Separation of Lipoproteins for Quantitative Analysis of 14C-Labeled Lipid-Soluble Compounds by Accelerator Mass Spectrometry.

To date, 14C tracer studies using accelerator mass spectrometry (AMS) have not yet resolved lipid-soluble analytes into individual lipoprotein density subclasses. The objective of this work was to develop a reliable method for lipoprotein separation and quantitative recovery for biokinetic modeling purposes. The novel method developed provides the means for use of small volumes (10-200 µL) of frozen plasma as a starting material for continuous isopycnic lipoprotein separation within a carbon- and pH-stable analyte matrix, which, following post-separation fraction clean up, created samples suitable for highly accurate 14C/12C isotope ratio determinations by AMS. Manual aspiration achieved 99.2 ± 0.41% recovery of [5-14CH3]-(2R, 4'R, 8'R)-α-tocopherol contained within 25 µL plasma recovered in triacylglycerol rich lipoproteins (TRL = Chylomicrons + VLDL), LDL, HDL, and infranatant (INF) from each of 10 different sampling times for one male and one female subject, n = 20 total samples. Small sample volumes of previously frozen plasma and high analyte recoveries make this an attractive method for AMS studies using newer, smaller footprint AMS equipment to develop genuine tracer analyses of lipophilic nutrients or compounds in all human age ranges.

Carbon isotopes profiles of human whole blood, plasma, red blood cells, urine and feces for biological/biomedical 14C-accelerator mass spectrometry applications.

Radiocarbon ((14)C) is an ideal tracer for in vivo human ADME (absorption, distribution, metabolism, elimination) and PBPK (physiological-based pharmacokinetic) studies. Living plants peferentially incorporate atmospheric (14)CO(2) versus (13)CO(2) versus (12)CO(2), which result in unique signature. Furthermore, plants and the food chains they support also have unique carbon isotope signatures. Humans, at the top of the food chain, consequently acquire isotopic concentrations in the tissues and body fluids depending on their dietary habits. In preparation of ADME and PBPK studies, 12 healthy subjects were recruited. The human baseline (specific to each individual and their diet) total carbon (TC) and carbon isotope (13)C (δ(13)C) and (14)C (F(m)) were quantified in whole blood (WB), plasma, washed red blood cell (RBC), urine, and feces. TC (mg of C/100 µL) in WB, plasma, RBC, urine, and feces were 11.0, 4.37, 7.57, 0.53, and 1.90, respectively. TC in WB, RBC, and feces was higher in men over women, P < 0.05. Mean δ(13)C were ranked low to high as follows: feces < WB = plasma = RBC = urine, P < 0.0001. δ(13)C was not affected by gender. Our analytic method shifted δ(13)C by only ±1.0 ‰ ensuring our F(m) measurements were accurate and precise. Mean F(m) were ranked low to high as follows: plasma = urine < WB = RBC = feces, P < 0.05. F(m) in feces was higher for men over women, P < 0.05. Only in WB, (14)C levels (F(m)) and TC were correlated with one another (r = 0.746, P < 0.01). Considering the lag time to incorporate atmospheric (14)C into plant foods (vegetarian) and or then into animal foods (nonvegetarian), the measured F(m) of WB in our population (recruited April 2009) was 1.0468 ± 0.0022 (mean ± SD), and the F(m) of WB matched the (extrapolated) atmospheric F(m) of 1.0477 in 2008. This study is important in presenting a procedure to determine a baseline for a study group for human ADME and PBPK studies using (14)C as a tracer.

Calculating radiation exposures during use of (14)C-labeled nutrients, food components, and biopharmaceuticals to quantify metabolic behavior in humans.

(14)C has long been used as a tracer for quantifying the in vivo human metabolism of food components, biopharmaceuticals, and nutrients. Minute amounts (< or =1 x 10 (-18) mol) of (14)C can be measured with high-throughput (14)C-accelerator mass spectrometry (HT (14)C-AMS) in isolated chemical extracts of biological, biomedical, and environmental samples. Availability of in vivo human data sets using a (14)C tracer would enable current concepts of the metabolic behavior of food components, biopharmaceuticals, or nutrients to be organized into models suitable for quantitative hypothesis testing and determination of metabolic parameters. In vivo models are important for specification of intake levels for food components, biopharmaceuticals, and nutrients. Accurate estimation of the radiation exposure from ingested (14)C is an essential component of the experimental design. Therefore, this paper illustrates the calculation involved in determining the radiation exposure from a minute dose of orally administered (14)C-beta-carotene, (14)C-alpha-tocopherol, (14)C-lutein, and (14)C-folic acid from four prior experiments. The administered doses ranged from 36 to 100 nCi, and radiation exposure ranged from 0.12 to 5.2 microSv to whole body and from 0.2 to 3.4 microSv to liver with consideration of tissue weighting factor and fractional nutrient. In comparison, radiation exposure experienced during a 4 h airline flight across the United States at 37000 ft was 20 microSv.

