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1.
Clin Pharmacol Ther ; 102(3): 537-546, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28214288

ABSTRACT

14 C-labeled YH4808, a novel potassium-competitive acid blocker, was intravenously administered as a microtracer at 80 µg (11.8 kBq or 320 nCi) concomitantly with the nonradiolabeled oral drug at 200 mg to determine the absolute bioavailability and to assess the effect of pharmacogenomics on the oral absorption of YH4808. The absolute bioavailability was low and highly variable (mean, 10.1%; range, 2.3-19.3%), and M3 and M8, active metabolites of YH4808, were formed 22.6- and 38.5-fold higher after oral administration than intravenous administration, respectively. The product of the fraction of an oral YH4808 dose entering the gut wall and the fraction of YH4808 passing on to the portal circulation was larger in subjects carrying the variants of the CHST3, SLC15A1, and SULT1B1 genes. A combined LC+AMS is a useful tool to construct a rich and highly informative pharmacokinetic knowledge core in early clinical drug development at a reasonable cost.


Subject(s)
Anti-Ulcer Agents/administration & dosage , Drug Design , Mass Spectrometry/methods , Pharmacogenetics , Potassium Channel Blockers/administration & dosage , Administration, Intravenous , Administration, Oral , Adult , Anti-Ulcer Agents/pharmacokinetics , Biological Availability , Carbon Radioisotopes , Humans , Male , Potassium Channel Blockers/pharmacokinetics
2.
J Lipid Res ; 41(11): 1790-800, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11060348

ABSTRACT

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.


Subject(s)
beta Carotene/pharmacokinetics , Adult , Biological Availability , Carbon Dioxide , Carbon Radioisotopes , Feces/chemistry , Humans , Isotope Labeling/methods , Kinetics , Male , Photosynthesis , Spinacia oleracea , Tretinoin/blood , Vitamin A/blood , beta Carotene/blood , beta Carotene/urine
3.
Anal Biochem ; 283(2): 266-75, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10906248

ABSTRACT

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.


Subject(s)
Folic Acid/blood , Gas Chromatography-Mass Spectrometry/methods , Acids/chemistry , Adult , Calibration , Chromatography, High Pressure Liquid , Female , Humans , Hydrolysis , Luminescent Measurements , Male , Methotrexate/chemistry , Quality Control , Reference Standards
4.
Am J Clin Nutr ; 71(6): 1545-54, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10837297

ABSTRACT

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

Subject(s)
Vitamin A/blood , beta Carotene/administration & dosage , beta Carotene/pharmacokinetics , Absorption , Adult , Biological Availability , Deuterium , Diterpenes , Female , Humans , Kinetics , Retinyl Esters , Vitamin A/analogs & derivatives , beta Carotene/blood
5.
Article in English | MEDLINE | ID: mdl-10390055

ABSTRACT

Localized folate deficiency may be a risk factor for cancer. Since, folate binding proteins (FBP) and reduced folate carrier proteins (RFC) mediate cellular transport of folate, we compared FBP concentrations in several organs from tumor-bearing transgenic (TBT) mice and tumor-free non-transgenic controls (NTC) of the same strain, age, and fed identical diets. Liver, spleen, brain, small intestine and kidney were individually homogenized in phosphate-buffered saline (PBS) and separated into membrane, cytoplasmic, mitochondrial/lysomal and nuclear fractions (confirmed with marker enzymes). Homogenates and fractions was analyzed for total protein, and FBP. We used rabbit anti-bovine milk antibody and ELISA to measure FBP. FBP concentrations in kidney, small intestine, and spleen of TBT mice were higher than those of NTC mice; the opposite was true in liver and lung. FBP seemed to be upregulated in kidneys (all fractions), small intestine (all fractions), and spleen (cytoplasmic and nuclear fractions only) of TBT mice compared to NTC mice; the opposite appeared true in liver (all fractions) and lung (all fractions). FBP concentrations in brain, heart, and muscle of TBT mice were not different from those in brain, heart and muscle of NTC mice. A longitudinal study will determine if these changes in FBP concentrations precede tumor onset.


