Neutral resonant ionization in a H⁻ plasma source: Potential of doubly excited **H⁻.

Hydrogen plasmas are optically dense to Lyman-α radiation, maintaining *H(n = 2) neutral atoms that may undergo neutral resonant ionization to **H(-). One state, **H(-)(2p(2) (3)P(e)), is thought bound at 9.7 meV with a several nanosecond lifetime while all others are unbound resonances. Collision dynamics of two *H(2s) shows that an ionic pair of (p, **H(-)) resolves at least three long-standing collision experiments. The doubly excited anion also has a path to the unexcited ion pair whose only physical distinction is that both (p, H(-)) have energy of 3.7 eV.

Anion formation in sputter ion sources by neutral resonant ionization.

Focused Cs(+) beams in sputter ion sources create mm-diameter pits supporting small plasmas that control anionization efficiencies. Sputtering produces overwhelmingly neutral products that the plasma can ionize as in a charge-change vapor. Electron capture between neutral atoms rises as the inverse square of the difference between the ionization potential of the Cs state and the electron affinity of the sputtered atom, allowing resonant ionization at very low energies. A plasma collision-radiation model followed electronic excitation up to Cs(7d). High modeled Cs(7d) in a 0.5 mm recess explains the 80 µA/mm(2) C(-) current density compared to the 20 µA/mm(2) from a 1 mm recess.

Insecticide Transfer Efficiency and Lethal Load in Argentine Ants.

Trophallaxis between individual worker ants and the toxicant load in dead and live Argentine ants (Linepithema humile) in colonies exposed to fipronil and hydramethylnon experimental baits were examined using accelerator mass spectrometry (AMS). About 50% of the content of the crop containing trace levels of 14C-sucrose, 14C-hydramethylnon, and 14C-fipronil was shared between single donor and recipient ants. Dead workers and queens contained significantly more hydramethylnon (122.7 and 22.4 amol/µg ant, respectively) than did live workers and queens (96.3 and 10.4 amol/µg ant, respectively). Dead workers had significantly more fipronil (420.3 amol/µg ant) than did live workers (208.5 amol/µg ant), but dead and live queens had equal fipronil levels (59.5 and 54.3 amol/µg ant, respectively). The distribution of fipronil differed within the bodies of dead and live queens; the highest amounts of fipronil were recovered in the thorax of dead queens whereas live queens had the highest levels in the head. Resurgence of polygynous ant colonies treated with hydramethylnon baits may be explained by queen survival resulting from sublethal doses due to a slowing of trophallaxis throughout the colony. Bait strategies and dose levels for controlling insect pests need to be based on the specific toxicant properties and trophic strategies for targeting the entire colony.

Applications of accelerator MS in pediatric drug evaluation.

Accelerator MS (AMS) provides a novel method for obtaining and analyzing pharmacokinetics and pharmacodynamics in children. This paper reviews the scientific and ethical rationale for AMS in pediatric trials, the regulatory framework and general considerations with some specific examples of pediatric clinical trials using AMS. Microdosing in the context of this article refers to studies using a negligible amount (nanocuries) of (14)C as tracer, and AMS as a quantitative technique. The technology is by no means a panacea for the deficiency in pediatric clinical research; however, it lessens the challenges and provides the most quantitative tool for pediatric pharmacology studies.

Improving tritium exposure reconstructions using accelerator mass spectrometry.

Direct measurement of tritium atoms by accelerator mass spectrometry (AMS) enables rapid low-activity tritium measurements from milligram-sized samples and permits greater ease of sample collection, faster throughput, and increased spatial and/or temporal resolution. Because existing methodologies for quantifying tritium have some significant limitations, the development of tritium AMS has allowed improvements in reconstructing tritium exposure concentrations from environmental measurements and provides an important additional tool in assessing the temporal and spatial distribution of chronic exposure. Tritium exposure reconstructions using AMS were previously demonstrated for a tree growing on known levels of tritiated water and for trees exposed to atmospheric releases of tritiated water vapor. In these analyses, tritium levels were measured from milligram-sized samples with sample preparation times of a few days. Hundreds of samples were analyzed within a few months of sample collection and resulted in the reconstruction of spatial and temporal exposure from tritium releases. Although the current quantification limit of tritium AMS is not adequate to determine natural environmental variations in tritium concentrations, it is expected to be sufficient for studies assessing possible health effects from chronic environmental tritium exposure.

Attomole quantitation of protein separations with accelerator mass spectrometry.

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to subattomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5%. Micro-proton-induced X-ray emission quantifies elemental abundances in separated metalloprotein samples to nanogram amounts and is capable of quantifying phopsphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.

Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry.

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.

Long-term kinetic study of beta-carotene, using accelerator mass spectrometry in an adult volunteer.

