The biotransformation and urinary excretion of dexamethasone in equine male castrates

The pro-drugs of dexamethasone, a potent glucocorticoid, are frequently used as anti-inflammatory steroids in equine veterinary practice. In the present study the biotransformation and urinary excretion of tritium labelled dexamethasone were investigated in cross-bred castrated male horses after therapeutic doses. Between 40-50% of the administered radioactivity was excreted in the urine within 24 h; a further 10% being excreted over the next 3 days. The urinary radioactivity was largely excreted in the unconjugated steroid fraction. In the first 24 h urine sample, 26-36% of the total dose was recovered in the unconjugated fraction, 8-13% in the conjugated fraction and about 5% was unextractable from the urine. The metabolites identified by microchemical transformations and thin-layer chromatography were unchanged dexamethasone, 17-oxodexamethasone, 11-dehydrodexamethasone, 20-dihydrodexamethasone, 6-hydroxydexamethasone and 6-hydroxy-17-oxodexamethasone together accounting for approx 60% of the urinary activity. About 25% of the urinary radioactivity associated with polar metabolites still remains unidentified.     

Simple radioimmunological method for urinary 6-keto-PGF1 alpha measurement

A radioimmunoassay for the determination of 6-keto-PGF1 alpha (stable metabolite of Prostacyclin) in urine using a specific antiserum, is described. The antibody used, showed no significant cross-reactivity with other PGS. (3H)-6-keto-PGF1 alpha has been used as tracer and a 30% Hydroxyapatite suspension was utilized for the separation of bound and free fractions. Standard curve ranges between 2 and 1000 pg thus allowing measurements in urine. Aliquots of 0.2 to 1 ml of 24 hours urinary samples were submitted to a preextraction step with Hexane in order to eliminate neutral fats. After acidifying with 0.1 N HCl to pH = 3.5-4, an Ethyl Ether extraction step was performed showing a recovery of 66%. Repeated analysis of a rat urine pool, showed 7.7 intraassay and 9.8 interassay variation coefficients. Values obtained amounted to 38.3 +/- 4.6 ng/24 h in normal rats and 133 +/- 32 ng/24 h in 5% NaCl loaded rats (p less than 0.001). The values obtained in healthy men were 508 +/- 94 ng/24 h and in women 375 +/- 136 (p less than 0.05).     

Metabolism and pharmacokinetics of progesterone in the cynomolgus monkey (Macaca fascicularis)

[4-14C]Progesterone was administered to two cycling female monkeys during the luteal phase of the cycle, and blood and urine were sampled over a 24 h period. Progesterone had a volume of distribution of 1.75 +/- 0.3 L/kg, and a plasma elimination clearance of 0.06 +/- 0.03 L/kg/min. In comparison to the human, plasma progesterone binding was greater and progesterone clearance was slower in the cynomolgus monkey. The major unconjugated metabolite in plasma was 20 alpha-hydroxy-4-pregnen-3-one. In urine 6.2% of 14C-steroids were unconjugated, 2.3% of which were [14C]progesterone. Thin-layer chromatography (TLC) of conjugated metabolites in urine revealed that 24% had the mobility of sulfates, 19% that of glucuronides, and 52% were more polar. After hydrolysis of conjugates, a major fraction chromatographed with pregnanediol. However, despite evidence for the presence of a 20 alpha-hydroxyl group, none of the pregnanediol isomers could be identified among these 14C-steroids. Nevertheless, over 80% of urinary metabolites had sufficient analogy to pregnanediol to bind to an antiserum specific for ring D and the C-17 side-chain of pregnanediol.     

Estimation by gas chromatography-mass spectrometry with selected ion monitoring of urinary excretion rates of 3 alpha-androstanediol during/after i.v. administration of 13C-labelled testosterone in man

