Ermittlung der Konzentration von Folsäuremetaboliten in biologischen Proben mittels LC-MS/MS

Eine ausreichende Folataufnahme ist wichtig, um das Risiko für Neuralrohrdefekte, Herz-Kreislauf-Erkrankungen, einige Krebserkrankungen sowie Bluthochdruck bei Frauen zu reduzieren. Eine gesteigerte Zufuhr von Nahrungsfolaten, mit Folsäure angereicherten Lebensmitteln oder Folsäuresupplementen, kann zu höheren Erythrozyten-folatspiegeln führen und das Risiko für Neuralrohrdefekte minimieren. Studien über die Bestimmung der Folatkonzentrationen in den roten Blutkörperchen sind wesentlich seltener anzufinden, als über die Plasma- bzw. Serumfolatanalyse, da die Aufbereitung der Erythrozytenfolate viel komplexer ist. In dieser Arbeit wurde versucht, eine sensitive und aussagekräftige Flüssigkeitschromatographie-Massenspektrometrie-Methode (LC-MS), zur Bestimmung der empfindlichen Folate in wässrigen Standardlösungen und Erythrozyten, zu entwickeln. Die Folate wurden durch negative und positive Elektrosprayionisierung (ESI), an zwei unterschiedlichen MS-Typen – time of flight (TOF) und tandem/triplequadrupole, detektiert und quantifiziert. Zum Vergleich der Methoden, wurde zusätzlich versucht, die gerätespezifischen Nachweis- und Bestimmungsgrenzen, LOD (Limit of Detection) und LOQ (Limit of Quantification) zu ermitteln. Um die Stabilität der Folate zu ermitteln, wurden Lagerversuche bei 4 °C und -18 °C durchgeführt. Beide Experimente zeigten deutliche Intensitätsrückgänge, sowohl bei Lagerung im Autosampler (4 °C) als auch bei Lagerung im Tiefkühlschrank (-18 °C). Die Konditionen bei -18 °C waren für p-ABA, PteGlu2 und PteGlu3 vorteilhafter als bei 4 °C. Auf 5-MTHF und DHF zeigte die Art der Lagerung keinen signifikanten Einfluss, während sich die Lagerung bei -18 °C für THF als nachteilig herausstellte.An adequate folate intake is indispensable in lowering the risk of neural tube defects, cardiovascular disease, some cancers, and hypertension in women. Increased intakes of food folates, folic acid enriched food or folic acid supplements can reduce the risk of neural tube defects and enhance the folate concentrations in red blood cells (RBC). Methods for determination of folate levels in plasma or serum are still more common than methods for analyzing folate concentrations in red blood cells as the sample prepa-ration is considerably more complex. In this master thesis a sensitive and meaningful liquid chromatography-mass spectrometry (LC-MS) method to analyse and determine the sensitive folates - in aqueos buffer and red blood cells – was developed. HPLC triplequadrupole and HPLC-qTOF (time of flight) in positive and negative electrospray ionization (ESI) were used to detect and quantify the folate metabolites. Method validation was carried out by defining the device-specific limit of quantification (LOQ) and limit of detection (LOD). Since the stability of folates is known to be unstable, the influence of storage at 4 °C and -18 °C was tested. Both experiments showed a significant decrease in intensity. The results for the metabolites were fairly different: p-ABA, PteGlu2 and PteGlu3 indicated better stability within storage at -18°C, 5-MTHF and DHF showed similar results when stored at -18 °C pointed out adverse effects for THF

Equine Muscular Adaptations to Effective Use of Dietary Fat During Exercise

Feeding supplemental dietary fat compared to a high-starch ration could promote muscular adaptations that would enable greater fat utilization during exercise. Seventeen Quarter Horse yearlings were assigned a treatment diet of FAT or STARCH. Body weights and skeletal measurements were taken and a total fecal collection was performed to evaluate growth patterns and nutrient digestibility. Blood and muscle was evaluated to determine if the capacity to transport fatty acid substrates from the blood to the myocyte can be increased. The SAS/MIXED procedure was used for the analyses at P<0.05 significance. FAT horses had improved digestibility of crude fat, crude protein and calcium. STARCH horses had heavier body weights and higher plasma betahydroxybutyrate concentrations. FAT horses had higher fatty acid translocase (FAT/CD36) protein expression in the myocyte suggesting that 10% fat supplementation for growing horses subjected to aerobic conditioning could potentially enhance the capacity for transport of fatty acid substrates.Department of Animal Scienc