Subject(s)
Carrier Proteins/metabolism , Folic Acid/metabolism , Neoplasms, Experimental/metabolism , Receptors, Cell Surface , Animals , Cattle , Cell Fractionation , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Female , Folate Receptors, GPI-Anchored , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Transgenic , Rabbits , Subcellular Fractions , Tissue Distribution
6.
Anal Biochem ; 269(2): 348-52, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10222009

ABSTRACT

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.


Subject(s)
Erythrocytes/metabolism , Folic Acid/pharmacokinetics , Mass Spectrometry/methods , Adult , Carbon Radioisotopes , Folic Acid/administration & dosage , Folic Acid/blood , Humans , Male , Middle Aged
7.
Adv Exp Med Biol ; 445: 239-51, 1998.
Article in English | MEDLINE | ID: mdl-9781393

ABSTRACT

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.


Subject(s)
Folic Acid/pharmacokinetics , Models, Biological , Area Under Curve , Carbon Isotopes/analysis , Erythrocytes/chemistry , Feces/chemistry , Folic Acid/blood , Folic Acid/urine , Gas Chromatography-Mass Spectrometry , Hematocrit , Humans , Male , Middle Aged , Particle Accelerators , Sensitivity and Specificity
8.
Adv Exp Med Biol ; 445: 363-78, 1998.
Article in English | MEDLINE | ID: mdl-9781401

ABSTRACT

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.


Subject(s)
Chemistry Techniques, Analytical/methods , Gas Chromatography-Mass Spectrometry/instrumentation , Isotope Labeling/methods , Isotopes/analysis , Carbon Isotopes/analysis , Chemistry Techniques, Analytical/instrumentation , Chromatography, Gas , Chromatography, High Pressure Liquid , Deuterium/analysis , Humans , Isotope Labeling/economics , Kinetics , Vitamin A/chemistry , beta Carotene/analysis
9.
Am J Clin Nutr ; 67(5): 837-45, 1998 May.
Article in English | MEDLINE | ID: mdl-9583839

ABSTRACT

The reportedly inconsistent antioxidant protective effect of beta-carotene on plasma LDL may depend on LDL's beta-carotene concentration. We measured carbonyl production by CuSO4-challenged LDL from nine healthy women living at the US Department of Agriculture-Western Human Nutrition Research Center and consuming a natural food diet that provided only 0.14 micromol beta-carotene/d for 120 d. During the first 60 d, four women received a placebo and the remaining five women received too small a supplement (0.93 micromol beta-carotene/d) to increase plasma or LDL beta-carotene; therefore, the data for all nine women during this time were pooled. From days 61 to 120, all subjects received the small supplement. From days 101 to 120 they all received an additional, larger, mixed carotenoid supplement (6.16 micromol beta-carotene/d). Plasma beta-carotene dropped from 0.76 +/- 0.21 micromol/L (x +/- SEM) on day 2 to 0.33 +/- 0.08 on day 60 (P = 0.035) and rose to 1.73 +/- 0.18 (P = 0.001) on day 120. LDL beta-carotene dropped from 1.67 +/- 0.53 micromol/g LDL protein on day 2 to 1.27 +/- 0.28 micromol/g LDL protein on day 60 (P = 0.650) and rose to 10.04 +/- 1.07 micromol/g LDL protein (P = 0.001) on day 120. Plasma lycopene dropped from 0.20 micromol/L on day 2 to 0.02 micromol/L on day 60 and did not increase by day 120. Carbonyl production rose from 24 +/- 6 micromol/g LDL protein on day 2 to 42 +/- 4 micromol/g LDL protein (P = 0.001) on day 60 and dropped to 6 +/- 1 micromol/g LDL protein (P = 0.001) on day 120. LDL seemed fully protected with 9.7 +/- 2.5 micromol beta-carotene/g LDL protein, or 2.3 +/- 1.8 micromol beta-carotene/L plasma.


