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New therapeutic biological entities such as bispecific antibodies targeting tissue or specific cell populations form an increasingly important part of the drug development portfolio. However, these biopharmaceutical agents bear the risk of extensive target-mediated drug disposition or atypical pharmacokinetic properties as compared to canonical antibodies. Pharmacokinetics and bio-distribution studies become therefore more and more important during lead optimization. Biologics present, however, greater analytical challenges than small molecule drugs due to the mass and selectivity limitation of mass spectrometry and ligand-binding assay, respectively. Radiocarbon (14C) and its detection methods, such as the emerging 14C cavity ring down spectroscopy (CRDS), thus can play an important role in the large molecule quantitation where a 14C-tag is covalently bound through a stable linker. CRDS has the advantage of a simplified sample preparation and introduction system as compared to accelerator mass spectrometry (AMS) and can be accommodated within an ordinary research laboratory. In this study, we report on the labeling of an anti-IL17 IgG1 model antibody with 14C propionate tag and its detection by CRDS using it as nanotracer (2.1 nCi or 77.7 Bq blended with the therapeutic dose) in a pharmacokinetics study in a preclinical species. We compare these data to data generated by AMS in parallel processed samples. The derived concentration time profiles for anti-IL17 by CRDS were in concordance with the ones derived by AMS and γ-counting of an 125I-labeled anti-IL17 radiotracer and were well described by a 2-compartment population pharmacokinetic model. In addition, antibody tissue distribution coefficients for anti-IL17 were determined by CRDS, which proved to be a direct and sensitive measurement of the extravascular tissue concentration of the antibody when tissue perfusion was applied. Thus, this proof-of-concept study demonstrates that trace 14C-radiolabels and CRDS are an ultrasensitive approach in (pre)clinical pharmacokinetics and bio-distribution studies of new therapeutic entities.

Pharmacokinetic studies in the neonatal population are often limited by the small volume of blood that can be collected. The high sensitivity of (14) C-accelerator mass spectrometry (AMS) enables pharmacokinetic studies to be conducted with greatly reduced sample volumes. We demonstrated the utility of AMS in infants by studying the plasma pharmacokinetic behavior of nanogram doses of (14) C-ursodiol administered as a non-perturbing microdose or as a microtracer with therapeutic doses of non-labeled ursodiol in infants. Five non-cholestatic infants were administered 3 consecutive oral microdoses of (14) C-ursodiol: 8 ng (1.0 nCi), 26 ng (3.3 nCi), and 80 ng (10 nCi) 48 hours apart. Three additional infants with cholestasis were administered a single 80 ng (10.0 nCi) oral dose of (14) C-ursodiol together with a therapeutic dose of 40 mg/kg of non-labeled ursodiol. A pharmacokinetic model describing ursodiol concentrations was developed using nonlinear mixed-effects modeling. The pharmacokinetics of ursodiol in this pilot study were best described by a two-compartment model with first-order elimination. This study demonstrates the feasibility and utility of microdose and microtrace methodology in pediatric research.

This was a single dose mass balance and metabolite characterization study of the antimalarial agent pyronaridine. Six healthy male adults were administered a single oral dose of 720 mg pyronaridine tetraphosphate with 800 nCi of radiolabeled (14)C-pyronaridine. Urine and feces were continuously collected through 168 h post-dose, with intermittent 48 h collection periods thereafter through 2064 h post-dose. Drug recovery was computed for analyzed samples and interpolated for intervening time periods in which collection did not occur. Blood samples were obtained to evaluate the pharmacokinetics of total radioactivity and of the parent compound. Total radioactivity in urine, feces, and blood samples was determined by accelerator mass spectrometry (AMS); parent concentrations in blood were determined with LC/MS. Metabolite identification based on blood, urine, and feces samples was conducted using a combination of LC + AMS for identifying radiopeaks, followed by LC/MS/MS for identity confirmation/elucidation. The mean cumulative drug recovery in the urine and feces was 23.7 and 47.8 %, respectively, with an average total recovery of 71.5 %. Total radioactivity was slowly eliminated from blood, with a mean half-life of 33.5 days, substantially longer than the mean parent compound half-life of 5.03 days. Total radioactivity remained detectable in urine and feces collected in the final sampling period, suggesting ongoing elimination. Nine primary and four secondary metabolites of pyronaridine were identified. This study revealed that pyronaridine and its metabolites are eliminated by both the urinary and fecal routes over an extended period of time, and that multiple, varied pathways characterize pyronaridine metabolism.