Prospective Power Calculations for the Four Lab Study of A Multigenerational Reproductive/Developmental Toxicity Rodent Bioassay Using A Complex Mixture of Disinfection By-Products in the Low-Response Region

In complex mixture toxicology, there is growing emphasis on testing environmentally representative doses that improve the relevance of results for health risk assessment, but are typically much lower than those used in traditional toxicology studies. Traditional experimental designs with typical sample sizes may have insufficient statistical power to detect effects caused by environmentally relevant doses. Proper study design, with adequate statistical power, is critical to ensuring that experimental results are useful for environmental health risk assessment. Studies with environmentally realistic complex mixtures have practical constraints on sample concentration factor and sample volume as well as the number of animals that can be accommodated. This article describes methodology for calculation of statistical power for non-independent observations for a multigenerational rodent reproductive/developmental bioassay. The use of the methodology is illustrated using the U.S. EPA’s Four Lab study in which rodents were exposed to chlorinated water concentrates containing complex mixtures of drinking water disinfection by-products. Possible experimental designs included two single-block designs and a two-block design. Considering the possible study designs and constraints, a design of two blocks of 100 females with a 40:60 ratio of control:treated animals and a significance level of 0.05 yielded maximum prospective power (~90%) to detect pup weight decreases, while providing the most power to detect increased prenatal loss

An In Silico Approach for Evaluating a Fraction-Based, Risk Assessment Method for Total Petroleum Hydrocarbon Mixtures

Both the Massachusetts Department of Environmental Protection (MADEP) and the Total Petroleum Hydrocarbon Criteria Working Group (TPHCWG) developed fraction-based approaches for assessing human health risks posed by total petroleum hydrocarbon (TPH) mixtures in the environment. Both organizations defined TPH fractions based on their expected environmental fate and by analytical chemical methods. They derived toxicity values for selected compounds within each fraction and used these as surrogates to assess hazard or risk of exposure to the whole fractions. Membership in a TPH fraction is generally defined by the number of carbon atoms in a compound and by a compound's equivalent carbon (EC) number index, which can predict its environmental fate. Here, we systematically and objectively re-evaluate the assignment of TPH to specific fractions using comparative molecular field analysis and hierarchical clustering. The approach is transparent and reproducible, reducing inherent reliance on judgment when toxicity information is limited. Our evaluation of membership in these fractions is highly consistent (˜80% on average across various fractions) with the empirical approach of MADEP and TPHCWG. Furthermore, the results support the general methodology of mixture risk assessment to assess both cancer and noncancer risk values after the application of fractionation

Examining the feasibility of mixture risk assessment: A case study using a tiered approach with data of 67 pesticides from the Joint FAO/WHO Meeting on Pesticide Residues (JMPR)

The way in which mixture risk assessment (MRA) should be included in chemical risk assessment is a current topic of debate. We used data from 67 recent pesticide evaluations to build a case study using Hazard Index calculations to form risk estimates in a tiered MRA approach in line with a Framework proposed by WHO/IPCS. The case study is used to illustrate the approach and to add detail to the existing Framework, and includes many more chemicals than previous case studies.A low-tier MRA identified risk as being greater than acceptable, but refining risk estimates in higher tiers was not possible due to data requirements not being readily met. Our analysis identifies data requirements, which typically expand dramatically in higher tiers, as being the likely cause for an MRA to fail in many realistic cases. This forms a major obstacle to routine implementation of MRA and shows the need for systematic generation and collection of toxicological data. In low tiers, hazard quotient inspection identifies chemicals that contribute most to the HI value and thus require attention if further refinement is needed. Implementing MRA requires consensus on issues such as scope setting, criteria for performing refinement, and decision criteria for actions.Oak Foundation (Grant number OCAY-13-391), which is gratefully acknowledged, and partly in the context of an European Food Safety Authority contract (CFT/EFSA/PPR/2010/04