Subject(s)
Antioxidants/administration & dosage , Lipoproteins, LDL/drug effects , beta Carotene/administration & dosage , Adolescent , Adult , Aldehydes/chemistry , Aldehydes/metabolism , Antioxidants/pharmacology , Carotenoids/blood , Copper Sulfate/pharmacology , Diet , Dietary Supplements , Dose-Response Relationship, Drug , Double-Blind Method , Female , Free Radicals/chemistry , Free Radicals/metabolism , Humans , Ketones/chemistry , Ketones/metabolism , Lipid Peroxidation/drug effects , Lipoproteins, LDL/blood , Lipoproteins, LDL/metabolism , Lycopene , Menstrual Cycle/drug effects , Middle Aged , Placebos , Regression Analysis , Skin/drug effects , Skin/pathology , Time Factors , beta Carotene/blood , beta Carotene/pharmacology
10.
Anal Chem ; 70(7): 1369-74, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9553494

ABSTRACT

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 of < 1 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.


Subject(s)
Vitamin A/chemistry , Adult , Deuterium , Female , Gas Chromatography-Mass Spectrometry/methods , Humans , Isotope Labeling , Kinetics , Organosilicon Compounds/chemistry , Vitamin A/metabolism
12.
J Lipid Res ; 36(8): 1825-38, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7595103

ABSTRACT

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.


Subject(s)
Antioxidants/pharmacokinetics , Body Fluid Compartments , Carotenoids/pharmacokinetics , Dietary Fats/metabolism , Vitamin A/blood , Absorption , Carotenoids/blood , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Humans , Male , Middle Aged , Models, Biological , beta Carotene
13.
Clin Chem ; 41(7): 1028-32, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7600683

ABSTRACT

This noninvasive method for collection and analysis of a wide range of aldehydes and ketones in human breath may enable assessment of lipid peroxidation and metabolic status in vivo. Breath samples are drawn through silica cartridges impregnated with 2,4-dinitrophenylhydrazine, which traps carbonyls as their hydrazone derivatives. The hydrazone derivatives are eluted from the cartridges with acetonitrile, separated by reversed-phase HPLC, and quantified spectrophotometrically. Using this method, we have measured formaldehyde, acetaldehyde, acetone, propanal, 2-butanone, butanal, pentanal, and hexanal. Recoveries of carbonyls added to Douglas bags were > 90%, except for 2-butanone, which was 86.2%. The overall CVs for sampling plus analyzing duplicate aliquots of breath were < 11%. The results indicate that this protocol can be used to monitor changes of carbonyl production by analyzing expired air, which may, with further study, indicate physiological and pathological status.


Subject(s)
Aldehydes/analysis , Breath Tests , Chromatography, High Pressure Liquid/methods , Ketones/analysis , Specimen Handling/methods , Acetaldehyde/analysis , Acetone/analysis , Butanones/analysis , Formaldehyde/analysis , Humans , Hydrazones , Phenylhydrazines , Volatilization
14.
Arch Toxicol ; 69(10): 725-8, 1995.
Article in English | MEDLINE | ID: mdl-8572933

ABSTRACT

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.


Subject(s)
Pyrrolizidine Alkaloids/metabolism , Animals , Carboxylic Ester Hydrolases/metabolism , Chromatography, Gas/methods , Chromatography, High Pressure Liquid , Guinea Pigs , Hydrolysis , Pyrrolizidine Alkaloids/chemistry , Pyrrolizidine Alkaloids/isolation & purification
15.
Anal Chem ; 66(23): 4177-85, 1994 Dec 01.
Article in English | MEDLINE | ID: mdl-7847624

ABSTRACT

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.


