Major Metabolite In Blood Research Articles

Simultaneous quantification of cyclosporin A and its major metabolites by time‐of‐flight secondary‐ion mass spectrometry and matrix‐assisted laser desorption/ionization mass spectrometry utilizing data analysis techniques: Comparison with high‐performance liquid chromatography

Simultaneous quantification of cyclosporin A (CsA) and its major metabolite (AM1) in blood has been achieved using time‐of‐flight secondary‐ion mass spectrometry (TOF‐SIMS) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI/TOF‐MS). Previous investigations indicated that spectral interferences exist in the analysis of CsA blood samples by the above methods. In TOF‐SIMS, interference is caused by overlap of the Ag‐cationized internal standard, cyclosporin D (CsD), with the Ag‐cationized metabolite, AM1. To resolve this interference and obtain quantitative information, cross‐correlation analysis was applied to the TOF‐SIMS data. Application of damped non‐linear least squares curve‐fitting was carried out to resolve an interference in the MALDI/TOF‐MS data due to multiple cationization products (i.e. Na and K). Measurement of standard samples indicates that the minimum accuracy (95% confidence level) of the TOF‐SIMS method was better than 9% for CsA and 13% for AM1 using only one standard curve'. Similarly, the minimum accuracy of the MALDI/TOF‐MS method was determined to be 14% for CsA and better than 25% for AM1. Blood samples obtained from transplant patients receiving CsA were analyzed by polyclonal fluorescence polarization immunoassay, high‐performance liquid chromatography (HPLC), and by both TOF‐MS methods. Both TOF‐MS results for CsA and mono‐hydroxylated CsA are in good agreement with the HPLC results.

Absorption, tissue distribution and metabolism of chlorpyrifos in channel catfish following waterborne exposure

AbstractThe pharmacokinetics, tissue distribution, and metabolism of waterborne chlorpyrifos were investigated in channel catfish (Ictalurus punctatus) fitted with arterial and urinary catheters. Pharmacokinetic analysis indicated rapid absorption of parent chlorpyrifos from water into blood and slower distribution to peripheral tissues. Total 14C residue concentrations were highest in fat and lowest in muscle. Parent chlorpyrifos comprised > 90% of the total 14C residues in the whole fish. 14C residues were excreted by renal and biliary routes as metabolites of chlorpyrifos; excretion of the parent chemical was negligible. Trichloropyridinol (TCP) was the major metabolite in blood (approximately 40% of total residues), whereas the glucuronide conjugate of TCP was the major metabolite in urine (60–90%) and bile (> 90%). The pharmacokinetics and metabolism of water‐borne chlorpyrifos in the channel catfish were similar to the disposition of chlorpyrifos in other vertebrates.

Somatic development of the infant monkey following in utero exposure to caffeine

Caffeine has been associated with a number of reproductive and developmental effects in animals and humans. In an effort to characterize the potential effects of caffeine on the developing infant, 40 adult female monkeys ( Macaca fascicularis) were randomly divided into three groups and exposed at 0, 0.15, and 0.35 mg/ml (equivalent to 0, 10–15, and 25–30 mg/kg/day, respectively) of caffeine via their drinking water, before, during, and after pregnancy. Maternal blood and milk samples were collected following parturition. Infants were separated from their mothers within 12 hr of birth and were reared in a primate nursery. Blood samples were collected during the first week of life, and body weight, somatic measurements, and food consumption data were collected throughout the first 3 years. Maternal blood and milk concentrations and infant blood concentrations of caffeine and theophylline (the major blood metabolite of caffeine in the monkey) were similar to one another. Infant half-life of these methylxanthines was longer than that of the adult but not as long as that observed in human infants. Body weights and somatic measurements of male infants were significantly reduced over the first 30 days, as were a number of initial somatic measurements in both male and female infants. These deficits were not evident after 1 year of age. There were no treatment-related effects on infant tooth eruption or milk consumption. Results from this study support previously published results from this group of monkeys as well as studies by other researchers in rodents indicating that caffeine consumption during pregnancy can alter infant somatic development.

