose tissue (aSAT) samples were collected before the clamp (non-insulin stimulated). Measurements were made before and after a 12-week endurance exercise training program (4 days/week). Before training, capillary density (# of capillaries/mm2) in aSAT samples for LOW was significantly lower than HIGH (p=0.004). Collagen abundance in aSAT extracellular matrix (ECM) was lower in LOW (p=0.013). In response to training, the antilipolytic response to insulin was significantly improved in LOW (72±5% lipolytic suppression by insulin; p&amp;lt;0.001) but not HIGH. This was accompanied by an increased capillary density (p=0.03), and collagen abundance in ECM (p = 0.02), along with a slight reduction in fat mass (1±1kg; p=0.05). Adipocyte size was reduced in both groups (p=0.02). In summary, aSAT from obese adults with low antilipolytic sensitivity to insulin displayed lower capillarization and ECM collagen abundance compared with obese adults with high sensitivity to the antilipolytic effects of insulin. Importantly, morphological adaptations to exercise training accompanied an increased antilipolytic sensitivity to insulin in subjects with low antilipolytic sensitivity before training. Disclosure C. Ahn: None. M. W. Schleh: None. B. J. Ryan: None. A. Ludzki: None. P. Varshney: None. J. B. Gillen: None. S. Chen: None. J. F. Horowitz: None. Funding National Institutes of Health (R01DK077966, P30DK089503, T32DK007245, F32DK117522); Canadian Institutes of Health Research (DFS146190, 338735)

1222-P: Adipose Tissue Antilipolytic Insulin Sensitivity Protects against Whole-Body Insulin Resistance and Lowers Acylcarnitine Accumulation in Adults with Obesity

The aims of this study were; 1) to assess clinical and subclinical factors associated with insulin-stimulated glucose uptake and 2) compare skeletal muscle lipid profile (untargeted lipidomics) in a sub-cohort of insulin sensitive vs. insulin resistant subjects. 66 obese adults (F=46 [BMI=34±3kg/m2], M=20 [BMI=33±2.9 kg/m2]) with homogeneous body composition completed a 2h hyperinsulinemic-euglycemic clamp, and stable isotope dilution methods to assess glucose rate of disappearance (Glucose Rd: index of insulin sensitivity), fatty acid rate of appearance (FA Ra), and hepatic glucose production (Glucose Ra) under basal and hyperinsulinemic conditions. We also performed an array of metabolic measurements (e.g., hepatic and visceral fat accumulation [via MRI], fat oxidation, lipid profile, circulating cytokines). Covariate analysis (LASSO) identified insulin-mediated suppression of FA Ra, basal glucose Ra, and fat oxidation as the cluster of measurements best predicting Glucose Rd (r=0.76, p&amp;lt;0.01). Insulin-mediated suppression of FA Ra was also the strongest independent predictor of Glucose Rd (r=0.51; p&amp;lt;0.01). As a result of greater adipose tissue anti-lipolytic response to insulin, lower FA delivery to skeletal muscle may improve insulin-mediated glucose uptake via lower muscle lipid accumulation. Importantly, we found the accumulation of several long-chain acylcarnitine species (measure of incomplete β-oxidation: C18:0,18:1,20:1,20:3) to be lower (p&amp;lt;0.05) in muscle samples from an insulin sensitive sub-cohort (Glucose Rd &amp;gt; 8.5 µmol/(min•FFM•[Insulin]); n=10) compared with an insulin resistant sub-cohort (&amp;lt; 7.2 µmol/(min•FFM•[Insulin]); n=8). In conclusion, our findings indicate that greater adipose tissue anti-lipolytic response to insulin is an important predictor of insulin-mediated glucose uptake - and acylcarnitine accumulation in skeletal muscle may mediate this effect. Disclosure M. W. Schleh: None. B. J. Ryan: None. C. Ahn: None. A. Ludzki: None. P. Varshney: None. J. B. Gillen: None. J. F. Horowitz: None. Funding National Institutes of Health (R01DK077966, P30DK089503, T32DK007245, F32DK117522); Canadian Institutes of Health Research (DFS146190, 338735)

Direct assessment of body iron balance in women with and without iron supplementation using a long-term isotope dilution method in Benin and Switzerland