Subject(s)
Adjuvants, Immunologic/blood , Carotenoids/blood , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/isolation & purification , Administration, Oral , Calibration , Carotenoids/administration & dosage , Carotenoids/isolation & purification , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Humans , Isotope Labeling , Magnetic Resonance Spectroscopy , Mass Spectrometry , Reproducibility of Results , Spectrometry, Mass, Fast Atom Bombardment , Vitamin A/blood , Vitamin A/isolation & purification , beta Carotene
16.
Biochem Biophys Res Commun ; 198(2): 516-22, 1994 Jan 28.
Article in English | MEDLINE | ID: mdl-8297362

ABSTRACT

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.


Subject(s)
Glutathione Transferase/metabolism , Glutathione/analogs & derivatives , Glutathione/metabolism , Liver/enzymology , Pyrrolizidine Alkaloids/metabolism , Animals , Cytosol/enzymology , Glutathione/biosynthesis , Guinea Pigs , Inactivation, Metabolic , Male , Rats , Rats, Sprague-Dawley , Spectrometry, Mass, Fast Atom Bombardment
17.
Clin Chem ; 39(11 Pt 1): 2318-22, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8222228

ABSTRACT

Solid-phase extraction permits the parallel processing of samples in large numbers. We have applied this technique to the isolation of retinol isotopomers from plasma of humans participating in a study of vitamin A stable isotope dilution. The isotopomers were analyzed by gas chromatography/mass spectrometry. The extraction involves the separation of retinol from its aqueous matrix with a C18 silica-based sorbent followed by removal of lipid contaminants with an aminopropyl silica-based sorbent. Overall recovery of retinol from plasma was 47.2% +/- 1.8%. Purity of the retinol isolated from plasma is comparable with that obtained with a single HPLC method. This method permits the preparation of 32 samples per day by one analyst. Elimination of the need for HPLC permits sample preparation in the field with a minimum of equipment and technical skill.


Subject(s)
Vitamin A/blood , Acetonitriles , Adsorption , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Humans , Indicator Dilution Techniques , Male , Middle Aged , Propylamines , Silicon Dioxide
18.
Toxicol Appl Pharmacol ; 117(1): 116-21, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1440605

ABSTRACT

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.


Subject(s)
Carboxylic Ester Hydrolases/metabolism , Isoenzymes/metabolism , Liver/enzymology , Pyrrolizidine Alkaloids/metabolism , Animals , Carboxylic Ester Hydrolases/antagonists & inhibitors , Carboxylic Ester Hydrolases/isolation & purification , Guinea Pigs , Hydrolysis , Kinetics , Male , Microsomes, Liver/enzymology , Monocrotaline/metabolism
19.
Drug Metab Dispos ; 20(2): 275-80, 1992.
Article in English | MEDLINE | ID: mdl-1352221

ABSTRACT

The comparative metabolism of the pyrrolizidine alkaloid, [14C]monocrotaline, was studied using rat and guinea pig hepatic microsomes. Metabolites were quantified to the nanomole level using HPLC and radiometric detection. Triorthocresylphosphate and carbon monoxide were used to assess the involvement of carboxylesterases and cytochrome P-450 in the hepatic microsomal metabolism of monocrotaline, respectively. Esterase hydrolysis accounted for 92% of the metabolism in the guinea pig; the rat displayed no esterase activity. This result may explain the guinea pig's resistance to pyrrolizidine alkaloid toxicity. Dehydropyrrole was found to be the major pyrrolic metabolite in the guinea pig, although colorimetric analysis indicated multiple pyrrolic moieties in the rat microsomal incubations.


Subject(s)
Microsomes, Liver/metabolism , Monocrotaline/metabolism , Tritolyl Phosphates/metabolism , Animals , Carbon Monoxide/pharmacology , Carboxylic Ester Hydrolases/metabolism , Colorimetry , Guinea Pigs , Inactivation, Metabolic , Male , Radiometry , Rats , Rats, Inbred Strains
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