John S. Vogel

Rapid Production of Graphite without Contamination for Biomedical AMS

The application of AMS to the detection of [sup 14]C makes possible a new class of sensitive experiments in molecular biology. Such experiments inherently produce large numbers of samples for the determination of biological variability in molecular interactions. The samples vary in [sup 14]C concentration over many orders of magnitude. The author added TiH[sub 2] to aid the reduction of CO[sub 2] by zinc in a sealed tube to reproducibly make graphite without sample cross-contamination. The CO[sub 2] is transferred from a combustion tube to the reaction tube through a disposable plastic manifold. The sealed tubes are heated to a single-reaction temperature in a muffle furnace. The process is complete within 5 h. Bulk isotopic fractionation in the finished graphite is less than 0.5%.

LLNL/UC AMS facility and research program

Abstract The Lawrence Livermore National Laboratory (LLNL) and the University of California (UC) now have in operation a large AMS spectrometer built as part of a new multiuser laboratory centered on an FN tandem. AMS measurements are expected to use half of the beam time of the accelerator. LLNL use of AMS is in research on consequences of energy usage. Examples include global warming, geophysical site characterization, radiation biology and dosimetry, and study of mutagenic and carcinogenic processes. UC research activities are in clinical applications, archaeology and anthropology, oceanography, and geophysical and geochemical research. Access is also possible for researchers outside the UC system. The technological focus of the laboratory is on achieving high rates of sample throughput, unattended operation, and advances in sample preparation methods. Because of the expected growth in the research programs and the other obligations of the present accelerator, we are designing a follow-on dedicated facility for only AMS and microprobe analysis that will contain at least two accelerators with multiple spectrometers.

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. Scatterplot smoothing methods are used to estimate the mean function and covariance surface of this model. We propose improved estimation in the neighborhood of and at the diagonal of the covariance surface, where the measurement errors are reflected. The presence of additive measurement errors motivates shrinkage estimates for the functional principal component scores. Shrinkage estimates are developed through best linear prediction and in a generalized version, aiming at minimizing one-curve-leave-out prediction error. The estimation of individual trajectories combines data obtained from that individual as well as all other individuals. We apply our methods to new data regarding the analysis of the level of 14C-folate in plasma as a function of time since dosing of healthy adults with a small tracer dose of 14C-folic acid. A time transformation was incorporated to handle design irregularity concerning the time points on which the measurements were taken. The proposed methodology, incorporating shrinkage and data-adaptive features, is seen to be well suited for describing population kinetics of 14C-folate-specific activity and random effects, and can also be applied to other functional data analysis problems.

Gerardia: Bristlecone pine of the deep-sea?

We measured carbon isotope abundances in the layered, proteinaceous skeleton of a zoanthid Gerardia collected from 620 m depth off the Little Bahama Bank (27°N, 79°W). The δ 14C values decreased from −76% in the outer growth edge to an average of −267% in the center of three portions of the skeleton. These δ 14C data suggest an age for this living organism of 1800 ± 300 years. The possibility that the large decrease in δ 14C reflects the gradual input of bomb 14C over the entire growth of the organism is inconsistent with the post-bomb δ 14C values obtained for the most recent growth tips. If the age estimate of two millennia is correct, it may be the longest-lived animal yet observed in the ocean. Gerardia may serve as a long-lived recorder of ocean chemistry, similar to the Bristlecone pine tree that has served as a millennial-timescale recorder for atmospheric 14CO2 (Suess, 1980) and climate. In particular, there is potential for Gerardia to serve as a millennial-scale integrator of upper ocean particle flux, and possibly reveal past changes in the productivity of the surface ocean.

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.