Long-term isotopic dilution measurements of body iron may allow quantification of basal body iron balance and iron gains during an iron intervention with higher precision and accuracy than conventional iron indices. We compared body iron balance before, during, and after oral iron supplementation in women in Benin and in Switzerland. In prospective studies, Beninese (n=11) and Swiss (n=10) women previously labeled with stable iron isotopes were followed preintervention for 90-120 d, then received 50-mg iron daily for 90-120 d and were followed postintervention for 90-120 d. We used changes in blood isotopic composition to calculate iron absorption (Feabs), iron loss (Feloss), and net iron balance (Fegain). Compliance with supplementation was>90%. In Benin, during the preintervention, intervention, and postintervention periods, Fe means±SDs were as follows: 1) Feabs: 0.92±1.05, 3.75±2.07, and 0.90±0.93 mg/d; 2) Feloss: 1.46±1.95, 1.58±1.57, and 1.84±1.61 mg/d; and 3) Fegain: -0.55±1.56 mg/d, 2.17±1.81 mg/d, and -0.94±1.13 mg/d. In Switzerland, the corresponding values were: 1) 1.51±0.37, 4.09±1.52, and 0.97±0.41 mg/d; 2) 0.76±1.37, 2.54±1.43, and 2.08±1.05 mg/d; and 3) 0.75±1.37, 1.55±1.75, and -1.11±1.06 mg/d. Inflammation was low in both settings, and isotopically calculated iron balance was comparable to that calculated from changes in conventional iron indices. Without iron supplementation, Beninese women had lower long-term dietary iron absorption and higher iron losses in the preintervention period than Swiss women. During iron supplementation, both groups had high iron absorption and similar iron gains. However, there was a 3-fold increase in iron losses in the Swiss women during the supplementation and postintervention period compared with the preintervention period. Body iron isotope dilution is a promising new method for quantifying long-term body iron balance and for assessing the impact of iron interventions. The studies were registered at clinicaltrials.gov as NCT02979080 and NCT02979132, respectively.

Determination of Vitamin A Total Body Stores in Children from Dried Serum Spots: Application in a Low- and Middle-Income Country Community Setting

BackgroundThe retinol isotope dilution (RID) method has been used to evaluate vitamin A (VA) status in healthy adults and children in low- and middle-income countries (LMIC) and to assess the efficacy of various VA interventions. ObjectiveThe study was designed to examine whether dried serum spots (DSS) can be applied to RID when conducting VA total body store (TBS) assessments in community settings. MethodsFour days after an oral dose of 0.4 mg [13C10]retinyl acetate was administered to Filipino children (12–18 mo), a single blood draw was divided to isolate both serum and plasma. Serum (40 μL) was spotted and dried on Whatman 903 cards and shipped at ambient temperature whereas liquid plasma (LP) was frozen at –80°C and shipped on dry ice. The VA tracer to tracee ratio from DSS and LP was quantified by LC-MS/MS. Comparisons between DSS and LP paired samples (n = 72) were made for [13C10]retinol specific activity (SAp) by Pearson’s correlation and for VA TBS by Bland–Altman analysis. ResultsThe sum of 3 coextracted DSS were required to consistently detect [13C10]retinol above the LC-MS/MS limit of quantitation (LOQ). [13C10]retinol SAp from DSS was highly correlated with SAp from LP (r = 0.945; P < 0.01). A comparison of methods for TBS determination using Bland–Altman analysis indicated agreement with an intraindividual difference of 24.7 μmol (4.6%). Mean total liver reserve (TLR) values from DSS and LP were 1.7 μmol/g (± 0.6 SD) and 1.6 μmol/g (± 0.6 SD), respectively. ConclusionsVA TBS can be determined from DSS thereby reducing the logistics and cost of maintaining a cold chain by shipping samples at ambient temperature and, thus, making the RID technique more feasible in LMIC community settings. This trial was registered at https://clinicaltrials.gov as NCT03030339.

Use of Model-Based Compartmental Analysis and Theoretical Data to Further Explore Choice of Sampling Time for Assessing Vitamin A Status in Groups and Individual Human Subjects by the Retinol Isotope Dilution Method.

