Stephen R. Dueker

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.

Carotenoid values of selected plant foods common to Southern Burkina Faso, West Africa

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).

Early human ADME using microdoses and microtracers: bioanalytical considerations.

Quantitative assessment of metabolites of drug candidates in early-phase clinical development presents an analytical challenge when methods, standards and assays are not yet available. Radioisotopic labeling, principally with radiocarbon ((14)C), is the preferred method for discovering and quantifying the absolute yields of metabolites in the absence of reference material or a priori knowledge of the human metabolism. However, the detection of (14)C is inefficient by decay counting methods and, as a result, high radiological human (14)C-doses had been needed to assure sensitive detection of metabolites over time. High radiological doses and the associated costs have been a major obstacle to the routine (and early) use of (14)C despite the recognized advantages of a (14)C-tracer for quantifying drug metabolism and disposition. Accelerator mass spectrometry eliminates this long-standing problem by reducing radioactivity levels while delivering matrix-independent quantitation to attomole levels of sensitivity in small samples or fractionated isolates. Accelerator mass spectrometry and trace (14)C-labeled drugs are now used to obtain early insights into the human metabolism of a drug candidate in ways that were not previously practical. With this article we describe some of our empirically based approaches for regualted bioanalysis and offer perspectives on current applications and opportunities for the future.

Accelerator Mass Spectrometry Can Be Used to Assess Vitamin A Metabolism Quantitatively in Boys in a Community Setting

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.

Chapter 2 - Mathematical Modeling in Nutrition: Constructing a Physiologic Compartmental Model of the Dynamics of β-Carotene Metabolism

This chapter discusses the mathematical modeling in nutrition: constructing a physiologic compartmental model of the dynamics of β-carotene metabolism. The chapter presents a model that is specific for β-carotene, and the general techniques and rationale used are typical of the compartmental modeling process. The model develops an analogy of the system under investigation, and obtains values for critical parameters of the model so that prediction is possible for the unobserved portions of the dynamic and kinetic behavior of the system under investigation. The first step in constructing a compartmental model is to examine the experimental observations for clues concerning the functionality of the system. Each alteration to the model parameters and associated iteration leads to the creation of a new model and a new prediction of the systems behavior. Once the model provides a good fit of experimental observations, the statistical certainties of the model parameters are inspected. As the mass of analyte in the system changes, the flows also changes, but the FTCs are expected to remain constant unless alternate pathways for metabolism of the tracee are induced by the large dose of tracer.

Overcoming bioanalytical challenges in an Onglyza® intravenous [14C]microdose absolute bioavailability study with accelerator MS

BACKGROUND An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range. RESULTS A technique-appropriate validation demonstrated the accuracy, precision, specificity, stability and recovery of the AMS methodology across the concentration range of 0.025 to 15.0 dpm/ml (disintegration per minute per milliliter), the equivalent of 1.91-1144 pg/ml. Based on the study sample analysis, the mean absolute bioavailability of saxagliptin was 50% in the eight subjects with a CV of 6.6%. Incurred sample reanalysis data fell well within acceptable limits. CONCLUSION This study demonstrated that the optimized sample pretreatment and chromatographic separation procedures were critical for the successful implementation of an UPLC plus AMS method for [(14)C]saxagliptin. The use of multiple-point standards are useful, particularly during method development and validation, to evaluate and correct for concentration-dependent recovery, if observed, and to monitor and control process loss and operational variations.

Methods and applications of HPLC–AMS

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

Protocol Development for Biological Tracer Studies

Improved instrumentation and the increased availability of labeled compounds have democratized the application of isotope-dilution (tracer) methodology in nutrient metabolism. Still, the most challenging aspects of tracer experimentation reside in the steps that precede the measurement of an isotopically labeled tracer, i.e. the design of a suitably labeled tracer and its isolation and purification from complex biological matrices. Construction of useful mathematical models of nutrient dynamics require methodologies that guarantee that the integrity of the tracer is maintained across the entire sampling and analyte isolation protocol. The ability to provide accurate and reliable data highlights a need for analytical chemists to play a central role in these studies. In this regard, examples and discussion of issues relevant to stable-isotope experimentation are provided.

Quantifying exploratory low dose compounds in humans with AMS

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.

Hydrolysis of pyrrolizidine alkaloids by guinea pig hepatic carboxylesterases

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 pigs susceptibility to PA intoxication can differ toward various PAs.