We present a sensitive tracer method, suitable for in vivo human research, that uses beta-[(14)C]carotene coupled with accelerator mass spectrometry (AMS) detection. Using this approach, the concentration-time course of a physiological (306 microgram 200 nCi) oral dose of beta-[(14)C]carotene was determined for 209 days in plasma. Analytes included beta-[(14)C]carotene, [(14)C]retinyl esters, [(14)C]retinol, and several [(14)C]retinoic acids. There was a 5.5-h lag between dosing and the appearance of (14)C in plasma. Labeled beta-carotene and [(14)C]retinyl esters rose and displayed several maxima with virtually identical kinetic profiles over the first 24-h period; elevated [(14)C]retinyl ester concentrations were sustained in the plasma compartment for >21 h postdosing. The appearance of [(14)C]retinol in plasma was also delayed 5.5 h postdosing and its concentration rose linearly for 28 h before declining. Cumulative urine and stool were collected for 17 and 10 days, respectively, and 57.4% of the dose was recovered in the stool within 48 h postdosing. The stool was the major excretion route for the absorbed dose. The turnover times (1/k(el)) for beta-carotene and retinol were 58 and 302 days, respectively. Area under the curve analysis of the plasma response curves suggested a molar vitamin A value of 0.53 for beta-carotene, with a minimum of 62% of the absorbed beta-carotene being cleaved to vitamin A.In summary, AMS is an excellent tool for defining the in vivo metabolic behavior of beta-carotene and related compounds at physiological concentrations. Further, our data suggest that retinyl esters derived from beta-carotene may undergo hepatic resecretion with VLDL in a process similar to that observed for beta-carotene.

Persian leopard (Panthera pardus) attack in Oklahoma: case report.

The authors report a fatal case of a Persian leopard (Panthera pardus) attack in an animal sanctuary in Oklahoma. The victim was a 53-year-old Costa Rican woman who was attempting to feed the animal when she was attacked and killed. Autopsy, radiography, fingerprint analysis, microbiologic cultures, and dental impressions were used to evaluate the case. These simple techniques can be applied to similar cases involving wild and domestic animal attacks.

Bioanalytical applications of accelerator mass spectrometry for pharmaceutical research.

Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying isotopes. It has had great impact in the geosciences and is now being applied in the biomedical fields. AMS measures radioisotopes such as 14C, 3H, 41Ca, and 36Cl, and others, with attomole sensitivity and high precision. Its use is allowing absorption, distribution, metabolism and elimination studies, as well as detailed pharmacokinetics, to be carried out directly in humans with very low chemical or radiological hazard. It is used in combination with standard separation methodologies, such as chromatography, in identification of metabolites and molecular targets for both toxicants and pharmacologic agents. AMS allows the use of very low specific activity chemicals (< 1 mCi/mmol), creating opportunities to use compounds not available in a high specific activity form, such as those that must be biosynthesized, produced in combinatorial libraries, or made through inefficient synthesis. AMS is allowing studies to be carried out with agents having low bioavailability, low systemic distributions, or high toxicity where administered doses must be kept low (<1 microg/kg). It may have uses in tests for idiosyncratic metabolism, drug interaction, or individual susceptibility, among others. The ability to use very low chemical doses, low radiological doses, small samples and conduct multiple dose studies may help move drug candidates into humans faster and safer than before. The uses of AMS are growing and its potential for drug development is only now beginning to be realized.

Isotope-labeled immunoassays without radiation waste.

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.

Species and strain comparisons in the macromolecular binding of extremely low doses of [14C]benzene in rodents, using accelerator mass spectrometry.

The kinetics of macromolecular binding of a 5 micrograms/kg body wt dose of [14C]benzene was studied over 48 h in B6C3F1, DBA/2, and C57BL/6 mice and Fischer rats to determine if adduct levels reflect known differences in metabolic capacity, genotoxicity, and carcinogenic potency. Previous studies have suggested that differences in benzene toxicity among strains result from differences in metabolism. Rats and mice were administered [14C]benzene (i.p.), followed by removal of liver and bone marrow at time intervals up to 48 h postexposure. Protein and DNA were isolated and analyzed by accelerator mass spectrometry. Area under the curves for protein and DNA adducts in bone marrow were greatest in B6C3F1 mouse > DBA/2 mouse > C57BL/6 mouse > Fischer rat. These data are consistent with the hypothesis that metabolic capacity contributes to the difference in benzene's carcinogenicity among species. Additionally, these data suggest that target organ adduct levels correlate with tumorigenicity and thus may be indicative of an individuals risk.

HPLC-accelerator MS measurement of atrazine metabolites in human urine after dermal exposure.

