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Publications
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Absolute quantification without internal standards: Accelerator Mass Spectrometry (BioMICADAS) and microtracers for pharmacokinetics, metabolite discovery and mass balance
Publish date: Sep 01, 2009
Author: Stephen Dueker, Pete Lohstroh, Jason Giacomo, Saira Abidi, Dana Pan, Brad Keck and John Vogel
Metabolites can be both mediators of drug toxicity and efficacy and recent
FDA Guidance for Industry strongly recommends that studies leading to in vivo drug metabolism in humans be performed as early as feasible. In this light, a complete accounting of significant metabolite(s) in both safety species and humans during clinical testing is a high priority. The quantitative sensitivity of 14C-Accelerator Mass Spectrometry (AMS) empowers early human metabolism studies to be conducted within Phase I trials with microtrace (nCi) quantities of administered 14C-labled drug. Direct atom counting with AMS can reveal the full complement of metabolites with no requirement for internal standards and
quantifies dose recovery into excreta (mass balance) with a high degree of
reproducibility. These measurements facilitate informed decision-making in and around Phase 1 studies with incremental added cost to the program using trace radiolabel.
Presented at APA Boston 2009.
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Use of Accelerator Mass Spectrometry to Measure the Pharmacokinetics and Peripheral Blood Mononuclear Cell Concentrations of Zidovudine
Publish date: Jul 01, 2007
Author: Le T. Vuong, Jon L. Ruckle, Arlin B. Blood, Michael J. Reid, Richard D. Wasnich, Hans-Arno Synal, Stephen R. Dueker
Source: Journal Of Pharmaceutical Sciences
The remarkable sensitivity of accelerator mass spectrometry (AMS) is
finding many new applications in pharmacology. In this study AMS was used to
measure [14C]-Zidovudine (ZDV) concentrations at the drug's site of action (peripheral
blood mononuclear cells, PBMCs) following a dose of 520 ng (less than one-millionth of
the standard daily dose) to a healthy volunteer. In addition, the pharmacokinetics of
this microdose were determined and compared to previously published parameters for
therapeutic doses. Microdose ZDV pharmacokinetic parameters fell within reported 95%
confidence intervals or standard deviations of most previously published values for
therapeutic doses. Blood, urine, stool, saliva, and isolated PBMCs were collected
periodically through 96 h postdose and analyzed for ZDV and metabolite concentrations.
The results showed that ZDV is rapidly absorbed and eliminated, has one major
metabolite, and is sequestered in PBMCs. 14C mass balance assessments indicated a
significant portion of ZDV remained after 96 h with a much prolonged elimination half-
life. Results of this study demonstrate the usefulness of microdosing and AMS as a tool
for studying the pharmacokinetic characteristics, including PBMC concentrations, of
ZDV and underscore the value of AMS as a tool with which to perform pharmacokinetic
and mass balance studies using trace amounts of radiolabeled compound.
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Disposition of 14C-β-carotene following delivery with autologous triacylglyceride-rich lipoproteins
Publish date: Feb 03, 2007
Author: Stephen R. Dueker, Le Thuy Vuong, Brian Faulkner, Bruce A. Buchholz, John S. Vogel
Source: Nuclear Instruments and Methods in Physics Research B 259 (2007) 767–772
Following ingestion, a fraction of β-carotene is cleaved into vitamin A in the intestine, while another is absorbed intact and distributed
among tissues and organs. The extent to which this absorbed β-carotene serves as a source of vitamin A is unknown in vivo. In the
present study we use the attomole sensitivity of accelerator mass spectrometry (AMS) for 14C to quantify the disposition of 14C-β-carotene
(930 ng; 60.4 nCi of activity) after intravenous injection with an autologous triacylglyceride-rich lipoprotein fraction in a single
volunteer. Total 14C was quantified in serial plasma samples and also in triglyceride-rich, and low density lipoprotein, subfractions.
The appearance of 14C-retinol, the circulating form of vitamin A in plasma, was determined by chromatographic separation of plasma
retinol extracts prior to AMS analysis. The data showed that 14C concentrations rapidly decayed within the triglyceride-rich lipoprotein
fractions after injection, whereas low density lipoprotein 14C began a significant rise in 14C 5 h post dose. Plasma 14C-retinol also
appeared at 5 h post dose and its concentrations were maintained above baseline for >88 days. Based upon comparisons of 14C-retinol
concentrations following an earlier study with orally dosed 14C-β-carotene, a molar vitamin A value of the absorbed β-carotene of 0.19
was derived, meaning that 1 mole of absorbed β-carotene provides 0.19 moles of vitamin A. This is the first study to show that infused
β-carotene contributes to the vitamin A economy in humans in vivo.