Hemolytic activity of ethylene glycol phenyl ether (EGPE) in rabbits

Studies were conducted to characterize the hemolytic effects of EGPE in rabbits following oral and dermal exposure, and to evaluate the in vitro hemolytic potential of EGPE and its major metabolite using rabbit red blood cells (RBC). Gavage administration of EGPE to female New Zealand White rabbits at 100, 300, 600, or 1000 mg/kg/day for up to 10 consecutive days (one dose/day) resulted in a dose-related intravascular hemolytic anemia. The hemolytic anemia was characterized by decreased RBC count, hemoglobin concentration, packed cell volume, hemoglobinuria, splenic congestion, renal tubule damage, and a regenerative erythroid response in the bone marrow. The hemolytic anemia was observed without alterations in RBC glutathione or methemoglobin. Phenoxyacetic acid (PAA) was identified as a major blood metabolite of EGPE. In vitro exposure of female rabbit erythrocytes indicated EGPE to be considerably more hemolytic than PAA. In a 90-day dermal study in which EGPE was applied to the skin of male and female New Zealand White rabbits 6 hr/day, 5 days/week, at doses up to 500 mg/kg/day, there was no indication of a hemolytic response. The only treatment-related effects were sporadic occurrences of slight erythema and scaling of skin at the site of test material application in high dose group male and female rabbits. However, erythema and scaling were not associated with gross or histopathologic changes and were not considered toxicologically significant.

The kinetics of cyclosporine and its metabolites in bone marrow transplant patients.

1. The pharmacokinetics of cyclosporine (CsA) and the time course of CsA metabolites were studied in five bone marrow transplant patients after intravenous (i.v.) administration on two separate occasions and once after oral CsA administration. 2. Cyclosporine and cyclosporine metabolites were measured in whole blood by h.p.l.c. 3. Cyclosporine clearance after i.v. administration decreased from 3.9 +/- 1.7 ml min-1 kg-1 to 2.0 +/- 0.6 ml min-1 kg-1 after 14 days of treatment. The mean +/- s.d. absolute oral bioavailability of cyclosporine was 17 +/- 11%. 4. Hydroxylated CsA (M-17) was the major metabolite in blood. There were no significant differences in the mean metabolite/CsA AUC ratios between the first and second i.v. studies. 5. After oral administration, the metabolite to CsA AUC ratios were higher for most metabolites compared to those observed in the second i.v. study, suggesting a contribution of intestinal metabolism to the clearance of CsA.

Simultaneous determination of isbufylline and its major metabolites in rabbit blood and urine by reversed-phase high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay for isbufylline and its major metabolites in rabbit blood and urine is described. After extraction, samples were eluted by a linear reversed-phase gradient. Specimens obtained after intravenous administration of isbufylline to rabbits were analysed to identify and subsequently quantify the potential metabolites. Using the ultraviolet absorption trace on the recorder as a reference, elution fractions were collected and analysed by mass spectrometry with the direct inlet system and gas chromatography—mass spectrometry after derivatization. Seven metabolites were identified and another five quantified. The method is specific, accurate, reproducible and recommended for pharmacokinetic studies.

