Chromatographic analysis of endogenous retinoids in tissues and serum

We present a reliable, highly sensitive, and versatile method for the simultaneous determination of endogenous polar (acidic) and apolar (retinol, retinal, and retinyl esters) retinoids in various biological matrices. Following a single liquid extraction of retinoids from tissues or plasma with isopropanol, polar retinoids are separated from apolar retinoids and neutral lipids via automated solid-phase extraction using an aminopropyl phase. After vacuum concentration to dryness and reconstitution of the residue in appropriate solvents, the obtained fractions are injected onto two different high-performance liquid chromatography (HPLC)-systems. Polar retinoids are analyzed on a RP18 column (2.1mm ID) using a buffered gradient composed of methanol and water and on-column-focusing large-volume injection. Apolar retinoids are separated on a normal-bore RP18 column using a nonaqueous gradient composed of acetonitrile, chloroform, and methanol. Both HPLC systems are coupled with UV detection, and retinoids are quantitated against appropriate internal standards. The method was validated with regard to recovery, precision, robustness, selectivity, and analyte stability. Using 400 microl serum or 200mg tissue, the limits of detection for all-trans-retinoic acid were 0.15ng/ml or 0.3ng/g, respectively. The corresponding values for retinol were 1.2ng/ml or 2.4ng/g, respectively. This method was successfully applied to mouse, rat, and human tissue and serum samples.     

Selective and sensitive determination of amisulpride in human plasma by liquid chromatography-tandem mass spectrometry with positive electrospray ionisation and multiple reaction monitoring

The development of a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionisation (ESI(+)) and multiple reaction monitoring (MRM) for the selective and sensitive bioanalytical determination of amisulpride, a substituted benzamide derivative, in human plasma is described. Plasma was cleaned up using a liquid-liquid extraction (diisopropylether:dichloromethane=1:1 (v/v)) procedure. The chemically related drug sulpiride was used as internal standard (ISTD) and a primary calibration function was established in the concentration range of 0.50-500.52 ng/ml for amisulpride in plasma by triple analysis of the corresponding calibration standards. A linear relationship between concentration and signal intensity (given as peak area ratio analyte/ISTD) was obtained (linear regression: r=0.9999). A lower limit of quantification (LLQ) of 0.50 ng/ml was used during measurement of study plasma samples. Satisfying results of within-day precision (CV=0.79 to 1.98%) and accuracy (mean relative deviation: -1.68 to 3.58%) and between-day precision (CV=1.34 to 4.62%) and accuracy (mean relative deviation: -1.73 to -3.77%) as well as of recovery (amisulpride: 81.74 to 84.83%; sulpiride: 58.65%) and selectivity investigations confirmed the high reliability of this established LC-MS/MS method. Sufficient stability of amisulpride in plasma was achieved during freeze-thaw-cycles, for storage periods of 24h at room temperature and 20 days at <-20 degrees C as well as in extracts (storage conditions: <-20 degrees C, 6 days and 7 degrees C, 6 days) with mean relative deviations between - 2.83 and 2.91%. An example of a pharmacokinetic profile determined after administration of an amisulpride 200mg dose in a pilot study is given in this paper. A peak plasma concentration (C(max)) of 522.58 ng/ml was achieved after 3.55 h (t(max)). Corresponding values of areas under the plasma concentration-time curve (AUC) of 3405.35 ngh/ml (AUC(0-infinity)) and 3306.54 ngh/ml (AUC(0-tlast)) were obtained. The terminal plasma elimination half-life (t(1/2)) was 10.36 h.     

First LC-MS/MS electrospray ionization validated method for the quantification of perindopril and its metabolite perindoprilat in human plasma and its application to bioequivalence study

A high throughput bioanalytical method based on solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS), has been developed for the estimation of perindopril and its metabolite perindoprilat, an angiotensin-converting enzyme inhibitor in human plasma. Ramipril was used as internal standard (IS). The extraction of perindopril, perindoprilat and ramipril from the plasma involved treatment with phosphoric acid followed by solid phase extraction (SPE) using hydrophilic lipophilic balance HLB cartridge. The SPE eluate without drying were analyzed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode to quantify perindopril and perindoprilat in human plasma. The total chromatographic run time was 1.5 min with retention time for perindopril, perindoprilat and ramipril at 0.33, 0.35 and 0.30 min. The developed method was validated in human plasma matrix, with a sensitivity of 0.5 ng/ml (CV, 7.67%) for perindopril and 0.3 ng/ml (CV, 4.94%) for perindoprilat. This method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect especially because the pattern of elution of all the analytes appears as flow injection elution. Sample preparation by this method yielded extremely clean extracts with very good and consistent mean recoveries; 78.29% for perindopril, 76.32% for perindoprilat and 77.72% for IS. The response of the LC-MS/MS method for perindopril and perindoprilat was linear over the range 0.5-350.0 ng/ml for perindopril and 0.3-40 ng/ml for perindoprilat with correlation coefficient, r>/=0.9998 and 0.9996, respectively. The method was successfully applied for bioequivalence studies in human subjects samples with 4 mg immediate release (IR) formulations.     