Scanned Probe Oxidation onp-GaAs(100) Surface with an Atomic Force Microscopy

Locally anodic oxidation has been performed to fabricate the nanoscale oxide structures onp-GaAs(100) surface, by using an atomic force microscopy (AFM) with the conventional and carbon nanotube (CNT)-attached probes. The results can be utilized to fabricate the oxide nanodots under ambient conditions in noncontact mode. To investigate the conversion of GaAs to oxides, micro-Auger analysis was employed to analyze the chemical compositions. The growth kinetics and the associated mechanism of the oxide nanodots were studied under DC voltages. With the CNT-attached probe the initial growth rate of oxide nanodots is in the order of ~300 nm/s, which is ~15 times larger than that obtained by using the conventional one. The oxide nanodots cease to grow practically as the electric field strength is reduced to the threshold value of ~2 × 107 V cm−1. In addition, results indicate that the height of oxide nanodots is significantly enhanced with an AC voltage for both types of probes. The influence of the AC voltages on controlling the dynamics of the AFM-induced nanooxidation is discussed

Non-Chemical Stressors and Cumulative Risk Assessment: An Overview of Current Initiatives and Potential Air Pollutant Interactions

Regulatory agencies are under increased pressure to consider broader public health concerns that extend to multiple pollutant exposures, multiple exposure pathways, and vulnerable populations. Specifically, cumulative risk assessment initiatives have stressed the importance of considering both chemical and non-chemical stressors, such as socioeconomic status (SES) and related psychosocial stress, in evaluating health risks. The integration of non-chemical stressors into a cumulative risk assessment framework has been largely driven by evidence of health disparities across different segments of society that may also bear a disproportionate risk from chemical exposures. This review will discuss current efforts to advance the field of cumulative risk assessment, highlighting some of the major challenges, discussed within the construct of the traditional risk assessment paradigm. Additionally, we present a summary of studies of potential interactions between social stressors and air pollutants on health as an example of current research that supports the incorporation of non-chemical stressors into risk assessment. The results from these studies, while suggestive of possible interactions, are mixed and hindered by inconsistent application of social stress indicators. Overall, while there have been significant advances, further developments across all of the risk assessment stages (i.e., hazard identification, exposure assessment, dose-response, and risk characterization) are necessary to provide a scientific basis for regulatory actions and effective community interventions, particularly when considering non-chemical stressors. A better understanding of the biological underpinnings of social stress on disease and implications for chemical-based dose-response relationships is needed. Furthermore, when considering non-chemical stressors, an appropriate metric, or series of metrics, for risk characterization is also needed. Cumulative risk assessment research will benefit from coordination of information from several different scientific disciplines, including, for example, toxicology, epidemiology, nutrition, neurotoxicology, and the social sciences

International Frameworks Dealing with Human Risk Assessment of Combined Exposure to Multiple Chemicals

The development of harmonised terminology and frameworks for the human risk assessment of combined exposure to multiple chemicals (“chemical mixtures”) is an important area for EFSA and a number of activities have already been undertaken, i.e. in the fields of pesticides and contaminants. The first step prior to a risk assessment of combined exposure to multiple chemicals is problem formulation defining the relevant exposure, hazard and population to be considered. In practice, risk assessment of multiple chemicals is conducted using a tiered approach for exposure assessment, hazard assessment and risk characterisation. Higher tiers require increasing knowledge about the group of chemicals under assessment and the tiers can range from tier 0 (default values, data poor situation) to tier 3 (full probabilistic models). This scientific report reviews the terminology, methodologies and frameworks developed by national and international agencies for the human risk assessment of combined exposure to multiple chemicals and provides recommendations for future activities at EFSA in this area