Somatic Development of the Infant Monkey following in Utero Exposure to Caffeine

Somatic Development of the Infant Monkey following in Utero Exposure to Caffeine. GILBERT, S. G., AND RICE, D. C. (1991). Fundam. Appl Toxicol. 17, 454–465. Caffeine has been associated with a number of reproductive and developmental effects in animals and humans. In an effort to characterize the potential effects of caffeine on the developing infant, 40 adult female monkeys (Macaca fascicularis) were randomly divided into three groups and exposed at 0, 0.15, and 0.35 mg/ml (equivalent to0, 10–15, and 25–30 mg/kg/day, respectively) of caffeine via their drinking water, before, during, and after pregnancy. Maternal blood and milk samples were collected following parturition. Infants were separated from their mothers within 12 hr of birth and were reared in a primate nursery. Blood samples were collected during the first week of life, and body weight, somatic measurements, and food consumption data were collected throughout the first 3 years. Maternal blood and milk concentrations and infant blood concentrations of caffeine and theophylline (the major blood metabolite of caffeine in the monkey) were similar to one another. Infant half-life of these methylxanthines was longer than that of the adult but not as long as that observed in human infants. Body weights and somatic measurements of male infants were significantly reduced over the first 30 days, as were a number of initial somatic measurements in both male and female infants. These deficits were not evident after 1 year of age. There were no treatment-related effects on infant tooth eruption or milk consumption. Results from this study support previously published results from this group of monkeys as well as studies by other researchers in rodents indicating that caffeine consumption during pregnancy can alter infant somatic development.

Hemolytic Activity of Ethylene Glycol Phenyl Ether (EGPE) in Rabbits

Hemolytic Activity of Ethylene Glycol Phenyl Ether (EGPE) in Rabbits. BRESLIN. W. J., PHILIPS, J. E., LOMAX, L. G., BARTELS, M. J., DITTENBER, D. A., CALHOUN, L. L., AND MILLER, R. R. (1991). Fundam. Appl. Toxicol. 17, 466–481. Studies were conducted to characterize the hemolytic effects of EGPE in rabbits following oral and dermal exposure, and to evaluate the in vitro hemolytic potential of EGPE and its major metabolite using rabbit red blood cells (RBC). Gavage administration of EGPE to female New Zealand White rabbits at 100, 300, 600, or 1000 mg/kg/day for up to 10 consecutive days (one dose/day) resulted in a dose-related intravascular hemolytic anemia. The hemolytic anemia was characterized by decreased RBC count, hemoglobin concentration, packed cell volume, hemoglobinuria, splenic congestion, renal tubule damage, and a regenerative erythroid response in the bone marrow. The hemolytic anemia was observed without alterations in RBC glutathione or methemoglobin. Phenoxyacetic acid (PAA) was identified as a major blood metabolite of EGPE. In Vitro exposure of female rabbit erythrocytes indicated EGPE to be considerably more hemolytic than PAA. In a 90-day dermal study in which EGPE was applied to the skin of male and female New Zealand White rabbits 6 hr/day, 5 days/week, at doses up to 500 mg/kg/day, there was no indication of a hemolytic response. The only treatment-related effects were sporadic occurrences of slight erythema and scaling of skin at the site of test material application in high dose group male and female rabbits. However, erythema and scaling were not associated with gross or histopathologic changes and were not considered toxicologically significant.

Determination of GX 071 and its major metabolite in rat blood by cold on-column injection capillary GC/ECD

This work was done as part of a study of the pharmacokinetics and toxicokinetics of GX-071 (N-ethylperfluoro-octanesulfonamide) to support its registration as a pesticide. GX-071 and its major metabolite were identified in the blood of rats and dogs that had been given oral doses of GX-071

HPLC Measurement of Cyclosporine in Blood Plasma and Urine and Simultaneous Measurement of its Four Metabolites in Blood

Abstract A high performance liquid chromatographic assay has been developed for the estimation of cyclosporine and its four major metabolites in blood and for cyclosporine alone in plasma and urine samples. This assay employs a rapid and very reproducible solid-liquid extraction system. Isocratic chromatographic conditions allow the simultaneous measurement of cyclosporine and its four major metabolites in blood. The method is linear up to 2500 ng/ml and the minimum quantifiable limit for cyclosporine is 30 ng/ml, when 1 ml of sample is analyzed.

No evidence of a genetic polymorphism in the oxidative metabolism of midazolam.