To study in vivo the conversion of testosterone (T) into its metabolites, dihydro-testosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta diol (3 alpha-Diol) the urinary excretion rates of these steroids were determined by mass spectrometry in 6 healthy men during/after the i.v. infusion (t = 4 h) of 20 mg [13C]testosterone. In addition, plasma concentrations of T, DHT and 3 alpha-Diol were determined by radioimmunoassay. During steady state conditions at the end of the 4-h infusion of [13C]T the increase in the plasma concentrations of T from, basal, 405 +/- 140 ng/dl to 4205 +/- 804 ng/dl was paralleled by an increase in the plasma concentrations of DHT to 106.4 +/- 62.5 ng/dl) (basal: 30.8 +/- 21.8 ng/dl), and of 3 alpha-Diol to 32.2 +/- 12.5 ng/dl (basal: 12.5 +/- 13.9 ng/dl). Plasma concentrations of T, DHT and 3 alpha-Diol then returned to basal concentrations within 24 hours. Using mass-spectrometry we found a cumulative renal excretion of 13C-labelled T of 15.6 +/- 9.6 micrograms/24 h, equivalent to 0.08 +/- 0.05% of the infused amount (20 mg) of [13C]T. Whereas urinary excretion of [13C]DHT was below the level of detection by mass-spectrometry the cumulative excretion of [13C]3 alpha-Diol was 67.7 +/- 19.9 micrograms/24 hours which is equivalent to 0.3 +/- 0.1% of the infused dose of 13C-labelled testosterone. These data suggest that the determination of urinary 3 alpha-Diol by mass-spectrometry during/after the infusion of stable-labelled testosterone represents an alternative to the use of radioactive label for turnover studies.     

Simultaneous determination of dexamethasone and 6 beta-hydroxydexamethasone in urine using solid-phase extraction and liquid chromatography: applications to in vivo measurement of cytochrome P450 3A4 activity

It has been demonstrated that the formation of the hydrophilic metabolites of dexamethasone, 6 alpha- and 6 beta-hydroxydexamethasone, correlated with cytochrome P450 (CYP) 3A4 enzyme levels. So, the 6 beta-hydroxydexamethasone/dexamethasone urinary ratio could be a specific marker for human CYP3A4 activity. We have developed a sensitive and specific high-performance liquid chromatographic method for the simultaneous quantification of urinary free dexamethasone and 6 beta-hydroxydexamethasone using 6 alpha-methylprednisolone as internal standard. This method involved a solid phase extraction of the three compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate (2 ml) followed by diethyl ether (1 ml). Separation of the three analytes was achieved within 24 min using a reversed-phase Nova-Pak C(18) analytical column (4 microm, 300 mm x 3.9 mm i.d.). An ultraviolet detector operated at 245 nm was used with a linear response observed from 10 to 100 ng/ml for dexamethasone and from 25 to 1000 ng/ml for 6 beta-hydroxydexamethasone. Obtained from the method validation, inter-assay precision was below 15% and accuracy ranged from 95.7 to 110%. The extraction efficiency of the assay was approximately of 99% and was constant across the calibration range. The lower limit of quantitation was 10 ng/ml for dexamethasone and 25 ng/ml for 6 beta-hydroxydexamethasone; at these levels, precision was below 16% and accuracy was 99-109%. This method was applied to in vivo measure of the CYP3A4 activity.     

Determination of estradiol-17-sulfate in human urine by a direct radioimmunoassay: urinary levels throughout the menstrual cycle

The measurement of urinary estradiol-17-sulfate concentration by direct radioimmunoassay was established. The urinary estradiol-17-sulfate levels measured by the radioimmunoassay correlated well with those determined by high-performance liquid chromatography equipped with electrochemical detector. Estradiol-17-sulfate concentrations in early morning urine of six healthy adult men was 8.2 +/- 2.0 ng/mL, or 5.7 +/- 1.8 ng/mg creatinine. The urinary levels in women throughout the menstrual cycle showed a characteristic three-peak excretion pattern: the first and the second peaks appeared just after and three days before the urinary LH peak, and the third peak appeared a few days before menstruation.     

Radioimmunoassay for plasma betamethasone 17-benzoate.

A sensitive radioimmunoassay for plasma betamethasone 17-benzoate has been developed. The antiserum used was obtained by immunizing rabbits with betamethasone 17-benzoate-21-hemisuccinate-bovine-serum-albumin conjugate. All of the endogenous steroids tested cross reacted less than 0.10%. A standard curve was established with a useful range from 0.05-5 ng. Reliability criteria were satisfactory. Measurement of plasma concentrations of betamethasone 17-benzoate was performed in patients and in rabbits following occlusive dressing of betamethasone 17-benzoate cream and gel base.     

A highly sensitive radioimmunoassay of atrial natriuretic peptide (ANP) in human plasma and urine

A highly sensitive radioimmunoassay has been established for measurement of human plasma and urine concentrations of atrial natriuretic peptide (ANP) and requires no extraction or concentration process such as Sep-Pak C-18 cartridge treatment. An antiserum was prepared from rabbits immunized with alpha-human ANP (alpha-hANP) coupled with bovine-thyroglobulin. The sensitivity of this method was 0.3 pg/tube of synthetic alpha-hANP utilized as authentic standard. Recovery of alpha-hANP spiked to plasma and urine was 97.7 +/- 15.4% and 97.1 +/- 9.5% (mean +/- SD), respectively. Plasma and urinary ANP concentrations versus assay data showed satisfactory linearity. In 124 healthy subjects, the plasma ANP-concentration was 31.7 +/- 12.0 pg/ml. Two different molecular forms of ANP in plasma and a single form in urine were found by gel permeation chromatography.     