Our AMS produced a ratio of instrument transmissions for 14C and 13C equal to 13/14 to 0.22 ± 0.06% accuracy at a specific charge-changing energy of 235 keV and giving absolute isotope ratios with that mass correction. The ratio for 13C and 12C transmissions was 12/13 at 215 keV. A differential equation model of energetic ion and atom transport through the gas collision cell was constructed with functions representing experimental cross-sections for all collision effects. This model showed cation yield dependencies that were linear with energy for the monoenergetic isotopic ions at the energies that we measured, explaining the inverse mass dependence. The model predicted multiple lower energies that have this dependence and, more importantly, two low energies at which cation yields were equal for pairs of isotopes, presaging potential absolute isotope ratio measurements at low MS energies.

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. In this paper, we review some of the exploratory applications of AMS (14)C in toxicological, nutritional, and pharmacological research. This body of research addresses the human physiochemistry of important compounds in their own right, but also serves as examples of the analytical methods and clinical practices that are available for studying low dose physiochemistry of candidate therapeutic compounds, helping to broaden the knowledge base of AMS application in pharmaceutical research.

A survey indicated that high-dose vitamin A (HD-VA) supplements had no apparent effect on vitamin A (VA) status, assessed by serum retinol concentrations, of Zambian children lt 5 y of age. To explore possible reasons for the lack of response, we quantified absorption, retention, and urinary elimination of either a single HD-VA supplement (209.8 micromol; 60 mg) or a smaller dose of stable isotope (SI)-labeled VA (17.5 micromol; 5 mg), which was used to estimate VA pool size, in 3- to 4-y-old Zambian boys (n = 4 for each VA dose). A tracer dose of [(14)C(2)]-labeled VA (0.925 kBq; 25 nCi) was coadministered with the HD-VA supplement or SI-labeled VA, and 24-h stool and urine samples were collected for 3 and 7 consecutive days, respectively, and 24-h urine samples at 4 later time points. Accelerator MS was used to quantify (14)C in stool and urine. Estimates of absorption, retention, and the urinary elimination rate (UER) were 83.8 +/- 7.1%, 76.3 +/- 6.7%, and 1.9 +/- 0.6%/d, respectively, for the HD-VA supplement and 76.5 +/- 9.5%, 71.1 +/- 9.4%, and 1.8 +/- 1.2%/d, respectively, for the SI-labeled VA. Mean estimates of absorption, retention, and the UER did not differ by size of the VA dose administered. Estimated absorption and retention were negatively associated with reported fever (r = minus 0.83; P = 0.011). The HD-VA supplement and SI-labeled VA were adequately absorbed, retained, and utilized in apparently healthy Zambian preschool-age boys; absorption and retention may be affected by recent fever.

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 14C (t½=5370 years.). ‘Low’ voltage accelerations to 200 kV allow laboratory-sized accelerator mass spectrometers instruments for bioanalytical quantitation of 14C to 2–3% precision and accuracy in isolated biochemical fractions. After demonstrating this accuracy and precision for our new accelerator mass spectrometer, we discuss the critical aspects of maintaining quantitative accuracy from the defined biological fraction to the accelerator mass spectrometry quantitation. These aspects include sufficient sample mass for routine rapid sample preparation, isotope dilution to assure this mass, isolation of the carbon from other sample combustion gasses and use of high-efficiency biochemical separations. This review seeks to address a bioanalytical audience, who should know that high accuracy data of physiochemical processes within living human subjects are available, as long as a 14C quantitation can be made indicative of the physiochemistry of interest.