The benzodiazepine midazolam is rapidly eliminated by oxidative metabolism. In young healthy volunteers elimination half-life (t1/2) is about 2.4 hours. A recent study showed a prolonged t1/2 from 8 to 22 hours in 6.5% of surgical patients, and a genetic polymorphism of midazolam's metabolism has been suggested. Therefore, we measured in 168 surgical patients the elimination of midazolam and its major hydroxylated metabolite (alpha-OH-midazolam) in blood and urine. Co-medication, disease status, smoking habits and alcohol intake were recorded; normal liver and kidney functions were assessed by routine laboratory tests. Midazolam was administered intravenously (0.1 to 0.2 mg/kg) for the induction of anaesthesia. Blood was drawn 1.5, 3, 4.5 and 6 hours after application and urine was collected for 6 hours. Plasma protein binding of midazolam was determined by equilibrium dialysis. Midazolam and alpha-OH-midazolam were measured in plasma by specific gas-liquid chromatography and in urine by high performance liquid chromatography. Data for the dose-corrected area under plasma-level curve of midazolam (AUC-midazolam/dose: 1.23 +/- 961 x 10(5) h/ml; mean +/- SD) and for the metabolic plasma ratio (AUC of alpha-OH-midazolam/AUC-midazolam: 0.52 +/- 0.28) demonstrated a log-normal distribution. Likewise, the percentage of the unbound fraction of midazolam in plasma (5.0 +/- 2.4%), urinary excretion of alpha-OH-midazolam (55.9 +/- 22.7% of dose) and the values for t1/2 (2.9 +/- 1.1 hours) did indicate a unimodal distribution. Age, comedication and smoking habits did not affect the disposition of midazolam. However, patients with regular intake of alcohol had a higher (p less than 0.05) metabolic ratio. Only in 3 patients could a prolonged t1/2 of midazolam from 7.5 to 10.2 hours be detected, but plasma levels and urinary excretion of alpha-OH-midazolam in those individuals were found to be normal. Therefore it is very unlikely that the oxidative metabolism of midazolam exhibits a genetic polymorphism.

Pharmacokinetics of 14C‐etretinate in healthy volunteers and two patients with biliary t‐tube drainage

The pharmacokinetic profile of 14C-etretinate, a retinoid that is effective in the treatment of psoriasis, was studied in six healthy male volunteers and two biliary T-tube patients. Following a 100 mg oral dose of 14C-etretinate (20 microcurie), etretinate and its major blood metabolites (etretin, isoetretin) were measured by HPLC and total carbon-14 was measured in blood, bile, urine, and feces by liquid scintillation counting. Etretinate was extensively metabolized in healthy volunteers and in T-tube patients. During the absorption phase, 75 per cent of the total radioactivity in the blood could be accounted for as etretinate, etretin, and isoetretin whereas these compounds accounted for only approximately 12 per cent of the blood radioactivity in T-tube patients over the same time period. The blood concentrations of etretinate, etretin, and isoetretin appeared to be substantially reduced in T-tube patients compared to those in healthy volunteers. A higher proportion of the total drug was excreted in the feces and bile of the T-tube patients (84 per cent) than in the feces of healthy volunteers (62 per cent). The major factor responsible for the observed decrease in etretinate blood concentrations following biliary cannulation appears to be the reduced absorption of etretinate due to the elimination of solubilizing bile salts in the duodenum. Carbon-14 related material was detected in urine and feces for as long as 3 weeks in healthy subjects supporting the previous observation that a long terminal elimination half-life exists for etretinate, even following a single dose of the compound.

Residues of macrolide antibiotic sedecamycin and its major metabolites in swine blood and tissues

Residues of macrolide antibiotic sedecamycin and its major metabolites in swine blood and tissues

The determination of propoxyphene, norpropoxyphene, and methadone in postmortem blood and tissues by high-performance liquid chromatography.