Solid-phase extraction-liquid chromatographic method for the determination and pharmacokinetic studies of albiflorin and paeoniflorin in rat serum after oral administration of Si-Wu decoction

A sensitive and rapid high-performance liquid chromatography (HPLC) method with solid-phase extraction (SPE) to simultaneously determine albiflorin and paeoniflorin in rat serum was described. Serum samples were pretreated with solid-phase extraction using Extract-Clean cartridges, and the extracts were analyzed by HPLC on a reversed-phase C(18) column and a mobile phase of acetonitrile-0.03% formic acid (17:83 (v/v)) with ultraviolet detection at 230 nm. Pentoxifylline was used as the internal standard (IS). The linear ranges of the calibration curves were 29-1450 ng/ml for albiflorin and 10-2000 ng/ml for paeoniflorin. The intra- and inter-day precisions (R.S.D.) were 相似文献   

Adrenal-testicular-pituitary relationships in the cheetah subjected to anesthesia/electroejaculation

The influence of electroejaculation on the acute response in serum cortisol, testosterone and luteinizing hormone (LH) was studied in the South African cheetah . Males were either anesthetized with CT-1341 and 1) serially bled only (controls, n = 7) or 2) serially bled during and following a regimented protocol of rectal probe electroejaculation (n = 14). In the control cheetahs , mean cortisol concentrations declined over time (P less than 0.05) and neither testosterone nor LH varied over the 145-min sampling interval. Serum cortisol rose immediately in electroejaculated cheetahs , peaked at the end of electroejaculation in 13 of 14 males and then declined during the next 90 min. Temporal profiles and serum levels of testosterone and LH were similar in the electroejaculated and control groups (P greater than 0.05). Within individual cheetahs , serum levels of LH and testosterone were highly correlated (r = 0.77, P less than 0.01). Awake (n = 2) and CT-1341 anesthetized (n = 2) cheetahs also were bled and then challenged with an i.m. injection of 25 IU adrenocorticotropic hormone (ACTH, Cortrosyn). Serial blood samples were collected during the next 2 h and assayed. Cortisol concentrations prior to ACTH administration were greater in awake than in anesthetized males. In all animals, cortisol rose immediately and peaked within 30-60 min of injection. Whereas all 4 ACTH-treated cheetahs produced cortisol titers in excess of 200 ng/ml, only 4 of 14 electroejaculated males produced cortisol levels comparable to this concentration range. Neither testosterone nor LH profiles were affected by ACTH-induced elevations in cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Liquid chromatography-tandem mass spectrometry validated method for the estimation of indapamide in human whole blood

A highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5 ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO(4) followed by liquid-liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC-MS/MS method for indapamide was linear over the range of 0.5-80.0 ng/ml with correlation coefficient, r>or=0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of +/-20% at 0.5 ng/ml and +/-15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies and also applied to subject-sample analysis of bioequivalence study for 1.5mg sustained-release (SR) formulations.     

Determination of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in human serum using liquid chromatography-tandem mass spectrometry

Concentrations of mono(2-ethylhexyl)phthalate (MEHP), and di(2-ethylhexyl)phthalate (DEHP), in serum of healthy volunteers were determined by high performance liquid chromatography (HPLC) with tandem mass spectrometry (LC/MS/MS). The serum was extracted with acetone, followed by hexane extraction under acidic conditions, and then applied to the LC/MS/MS. Recoveries of 20 ng/ml of MEHP and DEHP were 101+/-5.7 (n=6) and 102+/-6.5% (n=6), respectively. The limits of quantification (LOQ) of MEHP and DEHP in the method were 5.0 and 14.0 ng/ml, respectively. The concentration of MEHP in the serum was at or less than the LOQ. The concentration of DEHP in the serum was less than the LOQ. Contaminations of MEHP and DEHP from experimental reagents, apparatus and air during the procedure were less than the LOQ and were estimated to be <1.0 and 2.2+/-0.6 ng/ml, respectively. After subtraction of the contamination, the net concentrations of MEHP and DEHP in the serum were estimated at or <5 and <2 ng/ml, respectively. To decrease contamination by DEHP, the cleanup steps and the apparatus and solvent usage were minimized in the sample preparation procedures. The high selectivity of LC/MS/MS is the key for obtaining reliable experimental data from in the matrix-rich analytical samples and for maintaining a low level contamination of MEHP and DEHP in this experimental system. This method would be a useful tool for the detection of MEHP and DEHP in serum.     