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 14 C to quantify the disposition of 14 C-β-carotene (930 ng; 60.4 nCi of activity) after intravenous injection with an autologous triacylglyceride-rich lipoprotein fraction in a single volunteer. Total 14 C was quantified in serial plasma samples and also in triglyceride-rich, and low density lipoprotein, subfractions. The appearance of 14 C-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 14 C concentrations rapidly decayed within the triglyceride-rich lipoprotein fractions after injection, whereas low density lipoprotein 14 C began a significant rise in 14 C 5 h post dose. Plasma 14 C-retinol also appeared at 5 h post dose and its concentrations were maintained above baseline for >88 days. Based upon comparisons of 14 C-retinol concentrations following an earlier study with orally dosed 14 C-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 .

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 14 C-accelerator mass spectrometry (AMS) for human kinetic studies are presented. Additionally, recent developments with the intrinsic labeling of spinach folate from a [ 14 C]- para -aminobenzoic is discussed.

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 (

The ability of beta-carotene to deliver bioactive retinoids to tissues is highly variable. A clearer understanding of the environmental and genetic factors that modulate the vitamin A potential of beta-carotene is needed.Assess the vitamin A value of orally administered beta-carotene relative to a co-administered reference dose of preformed vitamin A.Equimolar doses (30 micromol) of hexadeuterated D6 beta-carotene and D6 retinyl acetate were orally co-administered in an emulsified formulation to a male subject. The plasma concentration time courses of D6 retinol (derived from D6 retinyl acetate) and bioderived D3 retinol (from D(6) beta-carotene) were determined for 554 h postdosing using gas chromatography/mass spectrometry. Intact D6 beta-carotene plasma concentrations were determined by high-pressure liquid chromatography. The ratio of the two forms of vitamin A, D6 retinol/D3 retinol, at any single time point is postulated to reflect the quantity of vitamin A derived from beta-carotene relative to preformed vitamin A. Additionally, a minute amount of 14C beta-carotene (50 nCi; 0.27 microg) was included in the oral dose and cumulative 24-h stool and urine samples were collected for two weeks to follow absorption and excretion of the b-carotene. The 14C nuclide was detected using accelerator mass spectrometry (AMS). Results During the absorption/distribution phase (3-11 h) the D6/D3 ratio of the two retinols was not stable and ranged between a value of 3 and 16. Between 11 and 98 h postdosing the ratio was relatively stable with a mean value of 8.5 (95 % CI: 7.5, 8.7). These data suggest that in this subject and under these conditions, 8.5 moles of beta-carotene would provide a vitamin A quantity equivalent to 1 mole of preformed vitamin A. On a mass basis, 15.9 microg of beta-carotene was equivalent to 1 microg of retinol. The total administered beta-carotene was found to be 55 % absorbed by AMS analysis of cumulative stool.The co-administration of D6 beta-carotene and D6 retinyl acetate provides a technique for assessing individual ability to process beta-carotene to vitamin A. The results indicate that a single time point taken between 11-98 h after dose administration may provide a reliable value for the relative ratio of the two forms of vitamin A. However, results from more subjects are needed to assess the general utility of this method.

We describe an improved whole blood folate analysis method that facilitates increased throughput compared to our previous method (Dueker et al. (2000) Anal. Biochem. 283, 266). Improvements include three items: first, a buffered solvent exchange to remove interfering amino acids, especially phenylalanine whose esters may interfere with the analysis because their retention times on the gas chromatography are close to those of the para-aminobenzoic acid (pABA) isotopomers; second, substituting an NH2 solid phase extraction step for an HPLC step permits the batch parallel processing of samples; third, replacing trifluoroacetyl derivatives of ethyl-esterified pABA isotopomers with heptafluorobutyl derivatives, which are better resolved on the GC column. The method measures pABA, a stable degradation product of folate. This simplifies sample handling and purification. Relative standard deviations are typically 5% or less and a single operator can process samples in batches of 40. Results from our GCMS method correlate (R = 0.98) with the Lactobacillus casei assay for whole blood folate. The modifications will facilitate the development of high throughput methods for whole blood folate. Our method holds promise for epidemiological and clinical studies, where accurate whole blood folate concentrations are needed. Because it is internally standardized, interlaboratory variation should be minimal.

