DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS.

Autocrine inhibition of the c-fms proto-oncogene reduces breast cancer bone metastasis assessed with in vivo dual-modality imaging.

Breast cancer cells preferentially home to the bone microenvironment, which provides a unique niche with a network of multiple bidirectional communications between host and tumor, promoting survival and growth of bone metastases. In the bone microenvironment, the c-fms proto-oncogene that encodes for the CSF-1 receptor, along with CSF-1, serves as one critical cytokine/receptor pair, functioning in paracrine and autocrine fashion. Previous studies concentrated on the effect of inhibition of host (mouse) c-fms on bone metastasis, with resulting decrease in osteolysis and bone metastases as a paracrine effect.

Analytical validation of accelerator mass spectrometry for pharmaceutical development.

The validation parameters for pharmaceutical analyses were examined for the accelerator mass spectrometry measurement of (14)C/C ratio, independent of chemical separation procedures. The isotope ratio measurement was specific (owing to the (14)C label), stable across samples storage conditions for at least 1 year, linear over four orders of magnitude with an analytical range from 0.1 Modern to at least 2000 Modern (instrument specific).

Accelerator mass spectrometry best practices for accuracy and precision in bioanalytical (14)C measurements.

Accelerator mass spectrometers have an energy acceleration and charge exchange between mass definition stages to destroy molecular isobars and allow single ion counting of long-lived isotopes such as (14)C (t½=5370 years.). 'Low' voltage accelerations to 200 kV allow laboratory-sized accelerator mass spectrometers instruments for bioanalytical quantitation of (14)C to 2-3% precision and accuracy in isolated biochemical fractions.

Early human ADME using microdoses and microtracers: bioanalytical considerations.

Quantitative assessment of metabolites of drug candidates in early-phase clinical development presents an analytical challenge when methods, standards and assays are not yet available. Radioisotopic labeling, principally with radiocarbon ((14)C), is the preferred method for discovering and quantifying the absolute yields of metabolites in the absence of reference material or a priori knowledge of the human metabolism. However, the detection of (14)C is inefficient by decay counting methods and, as a result, high radiological human (14)C-doses had been needed to assure sensitive detection of metabolites over time.

Quantifying exploratory low dose compounds in humans with AMS.

Accelerator Mass Spectrometry is an established technology whose essentiality extends beyond simply a better detector for radiolabeled molecules. Attomole sensitivity reduces radioisotope exposures in clinical subjects to the point that no population need be excluded from clinical study. Insights in human physiochemistry are enabled by the quantitative recovery of simplified AMS processes that provide biological concentrations of all labeled metabolites and total compound related material at non-saturating levels.

Identification of the RLBP1 gene promoter.

Purpose: Cellular retinaldehyde-binding protein (CRALBP), transcribed from the RLBP1 gene, is a 36-kDa water-soluble protein with 316 amino acids found in the retinal pigment epithelium (RPE) and in retinal Müller cells. It is thought to play a critical role in the visual cycle by functioning as an acceptor of 11-cis-retinol from the isomerohydrolase reaction. The goal here was to evaluate the functional promoter of this gene.

A common 1317TC polymorphism in MTHFR can lead to erroneous 1298AC genotyping by PCR-RE and TaqMan probe assays.

Multiple polymorphisms of the methylenetetrahydrofolate reductase gene (MTHFR) have been documented, and some are associated with decreased enzyme activity. One polymorphism, 677CT, is commonly tested in the context of thrombosis. Recently, consideration has also been extended to 1298AC, which is also associated with reduced catalytic activity.

Accelerator mass spectrometry for quantitative in vivo tracing.

Accelerator mass spectrometry (AMS) counts individual rare, usually radio-, isotopes such as radiocarbon at high efficiency and specificity in milligram-sized samples. AMS traces very low chemical doses (micrograms) and radiative doses (100 Bq) of isotope-labeled compounds in animal models and directly in humans for pharmaceutical, nutritional, or toxicological research. Absorption, metabolism, distribution, binding, and elimination are all quantifiable with high precision after appropriate sample definition.

Quantitating isotopic molecular labels with accelerator mass spectrometry.

Accelerator mass spectrometry (AMS) traces isotopically labeled biochemicals and provides significant new directions for understanding molecular kinetics and dynamics in biological systems. AMS traces low-abundance radioisotopes for high specificity but detects them with MS for high sensitivity. AMS reduces radiation exposure doses to levels safe for use in human volunteers of all ages.

41Ca and accelerator mass spectrometry to monitor calcium metabolism in end stage renal disease patients.

Background: Monitoring bone resorption with measurements of bone density and biochemical markers is indirect. We hypothesized that bone resorption can be studied directly by serial measurements of the ratio (41)Ca/Ca in serum after in vivo labeling of calcium pools with (41)Ca. We report the preparation of an intravenous (41)Ca dose suitable for humans, an analytical method for determining (41)Ca/Ca isotope ratios in biological samples, and studies in human volunteers.

Neuroscience and accelerator mass spectrometry.

Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying rare isotopes. It has had a great impact in geochronology and archaeology and is now being applied in biomedicine. AMS measures radioisotopes such as 3H, 14C, 26Al, 36Cl and 41Ca, with zepto- or attomole sensitivity and high precision and throughput, allowing safe human pharmacokinetic studies involving microgram doses, agents having low bioavailability or toxicology studies where administered doses must be kept low (<1 microg kg(-1)).