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Quantitating Isotopic Molecular Labels with Accelerator Mass Spectrometry
Publish date: Jan 07, 2006
Author: JOHN S. VOGEL and ADAM H. LOVE
Source: Methods in Enzymology 2005;402:402-22.
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. Total radiation exposures are equivalent to those obtained in very short airplane flights, a commonly accepted radiation risk. Waste products seldom reach the Nuclear Regulatory Commission (NRC) definition of radioactive waste material for (14)C and (3)H. Attomoles of labeled compounds are quantified in milligram-sized samples, such as 20 microl of blood. AMS is available from several facilities that offer services and new spectrometers that are affordable. Detailed examples of designing AMS studies are provided, and the methods of analyzing AMS data are outlined.
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Neuroscience and accelerator mass spectrometry
Publish date: Feb 01, 2005
Author: Magnus Palmblad, Bruce A. Buchholz, Darren J. Hillegonds and John S. Vogel
Source: J Mass Spectrom. 2005 Feb;40(2):154-9.
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)). It is used to study long-term pharmacokinetics, to identify biomolecular interactions, to determine chronic and low-dose effects or molecular targets of neurotoxic substances, to quantify transport across the blood-brain barrier and to resolve molecular turnover rates in the human brain on the time-scale of decades. We review here how AMS is applied in neurotoxicology and neuroscience.
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Traditional and accelerator mass spectrometry for quantitation of human folate pools
Publish date: 2005
Author: Stephen R. Dueker, Michael Lamé, Yumei Lin, Andrew J. Clifford, Bruce A. Buchholz and John S. Vogel
Source: Trends in Food Science & Technology 16 (2005) 267-270
Folate is a structurally diverse family of molecules that occur
in low concentrations in biological tissues and fluids and
therefore present complex challenges to the analytical
chemist. As a result, techniques that quantify folates in
biological specimens have emerged as a high scientific
priority. In this manuscript, isotopic methods using traditional
mass spectrometry for the measurement of whole
blood total folate and 14C-accelerator mass spectrometry
(AMS) for human kinetic studies are presented. Additionally,
recent developments with the intrinsic labeling of spinach
folate from a [14C]-para-aminobenzoic is discussed.
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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
Publish date: Nov 01, 2004
Author: Punam Sandhu, John S. Vogel, Mark J. Rose, Esther A. Ubick, Janice E. Brunner, Michael A. Wallace, Jennifer K. Adelsberger, Maribeth P. Baker, Paul T. Henderson, Paul G. Pearson, and Thomas A. Baillie
Source: Drug Metab Dispos. 2004 Nov;32(11):1254-9. Epub 2004 Jul 30.
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. Compound A displayed multiphasic kinetics and exhibited low plasma clearance (5.8 ml/min/kg), a long terminal elimination half-life (17.5 h), and high oral bioavailability (103%). Currently, there are no published comparisons of the kinetics of a pharmaceutical compound at pharmacological versus subpharmacological doses using microdosing strategies. The present study thus provides the first description of the full pharmacokinetic profile of a drug candidate assessed under these two dosing regimens. The data demonstrated that the pharmacokinetic properties of Compound A following dosing at 0.02 mg/kg were similar to those at 1 mg/kg, indicating that in the case of Compound A, the pharmacokinetics in the dog appear to be linear across this 50-fold dose range. Moreover, the exceptional sensitivity of AMS provided a pharmacokinetic profile of Compound A, even after a microdose, which revealed aspects of the disposition of this agent that were inaccessible by conventional techniques.
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Phytochemical Research Using Accelerator Mass Spectrometry
Publish date: Oct 01, 2004
Author: Le T. Vuong, PhD, Bruce A. Buchholz, PhD, Michael W. Lame, PhD, and Stephen R. Dueker, PhD
Source: Nutrition Reviews, Vol. 62, No. 10 pp 375 - 388
Vegetables and fruits provide an array of microchemicals in the form of vitamins and secondary metabolites (phytochemicals) that may lower the risk of chronic disease. Tracing these phytochemicals at physiologic concentrations has been hindered by a lack of quantitative sensitivity for chemically equivalent tracers that could be used safely in healthy people. Accelerator mass spectrometry is a relatively new technique that provides the necessary sensitivity (in attomoles) and measurement precision (<3%) towards 14C-labeled phytochemicals for detailed kinetic studies in humans at dietary levels.