13-cis-retinoic acid is an endogenous compound in human serum

The occurrence of 13-cis-retinoic acid as an endogenous component in human serum has been confirmed by cochromatography with standards in both normal-phase and reverse-phase high-performance liquid chromatographic (HPLC) system, by the lambda max of its UV spectrum recorded simultaneously with the HPLC run, and by chromatography of its methyl derivative. The method using solid-phase extraction followed by a gradient reverse-phase HPLC procedure with an internal standard and sensitive UV detector, provides an efficient and sensitive technique for the separation and quantification of serum 13-cis- and all-trans-retinoic acid. Serum levels of 13-cis- and all-trans-retinoic acid in 26 fasting volunteers ranged from 1.0 to 2.2 ng/ml (mean +/- SEM = 1.4 +/- 0.3 ng/ml) and from 1.1 to 1.9 ng/ml (mean +/- SEM = 1.4 +/- 0.2 ng/ml), respectively. The levels determined by a liquid-liquid double-phase extraction method were 90% higher in both 13-cis- and all- trans-retinoic acid than those from a solid-phase extraction. Human small intestine can isomerize all-trans-retinoic acid. 13-cis-Retinoic acid is the predominant cis isomer after incubation of intestinal mucosa homogenates with all-trans-retinoic acid. Moreover, the concentration of retinoic acid in serum is related to diet in that the level of total retinoic acid was 36% higher (n = 10) 2 h after a nonstandard breakfast than in fasting subjects.     

Plasma and uterine cortisol, progesterone and protein changes in pseudopregnant gilts treated with hydrocortisone acetate

To determine the effects of cortisol concentrations during pregnancy, gilts, made pseudopregnant through twice daily administration of 5 mg estradiol benzoate on Days 11 to 15 (Day 0 = first day of estrus), received either 5 mg/kg body weight of hydrocortisone acetate (HA) in sesame oil (n=5) or sesame oil alone (n=6) twice daily on Days 21 to 30. Blood samples (20 ml) were collected on Days 11, 21 and 31. Uterine flushings were obtained surgically on Day 31. The HA-treated gilts had higher (P<0.01) plasma cortisol (295.7 vs 35.6 ng/ml) and lower (P<0.01) plasma progesterone (8.9 vs 17.8 ng/ml) concentrations than did controls. Uterine flushings recovered from HA-treated gilts had significantly (P<0.01) higher cortisol (9.9 vs 5.6 ng/ml), lower progesterone (2.1 vs 6.8 ng/ml) and lower total protein (8.3 vs 21.4 mg/ml) levels than the control animals. Cortisol measured in the uterine flushings of the gilts was more than 85% unbound. Plasma corticosteroid binding globulin binding capacity was lower (P<0.05) in HA-treated gilts (7.4 nmol/l) than in the control (38.7 nmol/l) animals on Day 31. Corpora lutea (CL) number and weight were lower (P<0.05) in HA-treated than control gilts. However, progesterone concentration per CL did not differ between the 2 groups. These results indicate that elevated cortisol levels can alter endocrine and uterine functions related to pregnancy using the pseudopregnant gilt as a model.     

Simultaneous determination of 6beta-hydroxycortisol and cortisol in human urine by liquid chromatography with ultraviolet absorbance detection for phenotyping the CYP3A activity determined by the cortisol 6beta-hydroxylation clearance

This study describes a high-performance liquid chromatographic (HPLC) method for the simultaneous determination of 6beta-hydroxycortisol (6beta-OHF) and cortisol in human urine using either methylprednisolone or beclomethasone as internal standard. Separation was achieved on a reversed-phase phenyl column by a gradient elution of 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH with acetonitrile (4:6, v/v). 6beta-Hydroxycortisol and cortisol were monitored by UV absorption at 239 nm. The lower quantitation limits of the present HPLC method were 21.5 ng/ml for 6beta-OHF and 5.0 ng/ml for cortisol in urine. The within-day reproducibilities in the amounts of 6beta-OHF and cortisol determined were in good agreement with the actual amounts added, the relative error being less than 1.59%. The inter-assay precisions (R.S.D. values) were less than 7.91% for 6beta-OHF and cortisol. The method was compared with the GC/MS method by measuring 6beta-OHF in the same urine samples. A good correlation was found between the amounts determined by the two methods. The regression equations for the HPLC (y) and GC/MS (x) methods were: y=1.0701x+17.389 (r=0.9772) for methylprednisolone as internal standard and y=1.0827x+6.1364 (r=0.9794) for beclomethasone as internal standard.     