ABSTRACTBackgroundAn optimal blood sampling time for application of the retinol isotope dilution (RID) method for predicting vitamin A total body stores (TBS) (i.e., vitamin A status) has not been established.ObjectivesObjectives were to identify sampling times that provide accurate estimates of TBS by RID in groups and individuals by applying compartmental modeling to data for theoretical adults and children.MethodsWe selected previously generated hypothetical adults and children (20 per group) that had a wide range of assigned values for TBS and vitamin A kinetic parameters. We used the Simulation, Analysis and Modeling software to simulate individual kinetic responses; then we calculated geometric mean values for the RID equation coefficients and each individual's plasma retinol specific activity at various times, using those values to predict group mean and individual subject TBS. Predicted values for TBS were compared with assigned values.ResultsAccurate estimates of group mean TBS were obtained at all sampling times from 1 to 30 d in both adults and children. For individuals, correlations between RID-predicted TBS and assigned values increased with time in the adults (R2 = 0.80 at day 14, 0.96 at day 21, and 0.99 at day 28); a similar trend was observed for the children, with R2 = 0.82 at day 7 and increasing to 0.97 at days 21 and 28 (P < 0.001 for all comparisons).ConclusionsAlthough no single, unique time provided the most accurate prediction of TBS for all individuals within these groups, applying the RID method at 21 or 28 d yielded predictions that were within 25% of assigned values for 90% or 95% of adults, respectively; corresponding values for children were 80% from 10 to 20 d, and 85% at 21 and 28 d. For most subjects, early times (<14 d for adults and <10 d for children) provided less accurate predictions.

A stable isotope dilution method for a highly accurate analysis of karrikins

BackgroundKarrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported.ResultsWe present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the validation of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the successful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l).ConclusionsOur results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development.

Development of a Compartmental Model to Investigate the Influence of Inflammation on Predictions of Vitamin A Total Body Stores by Retinol Isotope Dilution in Theoretical Humans

BackgroundInflammation, both acute and chronic, is associated with reductions in the synthesis of retinol-binding protein (RBP) and the concentration of retinol in plasma. Consequently, it is currently recommended that the retinol isotope dilution (RID) method not be used to estimate vitamin A total body stores (TBS) in subjects with inflammation. ObjectivesTo apply compartmental analysis to study the effects of inflammation on hepatic apo-RBP input, plasma retinol pool size, and RID-predicted TBS in theoretical subjects with known steady state values for these parameters. MethodsWe selected 6 previously generated hypothetical subjects who ingested a dose of stable isotope–labeled vitamin A (day 0). Starting with each subject’s published steady state model for retinol tracer kinetics, we developed a parallel model for unlabeled retinol and RBP that incorporated links between these entities and tied liver retinol secretion to RBP availability. Then we perturbed the steady state model by initiating chronic or acute inflammation on day 0 or acute inflammation on day 3 or 9 and simulating results for RBP, plasma retinol, and TBS. ResultsChronic inflammation led to substantial reductions in RID-predicted TBS for at least 2 weeks after tracer administration. In contrast, acute inflammation induced on day 0 or 3 resulted in less dramatic impacts on TBS (37% or 20% reduction, respectively, from steady state levels, with levels rebounding by 14 days). When inflammation was induced 9 days after administration of the tracer, the effects on predicted TBS were minimal. Overall, for acute inflammation initiated at 0, 3, or 9 days, accurate predictions of TBS were obtained by 2 weeks. ConclusionsCompartmental analysis can be applied in the novel way described here to study the influence of perturbations such as inflammation on predictions of vitamin A status using RID. Such an approach has potential value for studying other perturbations and different nutrients.

Species Interactions and Nitrogen Use during Early Intercropping of Intermediate Wheatgrass with a White Clover Service Crop

Perennial grain crops intercropped with legumes are expected to use nitrogen (N) resources efficiently. A pot experiment using the 15N isotope dilution method demonstrated interspecific competition and use of N from the soil and N2 fixation in intermediate wheatgrass (Thinopyrum intermedium (Host) Barkworth &amp; D.R. Dewey, IWG) and white clover (Trifolium repens L., WC) intercrops at five species-relative frequencies and four levels of inorganic N fertilizer in a replacement series design. The proportion of N in WC derived from the atmosphere increased from 39.7% in a sole crop to 70.9% when intercropped with IWG, and 10.1% N in IWG transferred from WC. Intermediate wheatgrass showed high fitness with maintained high total dry matter production at low relative frequencies. Decreasing IWG-relative frequency only increased dry matter and N accumulation of WC, resulting in increased amounts of N2 fixed. Increased levels of N fertilization increased the proportion of N acquired from the fertilizer in IWG and WC but decreased the N fixed by WC and N absorbed by IWG from the soil. Our study indicates that WC supply sufficient fixed N2 for IWG intercrop biomass yields under appropriate levels of soil N fertility and species-relative frequencies.