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Accelerator Mass Spectrometry in Protein Analysis
Publish date: 2004
Author: John S.Vogel, Darren J. Hillegonds,Magnus Palmblad, Patrick G. Grant and Graham Bench
Source: R M Kamp; Juan J Calvete; T Choli-Papadopoulou, eds. Methods in proteome and protein analysis. Berlin; New York: Springer, ©2004.
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Tamoxifen DNA Damage Detected in Human Endometrium Using Accelerator Mass Spectrometry
Publish date: Dec 01, 2003
Author: Martin EA, Brown K, Gaskell M, Al-Azzawi F, Garner RC, Boocock DJ, Mattock E, Pring DW, Dingley K, Turteltaub KW, Smith LL, White IN.
Source: Cancer Res. 2003 Dec 1;63(23):8461-5.
This study was aimed to establish whether tamoxifen binds irreversibly to uterine DNA when given to women. Patients were given a single therapeutic dose of [(14)C]tamoxifen citrate orally (20 mg, 0.37 or 1.85 MBq) approximately 18 h prior to hysterectomy or breast surgery. Nonmalignant uterine tissue was separated into myometrium and endometrium. DNA and protein were isolated and bound radiolabel determined by the sensitive technique of accelerator mass spectrometry. Levels of irreversible DNA binding of tamoxifen in the endometrium of treated patients were 237 +/- 77 adducts/10(12) nucleotides (mean +/- SE, n = 10). In myometrial tissues, a similar extent of DNA binding was detected (492 +/- 112 adducts/10(12) nucleotides). Binding of tamoxifen to endometrial and myometrial proteins was 10 +/- 3 and 20 +/- 4 fmol/mg, respectively. In breast tissue, sufficient DNA could not be extracted but protein binding was an order of magnitude higher than that seen with endometrial proteins (358 +/- 81 fmol/mg). These results demonstrate that after oral administration, tamoxifen forms adducts in human uterine DNA but at low numbers relative to those previously reported in women after long-term tamoxifen treatment where levels, when detected, ranged from 15000 to 130000 adducts/10(12) nucleotides. Our findings support the hypothesis that the low level of DNA adducts in human uterus is unlikely to be involved with endometrial cancer development.
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Protein Binding of Isofluorophate in Vivo after Coexposure to Multiple Chemicals
Publish date: Dec 01, 2002
Author: John S. Vogel, Garrett A. Keating II, and Bruce A. Buchholz
Source: Environ Health Perspect 110(suppl 6):1031–1036 (2002)
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. Pyridostigmine shows an overall protective effect against tracer binding in plasma, red blood cells, muscle, and brain that is not explained as competitive protein binding. PTN and PER induce a significant 25–30% increase in the amount of tracer reaching the brain with or without pyridostigmine. The sensitivity of AMS for isotope-labeled tracer compounds can be used to probe the physiologic responses of specific biochemical pathways to multiple compound exposures.
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Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry
Publish date: Apr 10, 2001
Author: Masahiro Miyashita, Jack M. Presley, Bruce A. Buchholz, Kit S. Lam, Young Moo Lee, John S. Vogel, and Bruce D. Hammock
Source: Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4403-8. Epub 2001 Apr 3.
Edman degradation remains the primary method for determining the sequence of proteins. In this study, accelerator mass spectrometry was used to determine the N-terminal sequence of glutathione S-transferase at the attomole level with zeptomole precision using a tracer of (14)C. The transgenic transferase was labeled by growing transformed Escherichia coli on [(14)C]glucose and purified by microaffinity chromatography. An internal standard of peptides on a solid phase synthesized to release approximately equal amounts of all known amino acids with each cycle were found to increase yield of gas phase sequencing reactions and subsequent semimicrobore HPLC as did a lactoglobulin carrier. This method is applicable to the sequencing of proteins from cell culture and illustrates a path to more general methods for determining N-terminal sequences with high sensitivity.
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Isotope-labeled immunoassays without radiation waste
Publish date: Mar 14, 2000
Author: Guomin Shan, Wei Huang, Shirley J. Gee, Bruce A. Buchholz, John S. Vogel, and Bruce D. Hammock
Source: Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2445-9.