Quality of graphite target for biological/biomedical/environmental applications of 14C-accelerator mass spectrometry.

Catalytic graphitization for (14)C-accelerator mass spectrometry ((14)C-AMS) produced various forms of elemental carbon. Our high-throughput Zn reduction method (C/Fe = 1:5, 500 degrees C, 3 h) produced the AMS target of graphite-coated iron powder (GCIP), a mix of nongraphitic carbon and Fe(3)C. Crystallinity of the AMS targets of GCIP (nongraphitic carbon) was increased to turbostratic carbon by raising the C/Fe ratio from 1:5 to 1:1 and the graphitization temperature from 500 to 585 degrees C. The AMS target of GCIP containing turbostratic carbon had a large isotopic fractionation and a low AMS ion current. The AMS target of GCIP containing turbostratic carbon also yielded less accurate/precise (14)C-AMS measurements because of the lower graphitization yield and lower thermal conductivity that were caused by the higher C/Fe ratio of 1:1. On the other hand, the AMS target of GCIP containing nongraphitic carbon had higher graphitization yield and better thermal conductivity over the AMS target of GCIP containing turbostratic carbon due to optimal surface area provided by the iron powder. Finally, graphitization yield and thermal conductivity were stronger determinants (over graphite crystallinity) for accurate/precise/high-throughput biological, biomedical, and environmental (14)C-AMS applications such as absorption, distribution, metabolism, elimination (ADME), and physiologically based pharmacokinetics (PBPK) of nutrients, drugs, phytochemicals, and environmental chemicals.

Accelerator mass spectrometry targets of submilligram carbonaceous samples using the high-throughput Zn reduction method.

The high-throughput Zn reduction method was developed and optimized for various biological/biomedical accelerator mass spectrometry (AMS) applications of mg of C size samples. However, high levels of background carbon from the high-throughput Zn reduction method were not suitable for sub-mg of C size samples in environmental, geochronology, and biological/biomedical AMS applications. This study investigated the effect of background carbon mass (mc) and background 14C level (Fc) from the high-throughput Zn reduction method. Background mc was 0.011 mg of C and background Fc was 1.5445. Background subtraction, two-component mixing, and expanded formulas were used for background correction. All three formulas accurately corrected for backgrounds to 0.025 mg of C in the aerosol standard (NIST SRM 1648a). Only the background subtraction and the two-component mixing formulas accurately corrected for backgrounds to 0.1 mg of C in the IAEA-C6 and -C7 standards. After the background corrections, our high-throughput Zn reduction method was suitable for biological (diet)/biomedical (drug) and environmental (fine particulate matter) applications of sub-mg of C samples (> or = 0.1 mg of C) in keeping with a balance between throughput (270 samples/day/analyst) and sensitivity/accuracy/precision of AMS measurement. The development of a high-throughput method for examination of > or = 0.1 mg of C size samples opens up a range of applications for 14C AMS studies. While other methods do exist for > or = 0.1 mg of C size samples, the low throughput has made them cost prohibitive for many applications.

Biological/biomedical accelerator mass spectrometry targets. 1. optimizing the CO2 reduction step using zinc dust.