A plasma dexamethasone radioimmunoassay

A double antibody radioimmunoassay for estimation of plasma dexamethasone is reported. Dexamethasone antiserum was produced by immunization of rabbits with dexamethasone-3-carboxymethyloxime-bovine serum albumin conjugate. All the endogenous steroids tested cross reacted less than 1%. Cortisol with a cross reaction of 0.4% gave significant interference in some plasma samples. This Interference could be removed by chromatography. The recoveries of dexamethasone added to plasma and corrected for procedural losses were 99 ± 9% after dichloromethane extraction and 98 ± 10% after paper chromatography. After dichloromethane extraction and after paper chromatography, the intraassay and inter-assay coefficients of variation were less than 11%. The peak dexamethasone levels were observed between 30 and 60 minutes after a single 1 mg oral dose in two normal subjects. The half-times of disappearance from plasma were 4 and 4.5 hours. During a constant infusion (50 μg/70 kg BW/hr) of dexamethasone phosphate, the plasma dexamethasone level reached a level of 250 ng/dl at 8 hours. It is concluded that plasma dexamethasone levels after either oral or intravenous administration may be measured specifically by radioimmunoassay.     

An imbalance between prostacyclin and thromboxane in relation to cerebral blood flow in neonates with maternal preeclampsia.

OBJECTIVE: A disturbance of prostacyclin (PGI2) and thromboxane A2 (TXA2) balance has been reported in preeclampsia. However, little is known about the concentrations of these prostanoids in neonates born to preeclamptic pregnant women. The purpose of this study is to determine whether the PGI2 and TXA2 concentrations are altered and whether the prostanoid balance correlates to the cerebral blood flow in neonates born to preeclampsia. METHODS: Spontaneously voided urine samples were collected from 20 neonates of normotensive and 16 neonates of preeclamptic women during the first 24 h after birth. We measured by radioimmunoassay the concentrations of urinary 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) and 11-dehydro-thromboxane B2 (11-dehydro-TXB2), respectively. Blood flow velocity in the middle cerebral artery was studied by pulsed Doppler ultrasonography in the neonates between 17 and 38 h after birth. RESULTS: There was no significant difference between the urinary 6-keto-PGF1alpha in the neonates of mothers with and without preeclampsia (median, 5.3 vs. 3.6 ng/mg of creatinine). In contrast, the urinary 11-dehydro-TXB2 and the ratio of 11-dehydro-TXB2 to 6-keto-PGF1alpha in the neonates of mothers with preeclampsia were significantly lower as compared with the neonates without preeclampsia, respectively (13.7 vs. 20.6 ng/mg of creatinine and 3.0 vs. 5.2, median). The resistance index in the middle cerebral artery was significantly reduced in the neonates with preeclampsia than without preeclampsia (0.67 +/- 0.01 vs. 0.74 +/- 0.02, mean +/- SEM). CONCLUSIONS: There was an association between maternal preeclampsia and the imbalance in the neonatal urinary excretion of PGI2 and TXA2 metabolites. This imbalance may contribute to the regulation of cerebral blood flow.     

Non-equilibrium method for the radioimmunoassay of clozapine in the presence of metabolites

Cross-reactions with metabolites are an ever-recurring problem encountered in the use of radioimmunoassay techniques to determine active compounds in biological material. Metabolites may interfere with the assay of the parent drug to a variable extent. Taking 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine (clozapine as an example, it was shown that the extent to which the antiserum produced interacts with the parent drug and the metabolites can be estimated by determining the equilibrium constants and the kinetics. In the present case, therefore, it was advantageous to carry out the radioimmunoassay in disequilibrium, i.e. in order to differentiate the metabolites from the parent drug, the sample was incubated with the antiserum for 10 min, after which the labelled antigen was added and the reaction mixture again incubated for a brief, exactly timed interval. It was shown that cross-reactions did not occur in mixtures of clozapine and its N-demethyl and N-oxide metabolites in the propor tions 1:1:2 over a range of concentration of 1.5-48 ng clozapine per 100 microliter human plasma. The equilibrium constants measured with the clozapine goat antiserum were as follows: clozapine 1.2 X 10(8) M-1, the N-demethyl metabolite 4.6 X 10(7) M-1 and the N-oxide metabolite 3.7 X 10(7) M-1 (pH 7.5 and 20 degrees C).     

