Accelerator mass spectrometry as a bioanalytical tool for nutritional research

Accelerator Mass Spectrometry is a mass spectrometric method of detecting long-lived radioisotopes without regard to their decay products or half-life. The technique is normally applied to geochronology, but recently has been developed for bioanalytical tracing. AMS detects isotope concentrations to parts per quadrillion, quantifying labeled biochemicals to attomole levels in milligram- sized samples. Its advantages over non-isotopeic and stable isotope labeling methods are reviewed and examples of analytical integrity, sensitivity, specificity, and applicability are provided

Technology Assessment and Roadmap for the Emergency Radiation Dose Assessment Program

A Joint Interagency Working Group (JIWG) under the auspices of the Department of Homeland Security Office of Research and Development conducted a technology assessment of emergency radiological dose assessment capabilities as part of the overall need for rapid emergency medical response in the event of a radiological terrorist event in the United States. The goal of the evaluation is to identify gaps and recommend general research and development needs to better prepare the Country for mitigating the effects of such an event. Given the capabilities and roles for responding to a radiological event extend across many agencies, a consensus of gaps and suggested development plans was a major goal of this evaluation and road-mapping effort. The working group consisted of experts representing the Departments of Homeland Security, Health and Human Services (Centers for Disease Control and the National Institutes of Health), Food and Drug Administration, Department of Defense and the Department of Energy's National Laboratories (see appendix A for participants). The specific goals of this Technology Assessment and Roadmap were to: (1) Describe the general context for deployment of emergency radiation dose assessment tools following terrorist use of a radiological or nuclear device; (2) Assess current and emerging dose assessment technologies; and (3) Put forward a consensus high-level technology roadmap for interagency research and development in this area. This report provides a summary of the consensus of needs, gaps and recommendations for a research program in the area of radiation dosimetry for early response, followed by a summary of the technologies available and on the near-term horizon. We then present a roadmap for a research program to bring present and emerging near-term technologies to bear on the gaps in radiation dose assessment and triage. Finally we present detailed supporting discussion on the nature of the threats we considered, the status of technology today, promising emerging technologies and references for further reading

Statistical Analysis of Variation in the Human Plasma Proteome

Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where one human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery

Pathomics: Final Report

Pathomics is a research project to explore the feasibility for developing biosignatures for early infectious disease detection in humans, particularly those that represent a threat from bioterrorism. Our goal is to use a science-based approach to better understand the underlying molecular basis of disease and to find sensitive, robust, and specific combinations of biological molecules (biosignatures) in the host that will indicate the presence of developing infection prior to overt symptoms (pre-syndromic). The ultimate goal is develop a national surveillance system for monitoring for the release and managing the consequences of a biothreat agent or an emerging disease. Developing the science for a more comprehensive understanding of the molecular basis of infectious disease and the development of biosignature-based diagnostics could help detect both emerging and engineered treats to humans

A Novel 14C-Postlabeling Assay Using Accelerator Mass Spectrometry For the Detection of O6-Methyldeoxyguanosine Adducts

Accelerator mass spectrometry (AMS) is currently one of the most sensitive methods available for the trace detection of DNA adducts and is particularly valuable for measuring adducts in humans or animal models. However, the standard approach requires administration of a radiolabeled compound. As an alternative, we have developed a preliminary {sup 14}C-postlabeling assay for detection of the highly mutagenic O{sup 6}-MedG, by AMS. Procedures were developed for derivatizing O{sup 6}-MedG using unlabeled acetic anhydride. Using conventional LC-MS analysis, the limit of detection for the major product, triacetylated O{sup 6}-MedG, was 10 fmoles. On reaction with {sup 14}C-acetic anhydride, using a specially designed enclosed system, the predominant product was {sup 14}C-di-acetyl O{sup 6}-MedG. This change in reaction profile was due to a modification of the reaction procedure, introduced as a necessary safety precaution. The limit of detection for {sup 14}C-diacetyl O{sup 6}-MedG by AMS was determined as 79 attomoles, {approx}18,000 fold lower than that achievable by LSC. Although the assay has so far only been carried out with labeled standards, the degree of sensitivity obtained illustrates the potential of this assay for measuring O{sup 6}-MedG levels in humans

Accelerator Mass Spectrometry Allows for Cellular Quantification of Doxorubicin at Femtomolar Concentrations

Accelerator mass spectrometry (AMS) is a highly sensitive analytical methodology used to quantify the content of radioisotopes, such as {sup 14}C, in a sample. The primary goals of this work were to demonstrate the utility of AMS in determining cellular [{sup 14}C]doxorubicin (DOX) concentrations and to develop a sensitive assay that is superior to high performance liquid chromatography (HPLC) for the quantification of DOX at the tumor level. In order to validate the superior sensitivity of AMS versus HPLC with fluorescence detection, we performed three studies comparing the cellular accumulation of DOX: one in vitro cell line study, and two in vivo xenograft mouse studies. Using AMS, we quantified cellular DOX content up to 4 hours following in vitro exposure at concentrations ranging from 0.2 pg/ml (345 fM) to 2 {micro}g/ml (3.45 {micro}M) [{sup 14}C]DOX. The results of this study show that, compared to standard fluorescence-based HPLC, the AMS method was over five orders of magnitude more sensitive. Two in vivo studies compared the sensitivity of AMS to HPLC using a nude mouse xenograft model in which breast cancer cells were implanted subcutaneously. After sufficiently large tumors formed, DOX was administered intravenously at two dose levels. Additionally, we tested the AMS method in a nude mouse xenograft model of multidrug resistance (MDR) in which each mouse was implanted with both wild type and MDR+ cells on opposite flanks. The results of the second and third studies showed that DOX concentrations were significantly higher in the wild type tumors compared to the MDR+ tumors, consistent with the MDR model. The extreme sensitivity of AMS should facilitate similar studies in humans to establish target site drug delivery and to potentially determine the optimal treatment dose and regimen

Characterizatio o a peptide adduct fouct by N

AminoRIRIoIRIooI8R--I88RI888I b]pyridine (PhIP) is a membero a classo cossI0;q knos as theheteroNIH38 amines (HCAs) that arefo8RA in meat duringcoingIM It is amulti-o3RM carcino3R inro0AR-- fo0A adducts and with DNA and proI;33 Altho33 pro33 adducts are no tho8qq to beinvoq3M in cancerdevelo0R;3I they may be useful as internaldornalI;R o PhIPexpoAM8 andbioM;N8IH0qRM TooM; thegoIM o characterizing the adductsfouct in humans and thedevelo0IH0 o an assayfo quantitatio o adduct levels, we have characterized a peptide adduct fouct by the putative genoiveI metaboI33; N - aceto0IH0N--0 AmoN8 peptide with the internal sequenceLeu--Gln--Lys--Cys--ProIo-- which isho830MIH0 to apoqMNRIH target sequencefo HCAs in human serum albumin, was reacted with N-acetoH03A0N and an adduct was identified and further characterized by LC--ESI-MS/MS