Biological and biomedical applications of accelerator mass spectrometry (AMS) use isotope ratio mass spectrometry to quantify minute amounts of long-lived radioisotopes such as (14)C. AMS target preparation involves first the oxidation of carbon (in sample of interest) to CO 2 and second the reduction of CO 2 to filamentous, fluffy, fuzzy, or firm graphite-like substances that coat a -400-mesh spherical iron powder (-400MSIP) catalyst. Until now, the quality of AMS targets has been variable; consequently, they often failed to produce robust ion currents that are required for reliable, accurate, precise, and high-throughput AMS for biological/biomedical applications. Therefore, we described our optimized method for reduction of CO 2 to high-quality uniform AMS targets whose morphology we visualized using scanning electron microscope pictures. Key features of our optimized method were to reduce CO 2 (from a sample of interest that provided 1 mg of C) using 100 +/- 1.3 mg of Zn dust, 5 +/- 0.4 mg of -400MSIP, and a reduction temperature of 500 degrees C for 3 h. The thermodynamics of our optimized method were more favorable for production of graphite-coated iron powders (GCIP) than those of previous methods. All AMS targets from our optimized method were of 100% GCIP, the graphitization yield exceeded 90%, and delta (13)C was -17.9 +/- 0.3 per thousand. The GCIP reliably produced strong (12)C (-) currents and accurate and precise F m values. The observed F m value for oxalic acid II NIST SRM deviated from its accepted F m value of 1.3407 by only 0.0003 +/- 0.0027 (mean +/- SE, n = 32), limit of detection of (14)C was 0.04 amol, and limit of quantification was 0.07 amol, and a skilled analyst can prepare as many as 270 AMS targets per day. More information on the physical (hardness/color), morphological (SEMs), and structural (FT-IR, Raman, XRD spectra) characteristics of our AMS targets that determine accurate, precise, and high-hroughput AMS measurement are in the companion paper.

Biological/biomedical accelerator mass spectrometry targets. 2. Physical, morphological, and structural characteristics.

The number of biological/biomedical applications that require AMS to achieve their goals is increasing, and so is the need for a better understanding of the physical, morphological, and structural traits of high quality of AMS targets. The metrics of quality included color, hardness/texture, and appearance (photo and SEM), along with FT-IR, Raman, and powder X-ray diffraction spectra that correlate positively with reliable and intense ion currents and accuracy, precision, and sensitivity of fraction modern ( F m). Our previous method produced AMS targets of gray-colored iron-carbon materials (ICM) 20% of the time and of graphite-coated iron (GCI) 80% of the time. The ICM was hard, its FT-IR spectra lacked the sp (2) bond, its Raman spectra had no detectable G' band at 2700 cm (-1), and it had more iron carbide (Fe 3C) crystal than nanocrystalline graphite or graphitizable carbon (g-C). ICM produced low and variable ion current whereas the opposite was true for the graphitic GCI. Our optimized method produced AMS targets of graphite-coated iron powder (GCIP) 100% of the time. The GCIP shared some of the same properties as GCI in that both were black in color, both produced robust ion current consistently, their FT-IR spectra had the sp (2) bond, their Raman spectra had matching D, G, G', D +G, and D '' bands, and their XRD spectra showed matching crystal size. GCIP was a powder that was easy to tamp into AMS target holders that also facilitated high throughput. We concluded that AMS targets of GCIP were a mix of graphitizable carbon and Fe 3C crystal, because none of their spectra, FT-IR, Raman, or XRD, matched exactly those of the graphite standard. Nevertheless, AMS targets of GCIP consistently produced the strong, reliable, and reproducible ion currents for high-throughput AMS analysis (270 targets per skilled analyst/day) along with accurate and precise F m values.

Energy-transfer dynamics of high-pressure rovibrationally excited molecular H2.

The energy-transfer dynamics of high-pressure molecular H(2) gas initially prepared in the |X (1)Sigma(g) (+),v = 1,J = 1 state using stimulated Raman pumping are probed with rotational Raman scattering. A computer simulation that incorporates the effects of collision-induced vibrational energy transfer is described and used to fit the experimental Raman scattering results obtained as a function of the pump/probe delay time. The 4.78 x 10(-14) +/- 3.85 x 10(-16) cm(3) s(-1) molecule(-1) vibrational energy-transfer rate for decay from the |X (1)Sigma(g) (+),v = 1,J = 1 >state compares well with other lower-pressure studies.

Resonant Raman-scattering measurements of trichloroethene in a thermal boundary layer.

Concentration profiles of trichloroethene were measured in a boundary-layer flow over a heated ceramic surface. Raman scattering was excited with the fifth harmonic of a Nd:YAG laser at 213 nm. This wavelength took advantage of a resonance in the trichloroethene molecule to significantly enhance the C2HCl3 scattering cross section. The resonant Raman system was calibrated in a heated flow. The optical system was optimized so that measurements could be obtained close to the solid surface, normally a significant challenge for a spontaneous Raman-scattering setup. Measured concentrations indicated the lack of catalytic activity on a bare alumina surface. However, the results showed that a surface that was coated with Cr2O3-based zeolite was catalytically active.