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF1 alpha, PGE-M, 2,3-dinor-TxB2 and 11-dehydro-TxB2) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE2 rose (p less than 0.05) from 14.2 +/- 4.0 to 86.2 +/- 20.7, PGF2 alpha from 60.0 +/- 4.9 to 119.8 +/- 24.0, 6-keto-PGF2 alpha from 7.2 +/- 1.3 to 51.5 +/- 17.0, and TxB2 from 11.2 +/- 3.3 to 13.6 +/- 3.6 ng/h/1.73 m2 (means +/- SEM) at the maximal urine flow. Except for 6-keto-PGF1 alpha and TxB2, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged period of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB2 one can assume that this prostanoid represents renal as well as systemic TxA2 activity.     

The development of a radioimmunoassay for formoterol

The development of a radioimmunoassay (RIA) for the beta 2-stimulant formoterol is described. The sensitivity of the method is 0.1 ng/ml in plasma and urine, when a 1-ml sample is used. The cross-reactivity of the antiserum with formoterol glucuronide was 30%. Since formoterol is metabolized extensively to formoterol glucuronide in rats, dogs and humans, extraction with ethyl ether prior to the radioimmunoassay was carried out. Satisfactory agreement was obtained for levels of formoterol in plasma and urine when they were determined by RIA and gas chromatography-mass spectrometry. The concentration of formoterol was determined in dog plasma and human urine after oral administration of formoterol fumarate to dogs (61 mcg/kg) and humans (40 mcg).     

Radioimmunoassay of 16alpha-hydroxy-dehydroepiandrosterone and its sulfate.

A simple and reliable radioimmunoassay for plasma 3beta, 16alpha-dihydroxy-5-androsten-17-one(16alpha-OH-DHEA) and its sulfate has been developed. The antiserum against 16alpha-OH-DHEA and its sulfate (16alpha-OH-DHEA-3-sulfate) was produced in rabbits immunized with 16alpha-OH-DHEA-3-succinate-bovine serum albumin. This antiserum reacted well with both 16alpha-OH-DHEA and its sulfate and only slightly cross reacted with DHEA and its sulfate. The coefficient of variation (C.V.) of the intra assay was 10.26% for 16alpha-OH-DHEA and 12.32% for 16alpha-OH-DHEA-S. The C.V. of the interassay were 14.34% for 16alpha-OH-DHEA and 15.64% for 16alpha-OH-DHEA-S. The umbilical artery concentrations for 16alpha-OH-DHEA and 16alpha-OH-DHEA-S were 7.20 +/- 6.71 ng/ml and 4490 +/- 2140 ng/ml, and the umbilical vein concentrations were 14.20 +/- 11.27 ng/ml and 2970 +/- 1450 ng/ml respectively.     

Use of highly specific antibodies against 17alpha-OH-progesterone in a simplified nonchromatographic RIA and in the simultaneous determination of four sex hormones in human plasma.

A highly specific antiserum raised against the 3(O-carboxymethyl)oxime of 17alpha-hydroxyprogesterone (17-OHP) was produced in rabbit and used in the development of a specific radioimmunoassay (RIA) for 17-OHP which included a celite chromatography. Methods allowing the measurement of plasma 17-OHP levels either separately or in combination with that of several plasma androgens (testosterone, delta4-androstenedione and dehydroepiandrosterone) are described. Moreover, RIA meabsurement of plasma 17-OHP levels with or without chromatographic (celite column) purification gave comparable results (mean +/- SD) in 29 normal adult males (118 +/- 34 ng/100 ml) and 35 normal adult females (follicular phase: 46+/- 16 ng/100 ml); luteal phase: 241 +/- 71 ng/100 ml).     

The kinetics of papillotoxic doses of 3H-N-phenylanthranilic acid in rats

N-phenylanthranilic acid (N-PAA; 4 mmol/kg/day p.o.) causes a diffuse renal papillary necrosis and a polyuria in 7 days. A single dose of 3H-N-PAA was widely distributed with second-order elimination kinetics, t1/2 +/- 50 h for stomach, heart, kidney, and bladder and t1/2 greater than or equal to 90 h for liver, spleen, muscle and lung. The estimated plasma t1/2 = 10.2 h, and over 75% was excreted via urine in 36 h and 13% via faeces in 72 h. In chronically cannulated animals 29% of N-PAA-derived material was in bile and 24% in urine at 36 h, which suggests enterohepatic circulation. Bile and urine contained several metabolites but no parent compound. Multiple doses for 8 and 16 days increased urinary N-PAA excretion to 90% in 36 h, but faecal contents decreased to 6-8% in 72 h and plasma t1/2 to less than or equal to 7.5 h.     