This paper describes the quantitative analysis of propoxyphene (PPX), its major metabolite, norpropoxyphene (NPPX), and methadone (METH) in blood and tissue specimens taken at autopsy in cases of suspected drug ingestion. Specimens are extracted into an organic solvent, back-extracted into acid, then reextracted into organic solvent and evaporated to dryness. The reconstituted extracts are subjected to analysis by reversed-phase ion-pair chromatography. The method is linear from 0.1 to 10 mg/L. Recoveries from blood are 86%, 93%, and 91% for PPX, NPPX, and METH respectively. Within-run coefficients of variation are 4.5%, 4.8%, and 2.6% and day-to-day coefficients of variation are 4.7%, 6.8%, and 3.7% for PPX, NPPX, and METH respectively at 1.0 mg/L for each drug.

Developmental effects of isotretinoin and 4-oxo-isotretinoin: the role of metabolism in teratogenicity.

Previous observations have indicated that isotretinoin (IT), a drug in common use for therapy of cystic acne, is teratogenic in humans but possesses low embryotoxicity in pregnant mice, probably because of its shorter half-life and limited placental transfer in rodents. In human volunteers and patients, one major blood metabolite of IT is 4-oxo-isotretinoin (4-oxo-IT) which undergoes slower elimination than IT and may itself be a participant in teratogenesis. To investigate the problem of species differences displayed by IT and the role of its metabolism, embryotoxic effects of 4-oxo-IT were examined after its single or repeated intubations into pregnant ICR mice and compared with the effects of a similar regimen of IT. The two compounds were also tested for their relative ability to suppress chrondrogenesis in the in vitro cell and organ culture assays. We found that a single dose of 4-oxo-IT, 100 mg/kg, given on day 11 of gestation (plug day = day 0 of gestation) produced a moderate incidence of limb reduction defects and cleft palate (39% and 27% of surviving fetuses, respectively), while a dose of 150 mg/kg affected virtually every fetus. IT, on the other hand, produced no defects in fetuses exposed to similar dose levels. Repeated intubations with IT, however, resulted in increasing the frequencies of limb reduction defects and cleft palate to levels obtained after 4-oxo-IT administration. We found that a 3-hour interval between IT intubations was more effective in this regard than an 8-hour interval. Repeated IT intubations also uncovered sharper stage-dependency of limb and palatal defects than obtained otherwise.(ABSTRACT TRUNCATED AT 250 WORDS)

3H]9-desglycinamide,8-arginine vasopressin: metabolism and in-vivo fate.

Half-lives based on the disappearance of [3H]9-desglycinamide,8-arginine vasopressin ([3H]DGAVP) following in-vitro incubation in plasma were 1.7 h (dog), 5.8 h (rat) and greater than 12 h (man). For all three species, and particularly dogs, biotransformation of the peptide in plasma occurred predominantly through carboxypeptidase activities, leading to the accumulation of AVP-(1-7). Disappearance of [3H]DGAVP from rat blood after a single i.v. injection followed a biphasic decay with half-lives of 2.2 +/- 0.8 (S.D.) min (distribution phase) and 14.4 +/- 1.2 min (elimination phase). The central and peripheral volumes of distribution were high and of the same order of magnitude, being 0.21 and 0.25 litres/kg respectively. Blood clearance values ranged from 36 to 45 ml/min per kg. In addition to [3H]AVP-(1-7), [3H]tyrosine was also found to be a major radioactive metabolite in blood. Compared with i.v. dosing, the s.c. route of administration for [3H]DGAVP resulted in longer-lasting peptide levels in blood which persisted for up to 4-5 h after injection. Maximal concentrations were reached at 7.5 min, whereafter they declined bi-exponentially with terminal half-lives of 31.1 +/- 8.7 min. The mean bioavailability for DGAVP was almost 100%, demonstrating virtually complete absorption from the s.c. injection site.