We present a sensitive tracer method, suitable for in vivo human research, that uses β-[14C]carotene coupled with accelerator mass spectrometry (AMS) detection. Using this approach, the concentration-time course of a physiological (306 μg; 200 nCi) oral dose of β-[14C]carotene was determined for 209 days in plasma. Analytes included β-[14C]carotene, [14C]retinyl esters, [14C]retinol, and several [14C]retinoic acids. There was a 5.5-h lag between dosing and the appearance of 14C in plasma. Labeled β-carotene and [14C]retinyl esters rose and displayed several maxima with virtually identical kinetic profiles over the first 24-h period; elevated [14C]retinyl ester concentrations were sustained in the plasma compartment for >21 h postdosing. The appearance of [14C]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/kel) for β-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 β-carotene, with a minimum of 62% of the absorbed β-carotene being cleaved to vitamin A. In summary, AMS is an excellent tool for defining the in vivo metabolic behavior of β-carotene and related compounds at physiological concentrations. Further, our data suggest that retinyl esters derived from β-carotene may undergo hepatic resecretion with VLDL in a process similar to that observed for β-carotene.—Dueker, S. R., Y. Lin, B. A. Buchholz, P. D. Schneider, M. W. Lamé, H. J. Segall, J. S. Vogel, and A. J. Clifford. Long-term kinetic study of β-carotene, using accelerator mass spectrometry in an adult volunteer. J. Lipid Res. 2000. 41: 1790–1800.

Pharmacokinetics of physiologic doses of nutrients, pesticides, and herbicides can easily be traced in humans using a 14 C-labeled compound. Basic kinetics can be monitored in blood or urine by measuring the elevation in the 14 C 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 profiles aAord 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 identified by co-elution with known standards and chemical tests that reveal functional groupings. Metabolites are quantified with the 14 C 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 14 C per HPLC fraction are typically achieved. Baselines are maintained by limiting injected 14 C activity

Six edible plants common to West African diet were analyzed for vitamin‐A precursors α‐carotene, s‐carotene, s‐cryptoxanthin, and cis‐isomers of s‐carotene. They were fresh leaves of Adansonia digitata, Ceiba pentandra. Hibiscus sabdarifa, and Vigna sp., dry leaves of Adansonia digitata, and flower and seed pulp of Adansonia digitata, Bixa orellano, Hibiscus sabdarifa, and Parkiia biglobosa. Plants were chosen because they were important to diet and were colored, with dark green, pink, orange, and yellow preferred. Color was assumed to reflect carotentoid concentrations. Four of the six were wild edible species, two were cultivated. Both fresh and dry samples were analyzed. On a weight basis, fresh samples contained twice the carotenoid content as dry samples. Colors did not reflect carotene content for the analyzed carotenoids. Fresh kapok leaves (Ceiba pentandra) contained twice the s‐carotene content of the reference food fresh spinach (Spinacea oleracea).

Folic acid plays a key role in nucleic acid biosynthesis, essential for normal cell proliferation and function. Localized folate deficiencies may be related to changes in cytology associated with cancer development ; analogs of folic acid, such as methotrexate, are potent chemotherapeutic agents and are widely used either alone or in combination therapy for many types of cancer. In this report we describe the synthesis of a terra-deuterated folic acid from perdeuterated toluene. The primary intermediate, N-(4-amino [2,3,5,6- 2 H 4 ]benzoyl)-L-glutamic acid diethyl ester was coupled to N(2')-acetyl-6-formylpterin to create [2',3',5',6'- 2 H 4 ]folic acid. A similar scheme can be used for the preparation of [1',2',3',4',5',6'- 13 C 6 ]folic acid from [ 13 C 6 ] ring labeled toluene.

Metabolism of a 73 mumol oral dose of beta-carotene-d8 in olive oil was determined from plasma beta-carotene-d8 and retinol-d4 concentration-time curves in an adult male. beta-Carotene-d8 and retinol-d4 concentrations in serial plasma were measured using high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), respectively. Plasma beta-carotene-d8 and retinol-d4 concentration-time curves were described by a 5-term and a 3-term polyexponential equation, respectively, using an empirical description of beta-carotene metabolism. A physiologic compartmental model of beta-carotene metabolism was also constructed and tested. This model suggests that 22% of the beta-carotene dose is absorbed: 17.8% as intact beta-carotene and 4.2% as retinoid. Also, it suggests that both liver and enterocyte are important in converting beta-carotene to retinoid; 43% is converted in liver and 57% in enterocyte. Finally, it suggests that the mean residence time for beta-carotene is 51 days and that the 73 mumole dose does not alter the fractional transfer coefficients of the system after absorption takes place. The issue of central versus eccentric cleavage of beta-carotene in humans can be studied with further modeling combined with use of appropriately labeled beta-carotene.