The practice of immunoassay has experienced a widespread transition from radioisotopic labeling to nonisotopic labeling over the last two decades. Radioisotope labels have drawbacks that hamper their applications: (i) perceived radiation hazards of reagents, (ii) regulatory requirements and disposal problems of working with radioactive materials, and (iii) short shelf-life of the labeled reagents. The advantage of isotopic labeling is the incorporation into analytes without altering structure or reactivity, as is often the case with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay with the long-life isotope (14)C as the label and accelerator mass spectrometer (AMS) as the detection system. AMS quantifies attomole levels of several isotopes, including (14)C. With this exquisite sensitivity, the sensitivity of an immunoassay is limited by the K(d) of the antibody and not the detection system. The detection limit of the assays for atrazine and 2,3,7,8-tetrachlorodibenzo-p-dioxin was 2.0 x 10(-10) M and 2.0 x 10(-11) M, respectively, approximately an order of magnitude below the standard enzyme immunoassay. Notably, <1 dpm (0.45 pCi) of (14)C-labeled compound was used in each assay, which is well below the limit of disposal (50 nCi per g) as nonradioactive waste. Thus, endogenous reporter ligands quantified by AMS provide the advantages of an RIA without the associated problems of radioactive waste.
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Methods and applications of HPLC-AMS
Publish date: 2000
Author: Bruce A. Buchholz, Stephen R. Dueker, Yumei Lin, Andrew J. Clifford, John S. Vogel
Source: Nuclear Instruments and Methods in Physics Research B 172 (2000) 910-914
Pharmacokinetics of physiologic doses of nutrients, pesticides, and herbicides can easily be traced in humans using a
14C-labeled compound. Basic kinetics can be monitored in blood or urine by measuring the elevation in the 14C content
above the control predose tissue and converting to equivalents of the parent compound. High performance liquid
chromatography (HPLC) is an excellent method for the chemical separation of complex mixtures whose profles afford
estimation of biochemical pathways of metabolism. Compounds elute from the HPLC systems with characteristic
retention times and can be collected in fractions that can then be graphitized for AMS measurement. Unknowns are
tentatively identifed by co-elution with known standards and chemical tests that reveal functional groupings. Metabolites
are quantifed with the 14C signal. Thoroughly accounting for the carbon inventory in the LC solvents, ionpairing
agents, samples, and carriers adds some complexity to the analysis. In most cases the total carbon inventory is
dominated by carrier. Baseline background and stability need to be carefully monitored. Limits of quantitation near
10 amol of 14C per HPLC fraction are typically achieved. Baselines are maintained by limiting injected 14C activity
<0.17 Bq (4.5 pCi) on the HPLC column.
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Dose-dependent binding of trichloroethylene to hepatic DNA and protein at low doses in mice
Publish date: Sep 01, 1997
Author: Kautiainen A, Vogel JS, Turteltaub KW.
Source: Chem Biol Interact. 1997 Sep 12;106(2):109-21.
Trichloroethylene (TCE) is a widely used industrial chemical and a low level contaminant of surface and ground water in industrialized areas. It is weakly mutagenic in several test systems and carcinogenic in rodents. However, the mechanism for its carcinogenicity is not known. We investigated the binding of [1,2-14C]TCE ([14C]TCE) to liver DNA and proteins in male B6C3F1 mice at doses more relevant to humans than used previously. The time course for the binding was studied in animals dosed with 4.1 micrograms [14C]TCE/kg body weight (b.w.) and sacrificed between 0.5 and 120 h after i.p. injection. A dose response study was carried out in mice given [14C]TCE at doses between 2 micrograms/kg and 200 mg/kg b.w. and sacrificed 2 h post-treatment. [14C]TCE associated with the DNA and protein extracts was measured using accelerator mass spectrometry. The highest level of protein binding (2.4 ng/g protein) was observed 1 h after the treatment followed by a rapid decline, indicating pronounced instability of the adducts and/or rapid turnover of liver proteins. DNA binding was biphasic with the first peak (75 pg/g DNA) at 4 h. However, the highest binding (120 pg/g DNA) was found between 24 and 72 h after the treatment. Dose response curves were linear for both protein and DNA binding. The binding of TCE metabolites to DNA was ca. 100-fold lower than to proteins when calculated per unit weight of macromolecules and when measured 2 h post-exposure. This study shows that TCE metabolites bind to DNA and proteins in a dose-dependent manner in liver, one of the target organs for its tumorigenicity. Thus, protein and DNA adduct formation should be considered as a factor in the tumorigenesis of TCE.
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