Identification of 11-dehydro-TXB 2 as a suitable parameter for monitoring thromboxane production in the human

In order to identify suitable parameters for measurement of thromboxane production in vivo the metabolism of TXB 2 was studied in the human. [ 3H 8]-TXB 2 was given intravenously to a healthy human volunteer. Blood samples were collected for 50 min after the injection, and urine was collected for 24 hours. The urinary and blood metabolic profiles were visualized by the use of two-dimensional TLC and autoradiography. Identification of metabolites was achieved with GC/MS and in some cases by cochromatography with reference compounds in TLC and GC. In blood, unmetabolized TXB 2 was the dominating compound during the first 30 min. Three less polar metabolites appeared, two of which were identified as 11-dehydro-TXB 2 and 11, 15-didehydro-13, 14-dihydro-TXB 2, respectively. The third compound was tentatively identified as 15-dehydro-13, 14-dihydro-TXB 2. Since 11-dehydro-TXB 2 was one of the major metabolites in blood as well as urine, it was deemed suitable as target for measurement of thromboxane production in vivo . The advantages of 11-dehydro-TXB 2 over its parent compound, TXB 2, were demonstrated in experiments where unlabeled TXB 2 was injected i.v. to a human volunteer, and the blood and urinary levels of both compounds were then followed by radioimmunoassay. Measured levels of 11-dehydro-TXB 2 were found to give a more reliable picture of metabolic events than TXB 2, the latter compound to a large extent reflecting technical difficulties during blood sample collection.

Selected ion monitoring analysis of pseudoracemic hexobarbital and its major metabolites in blood and urine of rats.

A stereospecific synthesis of N1-(2H3)-labelled R(-)-hexobarbital is described. A sensitive and rapid selected ion monitoring assay procedure for pseudoracemic hexobarbital, consisting of equal amounts of S(+)-hexobarbital (1a) and (2H3)-R(-)-hexobarbital (1b) in 100 microliters blood samples of rats was developed. Both the parent enantiomers in blood and three major metabolites excreted in urine were quantified. An application of the method in rats is described, and the results are compared to previously obtained data of separately administered enantiomers.

The metabolism of N-hydroxyphenacetin in vitro and in vivo.

N-Hydroxyphenacetin (100 mg/kg) injected i.p. into rats rapidly appeared in the blood and disappeared with a t1/2 of 14 min; phenacetin and 4-acetamidophenol were major metabolites in blood. Ferrihaemoglobin was formed, but 4-nitrosophenetole was not detected in blood. N-Hydroxyphenacetin injected i.p. into rats was excreted in the urine unchanged (partly conjugated 2.1% of the dose, 2% was excreted as phenacetin, 19% as 4-acetamidophenol) and 1.8% as 2-hydroxyphenacetin. In addition, small amounts of 3-hydroxyphenacetin (0.4%) and traces of N-[4-(2-hydroxyethoxy)phenyl]acetamide (beta-HAP) (0.05%) were found. Time-course kinetics have shown that N-hydroxyphenacetin is metabolized in vitro to phenacetin, 2- and 3-hydroxyphenacetin, and 4-acetamidophenol by microsomal and cytosolic preparations of rat and rabbit liver. However, after the initial reaction, the formation of phenacetin and 2- and 3-hydroxyphenacetin did not continue with time, indicating that these products were not formed enzymically. N-Hydroxyphenacetin incubated with rat erythrocytes formed ferrihaemoglobin; the relationship between ferrihaemoglobin, phenacetin and 4-nitrosophenetole concn indicated that N-hydroxyphenacetin was oxidized by oxyhaemoglobin to acetyl 4-ethoxyphenyl nitroxide, which yielded phenacetin and 4-nitrosophenetole spontaneously.

Determination of isotretinoin or etretinate and their major metabolites in human blood by reversed-phase high-performance liquid chromatography

A method is described for the quantitative analysis of isotretinoin and its 4-oxo metabolite, or of etretinate and its principal metabolites, in human blood in the range 10-2000 ng/ml. Following a simple one-step extraction, the compounds are determined by reversed-phase high-performance liquid chromatography (HPLC) with gradient elution and detection at 365 nm. This highly specific method separates the cis and trans isomers of the parent compounds and their metabolites. Examples are given of the application of this method to clinical studies of these two therapeutically important retinoids.