This report presents analytical methods for the isolation and quantification of all-trans-beta-carotene-d8 in human plasma following a 73 mumol oral dose. Retinol-d4 derived from beta-carotene-d8 was also determined in the same plasma. Plasma samples drawn over a 24 day period were analyzed. beta-Carotene and retinol were isolated and purified for analysis using a solid phase extraction protocol with aminopropyl-bonded silica sorbent. Ratios of beta-carotene-d8/beta-carotene were determined using reversed-phase HPLC with spectrophotometric detection, which fully resolved the isotopomers, and by tandem mass spectrometry (MS/MS) with electron ionization. Results obtained from MS/MS and HPLC analysis showed close agreement and demonstrated improved selectivity relative to analysis using a single mass analyzer. Retinol-d4 was converted to its tert-butyldimethylsilyl ether and analyzed by gas chromatography/mass spectrometry using selected ion monitoring. The ability to resolve the beta-carotene isotopomers by HPLC makes it feasible for investigators without mass spectrometers to investigate the dynamics of absorption and metabolism of beta-carotene-d8 in humans.

Two carboxylesterases (GPL1 and GPH1) were isolated from guinea pig hepatic microsomes and assayed for activity using the following pyrrolizidine alkaloids (PAs): seneciphylline (SNP), monocrotaline (MCT), and a mixture of senecionine (SEN) and integerrimine (INT) referred to as SEN-INT. GPH1 was able to effect the hydrolysis of all PAs, however, only minimal activity was seen for SEN-INT. The specific activity of GPL1 for p-nitrophenyl acetate was four times that of GPH1, but the former showed no activity toward PAs. The molecular weights and pIs were determined for both enzymes, and the Michaelis-Menten constants for two PAs, SNP and MCT were obtained using GPH1. The response to inhibitors confirmed GPH1 as a type B serine hydrolase although it was also inhibited by HgCl2. The isolation of a PA active esterase from the guinea pig may help to explain the resistance of this animal to PA intoxication, while enzyme substrate specificity may explain how the guinea pig's susceptibility to PA intoxication can differ toward various PAs.

The remarkable sensitivity of accelerator mass spectrometry (AMS) is finding many new applications in pharmacology. In this study AMS was used to measure [(14)C]-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. (14)C 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.

There is a need for an improved test of human ability to assimilate dietary vitamin B 12 . Assaying and understanding absorption and uptake of B 12 is important because defects can lead to hematological and neurological complications. Accelerator mass spectrometry is uniquely suited for assessing absorption and kinetics of carbon-14 ( 14 C)-labeled substances after oral ingestion because it is more sensitive than decay counting and can measure levels of 14 C in microliter volumes of biological samples with negligible exposure of subjects to radioactivity. The test we describe employs amounts of B 12 in the range of normal dietary intake. The B 12 used was quantitatively labeled with 14 C at one particular atom of the dimethylbenzimidazole (DMB) moiety by exploiting idiosyncrasies of Salmonella metabolism. To grow aerobically on ethanolamine, Salmonella enterica must be provided with either preformed B 12 or two of its precursors, cobinamide and DMB. When provided with 14 C-DMB specifically labeled in the C2 position, cells produced 14 C-B 12 of high specific activity (2.1 GBq/mmol, 58 mCi/mmol) (1 Ci = 37 GBq) and no detectable dilution of label from endogenous DMB synthesis. In a human kinetic study, a physiological dose (1.5 μg, 2.2 kBq/59 nCi) of purified 14 C-B 12 was administered and showed plasma appearance and clearance curves consistent with the predicted behavior of the pure vitamin. This method opens new avenues for study of B 12 assimilation.