Radioimmunoassay of human plasma corticosterone: method, measurement of episodic secretion and adrenal suppression and stimulation

A radioimmunoassay for human plasma corticosterone has been developed. Antisera were obtained by immunizing rabbits with corticosterone-21-hemisuccinate-BSA. An antiserum titer of 1:4000 was used for standard curves ranging from 0–1000 pg. Interfering steroids were removed from plasma extracts by paper chromatography. Plasma blanks obtained from adrenalectomized or Addisonian patients ranged from 29 to 42 ng/dl. Recovery of radioactive corticosterone through the entire method was 67.6 ± 5.2%. The coefficient of variation within assays was 19% and between assays 13%. The average 8 a.m. value in males was 396 ± 228 ng/dl and in females it was 655 ± 271 ng/dl. Corticosterone was found to be secreted episodically, in parallel with cortisol. Secretion of this steroid was suppressed by dexamethasone and stimulated by ACTH infusion.     

大熊猫生殖生物学研究:Ⅰ.大熊猫发情期血清和尿液中促黄体素、孕酮和17β-雌二醇含量的变化

作者用放射免疫法和生物测定法对4只大熊猫发情期血清和尿液中的促黄体素、孕酮和17β-雌二醇的含量进行分析。结果表明:3只大熊猫在发情高峰期均出现雌二醇和促黄体素高峰;LH峰值出现在E_2峰值之后。根据雌兽在发情期血清和尿液中这二种激素含量的变化可为选择人工授精的最佳时间提供可靠的理论依据。     

Thromboxane (TX)A3 and prostaglandin (PG)I3 are formed in man after dietary eicosapentaenoic acid: identification and quantification by capillary gas chromatography-electron impact mass spectrometry

The low incidence of myocardial infarction in Greenland Eskimos has been related to their traditional marine diet rich in eicosapentaenoic acid. However, whether dietary eicosapentaenoic acid is indeed transformed in man to antiaggregatory PGI3 and weakly proaggregatory TXA3 has not been clarified. In our studies we ingested either cod liver oil or mackerel both rich in eicosapentaenoic acid. Formation of TXB3, the hydrolysis product of TXA3, in platelet-rich plasma stimulated ex vivo with collagen was traced by capillary GC/EIMS. Via external standard, TXB3 formation in platelets was estimated to be 5-15% of TXB2 formation. From urine we extracted dinor metabolites of PGI according to a selective method. We utilized delta 17-2,3-dinor-6-keto-PGF1 alpha (PGI3-M) as an index of total body production of PGI3 in analogy to 2,3-dinor-6-keto-PGF1 alpha (PGI2-M), the major urinary metabolite of PGI2. We separated PGI2-M and PGI3-M as the Me, MO, Me3Si derivatives by capillary gas chromatography and identified PGI3-M by EI mass spectrometry. Excretion of PGI3-M, which was not detectable under control conditions, was 83 +/- 25 ng/24 h (SD) after ingestion of cod liver oil and 134 +/- 38 ng/24 h after mackerel ingestion, while excretion of PGI2-M was 162 +/- 52 ng/24 h and 236 +/- 32 ng/24 h, respectively. Our findings with diets rich in EPA show that it is possible in man to change in vivo the spectrum of biologically active prostanoids by nutritional means and alter it in a favourable direction.     

Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose

Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spectrometry (GC-MS). Fenspiride (A) and seven metabolites (B-->G) arising from oxidation on both the aromatic and heterocyclic substructures were detected in urine. The positive ion electron ionisation mass spectra of the TMS derivatives of fenspiride and its metabolites provided useful information on its metabolism. Positive ion methane chemical ionisation-GC-MS of the derivatives provided both derivatised molecular mass and structural information. Unchanged fenspiride can be detected in post-administration plasma and urine samples for up to 24 h. Maximum urinary levels of 100-200 ng ml(-1) were observed between 3 and 5 h after administration. After enzymatic deconjugation, the major phenolic metabolite (G) can be detected in urine for up to 72 h. This metabolite is the analyte of choice in the GC-MS screening of post-race equine urine samples for detection of fenspiride use. However, a distinct difference was observed in the urinary excretion of this metabolite between the thoroughbred horses used in UK study and the quarterbred and standardbred horses used for the USA administrations.