Mass by energy loss quantitation as a practical submicrogram balance.

A simple device integrating a thin film support and a standard microcentrifuge tube can be used for making solutions of accurately known concentration of any organic compound in a single step, avoiding serial dilution and the use of microgram balances. Nanogram to microgram quantities of organic material deposited on the thin film are quantified by ion energy loss and transferred to the microcentrifuge tube with high recovery.

Quantitation of binding, recovery and desalting efficiency of peptides and proteins in solid phase extraction micropipette tips.

Micropipette-tip solid phase extraction (SPE) systems are common in proteomic analyses for desalting and concentrating samples for mass spectrometry, removing interferences, and increasing sensitivity. These systems are inexpensive, disposable, and highly efficient. Here, we show micropipette-tip solid phase extraction is a direct sample preparation method for (14)C-accelerator mass spectrometry (AMS), removing salts or reagent from labeled macromolecules.

In vivo release from a drug delivery MEMS device.

A drug delivery microelectromechanical systems (MEMS) device was designed to release complex profiles of multiple substances in order to maximize the effectiveness of drug therapies. The device is based on micro-reservoirs etched into a silicon substrate that contain individual doses of drug. Each dose is released by the electrochemical dissolution of the gold membrane that covers the reservoir.

DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

A protocol is described for the isolation of DNA and subsequent preparation of samples for the measurement of adduct levels by accelerator mass spectrometry (AMS). AMS is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. However, special precautions must be taken to avoid cross-contamination of isotope between samples and to produce a sample that is compatible with AMS.

Quantitation of in vivo human folate metabolism.

Background: A quantitative understanding of human folate metabolism is needed.

Objective: The objective was to quantify and interpret human folate metabolism as it might occur in vivo.

Design: Adults (n = 13) received 0.

Evaluation of microdosing strategies for studies in preclinical drug development: demonstration of linear pharmacokinetics in dogs of a nucleoside analog over a 50-fold dose range.

The technique of accelerator mass spectrometry (AMS) was validated successfully and used to study the pharmacokinetics and disposition in dogs of a preclinical drug candidate (7-deaza-2'-C-methyl-adenosine; Compound A), after oral and intravenous administration. The primary objective of this study was to examine whether Compound A displayed linear kinetics across subpharmacological (microdose) and pharmacological dose ranges in an animal model, before initiation of a human microdose study. The AMS-derived disposition properties of Compound A were comparable to data obtained via conventional techniques such as liquid chromatography-tandem mass spectrometry and liquid scintillation counting analyses.

Alpha-particle energy loss measurement of microgram depositions of biomolecules.

A commercially available alpha-particle spectrometer and 210Po alpha-particle source were used to determine the mass of microgram quantities of biomolecules. Samples were deposited in microliter volumes on thin silicon nitride windows and dried. The energy loss of the alpha-particles after traversing the sample was converted to a mass using tabulated alpha-particle stopping powers.

Shrinkage estimation for functional principal component scores with application to the population kinetics of plasma folate.

We present the application of a nonparametric method to performing functional principal component analysis for functional curve data that consist of measurements of a random trajectory for a sample of subjects. This design typically consists of an irregular grid of time points on which repeated measurements are taken for a number of subjects. We introduce shrinkage estimates for the functional principal component scores that serve as the random effects in the model.

Absorption and retinol equivalence of beta-carotene in humans is influenced by dietary vitamin A intake.

The effect of vitamin A supplements on metabolic behavior of an oral tracer dose of [14C]beta-carotene was investigated in a longitudinal test-retest design in two adults. For the test, each subject ingested 1 nmol of [14C]beta-carotene (100 nCi) in an emulsified olive oil-banana drink. Total urine and stool were collected for up to 30 days; concentration-time patterns of [14C]beta-carotene, [14C]retinyl esters, and [14C]retinol were determined for 46 days.

A high-throughput method for the conversion of CO2 obtained from biochemical samples to graphite in septa-sealed vials for quantification of 14C via accelerator mass spectrometry.

The growth of accelerator mass spectrometry as a tool for quantitative isotope ratio analysis in the biosciences necessitates high-throughput sample preparation. A method has been developed to convert CO(2) obtained from carbonaceous samples to solid graphite for highly sensitive and precise (14)C quantification. Septa-sealed vials are used along with commercially available disposable materials, eliminating sample cross contamination, minimizing complex handling, and keeping per sample costs low.

Protein binding of isofluorophate in vivo after coexposure to multiple chemicals.

Full toxicologic profiles of chemical mixtures, including dose-response extrapolations to realistic exposures, is a prohibitive analytical problem, even for a restricted class of chemicals. We present an approach to probing in vivo interactions of pesticide mixtures at relevant low doses using a monitor compound to report the response of biochemical pathways shared by mixture components. We use accelerator mass spectrometry (AMS) to quantify [14C]-diisopropylfluorophosphate as a tracer at attomole levels with 1-5% precision after coexposures to parathion (PTN), permethrin (PER), and pyridostigmine bromide separately and in conjunction.

Semiparametric modeling of labeled-cell kinetics, with application to isotope labeling of erythrocytes.

We propose a stochastic model for the kinetics of cells that have been tagged with a chemical label. The proposed model consists of two components: a parametrically specified distribution for the time to incorporation of the label into the cells and a nonparametric survival function reflecting the survival time of the label-cell combination. The target quantity of this modeling approach is the fraction of labeled cells among all cells, viewed as a function of time.