MeIQx-DNA adduct formation in rodent and human tissues at low doses

Heterocyclic amines, such as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), are mutagenic/carcinogenic compounds formed during the cooking of protein-rich foods. Human exposure to MeIQx has been estimated to range from ng/person/day to a few microgram/person/day. In contrast, animal studies have been conducted at doses in excess of 10 mg/kg/day. In order to determine the relevance of high-dose animal data for human exposure, the dose-response curves for [14C]-MeIQx have been determined in rodents at low doses under both single-dose and chronic dosing regimens using the high sensitivity of accelerator mass spectrometry (AMS). To make a direct species comparison, rodent and human colonic MeIQx-DNA adduct levels have been compared following oral administration of [14C]-MeIQx. The results of these studies show: (1) total MeIQx levels are highest in the liver > kidney > pancreas > intestine > blood; (2) MeIQx levels in the liver plateau after 7 days of chronic feeding; (3) hepatic MeIQx-DNA adducts begin to plateau after 2-4 weeks and reach steady-state levels between 4 and 12 weeks on chronic exposures; (4) hepatic DNA adducts generally increase as a linear function of administered dose for a single-dose exposure and as a power function for chronic feeding over a dose range spanning 4 orders of magnitude; (5) human colon DNA adduct levels are approximately 10 times greater than in rodents at the same dose and time point following exposure; and (6) > or = 90% of the MeIQx-DNA adduct in both rodent and human colon appears to be the dG-C8-MeIQx adduct. These studies show that MeIQx is readily available to the tissues for both humans and rodents and that adduct levels are generally linear with administered dose except at high chronic doses where adduct levels begin to plateau slightly. This plateau indicates that linear extrapolation from high-dose studies probably underestimates the amount of DNA damage present in the tissues following low dose. Further, if adducts represent the biologically effective dose, these data show that human colon may be as sensitive to the genotoxic effects of MeIQx as rat liver. The significance of these endpoints to tumor response remains to be determined.

Absorption and retinol equivalence of β-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]β-carotene was investigated in a longitudinal test-retest design in two adults. For the test, each subject ingested 1 nmol of [14C]β-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]β-carotene, [14C]retinyl esters, and [14C]retinol were determined for 46 days. On Day 53, the subjects were placed on a daily vitamin A supplement (10,000 IU/day), and a second dose of [14C]β-carotene (retest) was given on Day 74. All 14C determinations were made using accelerator mass spectrometry. In both subjects, the vitamin A supplementation was associated with three main effects: 1) increased apparent absorption: test versus retest values rose from 57% to 74% (Subject 1) and from 52% to 75% (Subject 2); 2) an ∼10-fold reduction in urinary excretion; and 3) a lower ratio of labeled retinyl ester/β-carotene concentrations in the absorptive phase. The molar vitamin A value of the dose for the test was 0.62 mol (Subject 1) and 0.54 mol (Subject 2) vitamin A to 1 mol β-carotene. Respective values for the retest were 0.85 and 0.74. These results show that while less cleavage of β-carotene occurred due to vitamin A supplementation, higher absorption resulted in larger molar vitamin A values.

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.

Tips and traps in the 14C bio-AMS preparation laboratory

Maintaining a contamination free sample preparation lab for biological 14 C AMS requires the same or more diligence as a radiocarbon dating prep lab. Isotope ratios of materials routinely range over 4-8 orders of magnitude in a single experiment, dosing solutions contain thousands of DPM and gels used to separate proteins possess 14 C ratios of 1pMC. Radiocarbon contamination is a legacy of earlier tracer work in most biological laboratories, even if they were never hot labs. Removable surface contamination can be found and monitored using swipes. Contamination can be found on any surface routinely touched: door knobs, light switches, drawer handles, water faucets. In general, all surfaces routinely touched need to be covered with paper, foil, or plastic that can be changed frequently. Shared air supplies can also present problems by distributing hot aerosols throughout a building. Aerosols can be monitored for 14 C content using graphitized coal or fullerene soot mixed with metal powder as an absorber. The monitors can be set out in work spaces for 1-2 weeks and measured by AMS with regular samples. Frequent air changes help minimize aerosol contamination in many cases. Cross contamination of samples can be minimized by using disposable plastic or glassware in the prep lab, isolating samples from the air when possible and using positive displacement pipetters.

The LLNL AMS facility

The AMS facility at Lawrence Livermore National Laboratory (LLNL) routinely measures the isotopes 3H, 7Be, 10Be, 14C, 26Al, 36Cl, 41Ca, and 129I. During the past two years, over 30000 research samples have been measured. Of these samples, approximately 30% were for 14C bioscience tracer studies, 45% were 14C samples for archaeology and the geosciences, and the other isotopes constitute the remaining 25%. During the past two years at LLNL, a significant amount of work has gone into the development of the Projectile X-ray AMS (PXAMS) technique. PXAMS uses induced characteristic X-rays to discriminate against competing atomic isobars. PXAMS has been most fully developed for 63Ni but shows promise for the measurement of several other long lived isotopes. During the past year LLNL has also conducted an 129I interlaboratory comparison exercise. Recent hardware changes at the LLNL AMS facility include the installation and testing of a new thermal emission ion source, a new multi-anode gas ionization detector for general AMS use, re-alignment of the vacuum tank of the first of the two magnets that make up the high energy spectrometer, and a new cryo-vacuum system for the AMS ion source. In addition, we have begun design studies and carried out tests for a new high-resolution injector and a new beamline for heavy element AMS.