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 0.5 nmol [ 14 C]pteroylmonoglutamate (100 nCi radioactivity) plus 79.5 nmol pteroylmonoglutamate in water orally. 14 C was measured in plasma, erythrocytes, urine, and feces for40 d. Kinetic modeling was used to analyze and interpret the data. Results: According to the data, the population was healthy and had a mean dietary folate intake of 1046 nmol/d, and the apparent dose absorption of 14 C was 79%. The model predictions showed that only 0.25% of plasma folate was destined for marrow, mean bile folate flux was 5351 nmol/d, and the digestibility of the mix (1046 5351 nmol/d) was 92%. About 33% of visceral pteroylmonoglutamate was converted to the polyglutamate form, most of the body folate was visceral (99%), most of the visceral folate was pteroylpolyglutamate (98%), total body folate was 225 mol, and pteroylpolyglutamate synthesis, recycling, and catabolism were 1985, 1429, and 556 nmol/d, respectively. Mean residence times were 0.525 d as visceral pteroylmonoglutamate, 119 d as visceral pteroylpolyglutamate, 0.0086 d as plasma folate, and 0.1 d as gastrointestinal folate. Conclusions: Across subjects, folate absorption, bile folate flux, and body folate stores were larger than prior estimates. Marrow folate uptake and pteroylpolyglutamate synthesis, recycling, and catabolism are saturable processes. Visceral pteroylpolyglutamate was an immediate precursor of plasma p-aminobenzoylglutamate. The model is a working hypothesis with derived features that are explicitly model-dependent. It successfully quantitated folate metabolism, encouraging further rigorous testing. Am J Clin Nutr 2004;80:680 –91.

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. On Day 53, the subjects were placed on a daily vitamin A supplement (10000 IU/day), and a second dose of [14C]beta-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 approximately 10-fold reduction in urinary excretion; and 3). a lower ratio of labeled retinyl ester/beta-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 beta-carotene. Respective values for the retest were 0.85 and 0.74. These results show that while less cleavage of beta-carotene occurred due to vitamin A supplementation, higher absorption resulted in larger molar vitamin A values.

Nous proposons un modele stochastique pour la cinetiques de marquage de cellules avec un produit radiomarque. Ce modele est compose d'un modele parametrique pour la distribution du temps de penetration du marqueur dans les cellules et d'un modele non-parametrique de fonction de survie de la combinaison cellule-marqueur. L'objectif est de modeliser ainsi l'evolution au cours du temps de la fraction de cellules marquees parmi l'ensemble des cellules. Une experience recente avec des hematies et du folate radiomarque fournit des mesures longitudinales de cette fraction de cellules marquees. Le modele semi parametrique propose est ajuste a ces donnees et quelques unes des implications sont explorees. La methode proposee inclut egalement des inferences basees su le bootstrap.

BACKGROUND In rural Vietnam, vitamin A deficiency is a concern. Among the indigenous fruit and vegetables, Momordica cochinchinensis (gac) fruit has been identified as having the highest beta-carotene concentration. Locally, it is mixed with rice in a preparation called xoi gac. OBJECTIVE The purpose of this study was to assess this beta-carotene- rich rice preparation as a source of provitamin A for children in rural Vietnam. DESIGN Preschoolers (n = 185) participated in a 30-d controlled supplementation trial. Children with low hemoglobin concentrations were assigned to 1 of 3 groups: a fruit group, who received xoi gac that contained 3.5 mg beta-carotene per serving; a powder group, who received rice mixed with 5.0 mg synthetic beta-carotene powder; and a control group, who received rice without fortification. RESULTS The mean increase in plasma beta-carotene concentrations in the fruit and powder groups was significantly greater than that in the control group (P < 0.0001). After supplementation, the mean plasma retinol concentration in the fruit group was significantly higher than that in the control (P = 0.006) and powder (P = 0.0053) groups. Among the children with initial hemoglobin concentrations