Clinical use of unbound plasma cortisol as calculated from total cortisol and corticosteroid-binding globulin

A method to calculate unbound cortisol from total cortisol (measured by competitive protein binding) and CBG (measured by radial immunodiffusion) based on the binding equilibrium has been evaluated. The calculated results (y) correlate well with those (x) obtained by centrifugal ultrafiltration at 37 degrees C (y = 1.04 x - 2.11 ng/ml; r = 0.975; n = 150). The concentration of CBG is similar in normal men (37.7 +/- 3.5 (SD) micrograms/ml; n = 12) and women (39.5 +/- 3.7 (SD) micrograms/ml; n = 7) and shows no diurnal variation, but marked diurnal variation is observed for total cortisol (193.7 +/- 35.0 (SD) ng/ml at 08.00 h vs 43.2 +/- 23.3 (SD) ng/ml at 22.00 h; n = 19) and particularly for unbound cortisol (16.5 +/- 5.6 (SD) ng/ml at 08.00 h vs 2.3 +/- 1.8 (SD) ng/ml at 22.00 h; n = 19). The concentration of CBG (89.1 +/- 11.2 (SD) micrograms/ml) and of total cortisol (395.6 +/- 103.3 (SD) ng/ml at 08.00 h; 110.3 +/- 16.6 (SD) ng/ml at 22.00 h) are clearly elevated in estrogen treated women (n = 11) but unbound cortisol levels (17.2 +/- 7.7 (SD) ng/ml at 08.00 h; 2.5 +/- 0.5 (SD) ng/ml at 22.00 h) are similar to the control group. The concentration of CBG is significantly decreased in patients with Cushing's syndrome (33.2 +/- 5.6 micrograms/ml; n = 17) and unbound cortisol is relatively more elevated than total cortisol in these patients. In adrenal insufficiently CBG is normal, but total and unbound cortisol are markedly decreased. There is a significant decrease of CBG in hyperthyroidism (35.7 +/- 5.5 micrograms/ml; n = 22), in cirrhosis (32.0 +/- 8.0 micrograms/ml; n = 14) and in renal disease and a significant increase in patients treated with antiepileptic drugs (47.5 +/- 6.3 micrograms/ml; n = 14), but total and unbound cortisol are normal in all these conditions. We conclude that unbound cortisol can be calculated in a simple and reliable way from total cortisol and CBG and permits a better evaluation of adrenal function, particularly in patients with altered CBG concentrations.     

Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cortisol and cortisone in a single extract of 1 ml of serum is described. The method employs meprednisone as the internal standard. The steroids were analysed isocratically by reversed-phase HPLC with an octadecylsilane-bonded (ODS) column using ultraviolet detection. The matrix effect was reduced by lowering the sample pH by adding glacial acetic acid to the sera. The samples were then filtered through regenerated cellulose membranes at 4°C and extracted with diethyl ether. The dried eluates were redissolved in the mobile phase and injected into the column. The detection limit of the assay for both steroids was 500 ng/l. Cortisol was determined in twenty serum samples by both HPLC and radioimmunoassay (RIA). The results were similar. Interference by other steroids and certain steroid analogue drugs was also studied. The HPLC method yielded no cross-reactivity between the different steroids as may occur with the RIA technique. The HPLC method was technically easy to perform and it allowed us to quantify both cortisol and cortisone in a single serum extract with high specificity.     

Determination of dextromethorphan and its metabolite dextrorphan in human urine using high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry: a study of selectivity of a tandem mass spectrometric assay