Metabolites of atrazine were measured in human urine after dermal exposure using HPLC to separate and identify metabolites and accelerator mass spectrometry (AMS) to quantify them. Ring-labeled [14C]atrazine was applied for 24 h with a dermal patch to human volunteers at low (0.167 mg, 6.45 muCi) and high (1.98 mg, 24.7 muCi) doses. Urine was collected for 7 days. The urine was centrifuged to remove solids, and the supernatant was measured by liquid scintillation counting prior to injection on the HPLC to ensure that < 0.17 Bq (4.5 pCi) was injected on the column. A reversed-phase gradient of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile became less polar with increasing time and separated the parent compound and major atrazine metabolites over 31 min on an octadecylsilane column. Peaks were identified by coelution with known standards. Elution fractions were collected in 1-min increments; half of each fraction was analyzed by AMS to obtain limits of quantitation of 14 amol. Mercapturate metabolites of atrazine and dealkylated atrazine dominated the early metabolic time points, accounting for approximately 90% of the 14C in the urine. No parent compound was detected. The excreted atrazine metabolites became more polar with increasing time, and an unidentified polar metabolite that was present in all samples became as prevalent as any of the known ring metabolites several days after the dose was delivered. Knowledge of metabolite dynamics is crucial to developing useful assays for monitoring atrazine exposure in agricultural workers.

Dose-dependent binding of ortho-phenylphenol to protein but not DNA in the urinary bladder of male F344 rats.

ortho-Phenylphenol (OPP) is a widely used fungicide and antibacterial agent that is also known to be highly effective in inducing bladder tumors in male F344 rats. At present, neither the role of the urinary bladder in the bioactivation of OPP metabolites nor the nature of the molecular target is understood. To address these issues, we investigated the relationship between OPP dosage and macromolecular adduct formation in the urinary bladder of male F344 rats. Male F344 rats were treated with 0, 15, 50, 125, 250, 500, 1000 mg/kg of OPP and its radiocarbon analogue via oral gavage. The dosed rats were euthanized after 24 h, and the proteins were extracted from the liver, kidney, and bladder. The amount of radioactivity associated with the extracted protein was quantified using highly sensitive accelerator mass spectrometry. Protein binding in liver and kidney exhibited a linear or modest curvilinear relationship over the dose range studied. In the urinary bladder, however, a pronounced nonlinear relationship between protein adduct levels and administered dose was observed. The measured protein adduct levels were in agreement with the predicted concentrations of phenylbenzoquinone based on a proposed mechanism involving free phenylhydroquinone autoxidation in the urine. Unlike protein binding, DNA adducts measured from the same bladder samples did not show a significant difference from the control group. These data are consistent with the hypothesis that OPP is an indirect acting carcinogen, and that regenerative hyperplasia due to OPP-metabolite cytotoxicity and/or binding of OPP metabolites to protein targets may play an important role in OPP-induced bladder carcinogenesis.

Intrinsic erythrocyte labeling and attomole pharmacokinetic tracing of 14C-labeled folic acid with accelerator mass spectrometry.

Long-term physiologic tracing of nutrients, toxins, and drugs in healthy subjects is not possible using traditional decay counting of radioisotopes or stable isotope mass spectrometry due to radiation exposure and limited sensitivity, respectively. A physiologic dose of 14C-labeled folic acid (35 microg, 100 nCi) was ingested by a healthy adult male and followed for 202 days in plasma, erythrocytes, urine, and feces using accelerator mass spectrometry. All samples and generated wastes were classified nonradioactive and the subject received a lifetime-integrated radiological effective dose of only 11 microSv. Radiolabeled folate appeared in plasma 10 min after ingestion but did not appear in erythrocytes until 5 days later. Approximately 0.4% of the erythrocytes were intrinsically labeled with an average of 130 (14)C atoms during erythropoiesis from the pulse of plasma [14C]folate. An appropriate radiocarbon-labeled precursor can intrinsically label DNA or a specific protein during synthesis and obtain limits of quantitation several orders of magnitude below that of stable isotope methods.

Child homicide in Oklahoma: a continuing public health problem.

Homicide is a leading manner of injury to cause death in children. To assess this phenomenon in Oklahoma, the demographic characteristics and causes of death of the victims of child homicide in Oklahoma have been reviewed. One hundred eleven consecutive cases of homicide in children less than age 13 years were reviewed and the demographic characteristics of the victims were analyzed. The majority of homicides occurred in Tulsa and Oklahoma Counties (55.8%). The ratio of male to female victims was approximately equal. The races of the victims were 66.6 percent White, 24.3 percent Black, 8.1 percent Native American and 0.9 percent Asian. The most common cause of death was head injury (45.9%). An unexpected finding was that in 23.4 percent of cases, an additional fatality occurred in the family due to family violence. This fatality involved either suicide of the perpetrator or homicide of a sibling. These findings indicate a continuing family violence problem in Oklahoma.