{beta}-Carotene is a tetraterpenoid distributed widely throughout the plant kingdom. It is a member of a group of pigments referred to as carotenoids that have the distinction of serving as metabolic precursors to vitamin A in humans and many animals [1,2]. We used Accelerator Mass Spectrometry (AMS) [3] to determine the metabolic behavior of a physiologic oral dose of {beta}-[{sup 14}C]carotene (200 nanoCuries; 0.57 {micro}mol) in a healthy human subject. Serial blood specimens were collected for 210-d and complete urine and feces were collected for 17 and 10-d, respectively. Balance data indicated that the dose was 42% bioavailable. The absorbed {beta}-carotene was lost slowly via urine in accord with the slow body turnover of {beta}-carotene and vitamin A [4]. HPLC fractionation of plasma taken at early time points (0-24-h) showed the label was distributed between {beta}-carotene and retinyl esters (vitamin A) derived from intestinal metabolism.

Whole blood folate level is a superior indicator of folate nutritional status than serum/plasma level. Problems with and lack of confidence in results of current whole blood folate assays have limited its popularity for assessing folate nutritional status. Here, an acid extraction GCMS detection method that measures total folate whole blood is presented. Folates are released from the matrix of whole blood and cleaved to para-aminobenzoic acid (pABA) by acid hydrolysis in the presence of [(13)C(6)]pABA as internal standard (IS). The hydrolysate is passed over a C18 resin to remove heme. The pABA isotopomers are ethyl esterified, isolated on C18 resin, and trifluoroacetylated. Following normal-phase HPLC separation, the isotopomers are silylated to their tBDMS derivatives. The abundance of these derivatives are measured at m/z 324 for [(13)C(6)]pABA as IS and m/z 318 for pABA from whole blood folate. Our method uses readily available chemicals and our results agree well with those using Lactobacillus casei, the current gold standard reference assay. The presence of folate analogs (methotrexate) or antibacterials (sulfonamines) does not affect our method. This feature makes it useful in monitoring folate status of patients undergoing chemotherapy. Before using our method, pABA supplements must be discontinued for a few days.

BACKGROUND Blood beta-carotene and vitamin A responses to oral beta-carotene are variable in humans. Some individuals are characterized as responders and others as low- or nonresponders. A better understanding of the conditions that produce the variability is important to help design public health programs that ensure vitamin A sufficiency. OBJECTIVE Our objective was to assess variability in absorption and conversion of beta-carotene to vitamin A in vivo in humans by using a novel double-tracer ?hexadeuterated (D(6)) beta-carotene and D(6) retinyl acetate approach. DESIGN Eleven healthy women were housed at the US Department of Agriculture Western Human Nutrition Research Center metabolic unit for 44 d, where they consumed diets adequate in vitamins and minerals except for carotenoids. After an adaptation period, the women were given 30 micromol D(6) retinyl acetate orally, followed 1 wk later with 37 micromol D(6) beta-carotene (approximately equimolar doses). Time-dependent plasma concentration curves were determined for D(6) retinol, D(6) beta-carotene, and trideuterated (D(3)) retinol (derived from D(6) beta-carotene). RESULTS Mean (+/-SE) absorption of D(6) beta-carotene was 3.3 +/- 1.3% for all subjects. The mean conversion ratio was 0.81 +/- 0.34 mol D(3) retinol to 1 mol D(6) beta-carotene for all subjects. However, only 6 of the 11 subjects had plasma D(6) beta-carotene and D(3) retinol concentrations that we could measure. The mean absorption of D(6) beta-carotene in these 6 subjects was 6.1 +/- 0.02% and their conversion ratio was 1.47 +/- 0.49 mol D(3) retinol to 1 mol D(6) beta-carotene. The remaining 5 subjects were low responders with

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 C-14-labeled folic acid (35 mu g, 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 II mu Sv. 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 C-14 atoms during erythropoiesis from the pulse of plasma [C-14]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. (C) 1999 Academic Press.

The role of beta-carotene in chemoprevention of cancers and other chronic diseases generated controversy when subpopulations taking beta-carotene supplements showed increased mortality in clinical trials. Determination of the dynamics of beta-carotene in individual human subjects has emerged as a high priority. Stable isotope labeled beta-carotene tracers can be employed to determine rates of conversion to retinol (vitamin A), but tracer doses must be small to minimize perturbation of endogenous retinoid and carotenoid pools. In such cases, ratios of labeled tracer/endogenous retinol are often low, and quantitative analysis at enrichments of1 mol% are unreliable owing to ion-molecule reactions that generate ions at the same mass as the labeled tracer even when no tracer is present. The current study demonstrates improved gas chromatography/mass spectrometry quantification of retinol-d4 and unlabeled retinol, as their tert-butyldimethylsilyl ethers, at low enrichments using an ion trap mass spectrometer operated in selected ion storage mode. Electron ionization of analyte takes place in the ion trap using conditions that eject ions outside the range m/z 390-420, and molecular ions at m/z 400 and 404 from retinol and retinol-d4 are quantified. Using this approach, unlabeled retinol yields a signal close to values calculated from natural isotopic abundances (approximately 0.13%), whereas several quadrupole instruments operated using selected ion monitoring yielded 2-5 times greater signal when no labeled retinol was present.

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.

Many of the commonly studied pyrrolizidine alkaloids (PAs) are built upon the subgroup retronecine (RET), which is released from the parent molecule by either base catalyzed or enzymatic hydrolysis of the ester linkages. The rate of appearance of RET in a hydrolytic study would thus reflect the rate of hydrolysis for the PA being tested. We have developed a gas chromatographic (GC) method to measure the release of RET from incubations of PAs with the guinea pig carboxylesterase, GPH1. The PAs tested were the following: jacobine (JAB), jacozine (JAZ), retrorsine (RES), and seneciphylline (SNP). The KmS for SNP and JAZ were determined to be 64.9 and 349.2 microM, respectively. In addition, a qualitative assessment of hydrolytic activity toward a radiolabelled mixture of retrorsine/riddelliine (RES/RIL) was performed with HPLC and radiometric detection.

The reaction of glutathione with the epoxide containing pyrrolizidine alkaloid, jacobine, was catalyzed by guinea pig hepatic glutathione-S-transferase enzymes in in vitro experiments; the rate of the reaction in the presence of rat hepatic glutathione-S-transferases did not differ from the non-enzymatic rate. Using ion-pairing liquid chromatography we were able to isolate the conjugate and obtain a daughter ion spectrum using Fast Atom Bombardment Mass Spectrometry together with Collisionally Activated Dissociation/Mass Analyzed Kinetic Energy experiments.

Abstract 3018 Poster Board II-994 Recent evidence from our laboratory and others suggests that a variable portion of ingested cobalamin (Cbl), either crystalline or from food, is degraded in the gastrointestinal tract. We have developed a biosynthetic method to incorporate 14C into the lower axial ligand of cobalamin that has made it possible to study the fate of this vitamin during its passage through the gastrointestinal tract and to assess the presence of Cbl or its breakdown products in biological samples. Following oral administration of an aqueous physiological tracer dose of 14C-Cbl (1.3 μg, 50 nCi), blood, urine, and feces are analyzed for 14C by accelerator mass spectrometry. In 9 subjects, the plasma response was consistent with the expected behavior of peroral Cbl: 14C-Cbl first appeared in the plasma 3h post-dose reaching a peak level within 6-8h. Confirmation that this dose appears bound to the physiological transport protein transcobalamin (TC) was obtained in a subset of subjects by an immunoaffinity method using anti-human TC antibody-coated magnetic beads which selectively bound 95-98% of plasma 14C. Urinary excretion of 14C was maximal in the first 24h, with 14C first appearing in urine as early as 1.5h after dosing. Fecal excretion occurred variably over several days. The amount of 14C found in the urine (10-50% of the dose) was 100-fold greater than in previous reports using 57Co-labeled cyanocobalamin (0.1-0.5%), and fecal excretion was lower than expected (10-20% vs 30-70%). Urinary excretion of 14C was inversely correlated with the peak plasma level of 14C attained (r2=0.610; p Disclosures: Green: Vitalea Science: Research Funding. Miller:Vitalea Science: Research Funding. Lee:Vitalea Science: Research Funding. Sutter:Vitalea Science: Research Funding. Allen:Vitalea Science: Research Funding. Dueker:Vitalea Science: Employment.