The dynamics of folic acid metabolism in an adult given a small tracer dose of 14C-folic acid.

Folate is an essential nutrient that is involved in many metabolic pathways, including amino acid interconversions and nucleotide (DNA) synthesis. In genetically susceptible individuals and populations, dysfunction of folate metabolism is associated with severe illness. Despite the importance of folate, major gaps exist in our quantitative understanding of folate metabolism in humans. The gaps exist because folate metabolism is complex, a suitable animal model that mimics human folate metabolism has not been identified, and suitable experimental protocols for in vivo studies in humans are not developed. In general, previous studies of folate metabolism have used large doses of high specific activity tritium and 14C-labeled folates in clinical patients. While stable isotopes such as deuterium and 13C-labeled folate are viewed as ethical alternatives to radiolabeled folates for studying metabolism, the lack of sensitive mass spectrometry methods to quantify them has impeded advancement of the field using this approach. In this chapter, we describe a new approach that uses a major analytical breakthrough, Accelerator Mass Spectrometry (AMS). Because AMS can detect attomole concentrations of 14C, small radioactive dosages (nCi) can be safely administered to humans and traced over long periods of time. The needed dosages are sufficiently small that the total radiation exposure is only a fraction of the natural annual background radiation of Americans, and the generated laboratory waste may legally be classified non-radioactive in many cases. The availability of AMS has permitted the longest (202 d) and most detailed study to date of folate metabolism in a healthy adult human volunteer. Here we demonstrate the feasibility of our approach and illustrate its potential by determining empirical kinetic values of folate metabolism. Our data indicate that the mean sojourn time for folate is in the range of 93 to 120 d. It took > or = 350 d for the absorbed portion of small bolus dose of 14C-folic acid to be eliminated completely from the body.

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 is also available 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-isotopic and stable isotope labeling methods are reviewed and examples of analytical integrity, sensitivity, specificity, and applicability are provided.

Analytical performance of accelerator mass spectrometry and liquid scintillation counting for detection of 14C-labeled atrazine metabolites in human urine.

Accelerator mass spectrometry (AMS) has been applied to the detection of 14C-labeled urinary metabolites of the triazine herbicide, atrazine, and the analytical performance of AMS has been directly compared to that of liquid scintillation counting (LSC). Ten human subjects were given a dermal dose of 14C-labeled atrazine over 24 h, and urine from the subjects was collected over a 7-day period. Concentrations of 14C in the samples have been determined by AMS and LSC and range from 1.8 fmol/mL to 4.3 pmol/mL. Data from these two methods have a correlation coefficient of 0.998 for a linear plot of the entire sample set. Accelerator mass spectrometry provides superior concentration (2.2 vs 27 fmol/mL) and mass (5.5 vs 54,000 amol) detection limits relative to those of LSC for these samples. The precision of the data provided by AMS for low-level samples is 1.7%, and the day-to-day reproducibility of the AMS measurements is 3.9%. Factors limiting AMS detection limits for these samples and ways in which these can be improved are examined.

Initial uptake kinetics in human skin exposed to dilute aqueous trichloroethylene in vitro.

In vitro uptake of 14C-labeled trichloroethylene (TCE) from dilute (approximately 5-ppb) aqueous solutions into human surgical skin was measured using accelerator mass spectrometry (AMS). We analyzed 105 breast-tissue samples obtained from three subjects, representing 27 separate exposure experiments conducted at approximately 20 degrees C for 0, 1, 5, 15, 30, or 60 min. The AMS data obtained positively correlate with (p approximately 0) and vary significantly nonlinearly with (p = 0.0094) exposure duration. These data are inconsistent (p approximately 0) with predictions made for TCE by a proposed U.S. Environmental Protection Agency (USEPA) dermal-exposure model, even when uncertainties in its recommended parameter values for TCE are considered, but are consistent (p = 0.17) with a 1-compartment model for exposed skin-surface tissue governed in vitro by a maximum effective permeability of K*p = 0.28 cm h-1 (+/- 7.0%) and a first-order rate constant of k1 = 1.2 h-1 (+/- 16%). The apparent compartment depth is estimated to be approximately 40-100 microns, i.e., to comprise much or all of the epidermis. In contrast, the USEPA model implies only negligible TCE penetration beyond SC during a 1-h exposure. The K*p estimate based on the 1-compartment model fit is consistent with estimates for TCE based on in vivo studies, which supports the hypothesis that the USEPA model underpredicts short-term dermal uptake of TCE from water. It is shown that for humans, this fit also implies that normalized total uptake of TCE from water by short-term dermal contact in vivo is predicted to be fK*p, where f is approximately 80% for longer normothermic exposures and approximately 95% during a brief hot shower or bath. This study illustrates the power of AMS to facilitate analyses of contaminant biodistribution and uptake kinetics at very low environmental concentrations.