Analytical method for the simultaneous determination of dextromethorphan (1) and dextrorphan (2) in urine, based on solid-phase extraction of drug from acidified hydrolyzed biological matrix, were developed. The analytes (1 and 2) and the internal standard (levallorphan, 3, IS) were detected by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) in positive ionization mode using a heated nebulizer (HN) probe and monitoring their precursor-->product ion combinations of m/z 272-->215, 258-->201, and 284-->201 for 1, 2, and 3, respectively, in multiple reaction monitoring mode. The analytes and IS were chromatographed on a Keystone Prism reverse phase (50 mm x 2.0 mm) 5 microm column using a mobile phases consisting of a 35/65 or 27/73 mixtures of methanol/water containing 0.1% TFA adjusted to pH 3 with ammonium hydroxide pumped at 0.4 ml/min for 1 and 2, respectively. The limits of reliable quantification of 1 and 2 were 2 and 250 ng/ml, respectively, when 1 ml of urine was processed. The absence of matrix effect was demonstrated by analysis of neat standards and standards spiked into urine extracts originating from five different sources. The linear ranges of the assay were 2-200 and 250-20,000 ng/ml for 1 and 2, respectively. Assay selectivity was evaluated by monitoring the "cross-talk" effects from other metabolites into the MS/MS channels used for monitoring 1, 2, and 3. In addition, an interfering peak originating from an unknown metabolite of 1 into the quantification of dextromethorphan was detected, requiring an effective chromatographic separation of analytes from other metabolites of 1. The need for careful assessment of selectivity of the HPLC-MS/MS assay in the presence of metabolites, and the assessment of matrix effect, are emphasized.     

Simultaneous quantification of tiloronoxim and tilorone in human urine by liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3,000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3-->100.0, m/z 411.3-->100.0 and m/z 268.3-->116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1-100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.     

Simple method for the quantitative analysis of endogenous folate catabolites p-aminobenzoylglutamate (pABG) and its acetamido (apABG) derivative in human serum and urine by liquid chromatography-tandem mass spectrometry

OBJECTIVE: To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylglutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN AND METHODS: Urine, serum and aqueous standards were thawed. Two microliters of d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 x g for 10 min and the supernatant (10 microL) injected into a Biorad CAT/MET analytical column fitted to the LC-MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines. RESULTS: pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 microL injection). Limit of detection (LOD) was 1 nmol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 micromol/L for urines. Imprecision for spiked serum samples (n=10) was between 2.5 and 20% for apABG and 4.5 and 21% for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n=10) was between 2.9 and 4.0% for apABG and 6.0-12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n=5) are 2.9+/-2.3 umol/L (mean+/-S.D.). This group also had total serum catabolites of 11.9+/-7.6 nmol/L and serum folate of 35.3+/-5.8 nmol/L. Serum from patients being tested for PTH (n=11) had serum folate levels of 27.0+/-10.4 nmol/L with total serum catabolites of 20.4+/-23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n=18) were 33.9+/-22.7 and 11.4+/-8.7 nmol/L, respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n=19) was 581.8+/-368.4 nmol/L. CONCLUSION: A simple, reliable and highly specific method by LC-MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients.     

Inhibition of cortisol production in isolated guinea-pig adrenal cells

Cortisol, added to 1 ml incubation medium containing 3-4 X 10(5) isolated guinea-pig adrenal cells, provoked a decrease in basal and ACTH (250 pg)-stimulated cortisol production, in correlation with the amounts used (50 ng-2,000 ng). A decrease in aldosterone production could be seen when cortisol concentrations reached or exceeded 1,000 ng/ml. There were no variations in either androgens (delta 4-androstenedione, dehydropiandrosterone) or 17-hydroxyprogesterone. Only 11-deoxycortisol was slightly increased. Using increasing concentrations of ACTH (50-250 pg), both in the absence and in the presence of 1,000 ng cortisol, it was noted that the inhibition induced by cortisol was of a competitive type and could be overcome by ACTH. This decrease in cortisol was concomitant with an increase in 11-deoxycortisol. Neither corticosterone nor dexamethasone reduced cortisol production. In addition, it was shown that the conversion of tritiated 11-deoxycortisol to radioactive cortisol increased significantly under the influence of 250 pg ACTH (mean relative variation of 21.7% +/- 7.7 (SEM), n = 6, P less than 0.05); but decreased significantly under the combined effect of 1,000 ng exogenous cortisol and the same dose of ACTH: (mean relative variation of 4.3% +/- 1 (SEM), n = 8, P less than 0.005). There is therefore reason to believe that the concentrations of cortisol at the adrenal level modulate the stimulation induced by ACTH and that this self-adjustment forms part of the control mechanisms involved in corticosteroidogenesis.     

Cortisol concentrations in the perinatal and weaning periods of alpacas.

Cortisol concentrations were determined during the perinatal and weaning periods in alpacas. Fifty males and 50 females were chosen at random (25 at each period) for blood collection on day of parturition, 3 and 5 days after birth. For the weaning period, blood samples were collected 2 days before, on the day of weaning (0), and at days 3 and 5 after weaning. Cortisol was determined using an ELISA protocol validated for the alpaca. There was no difference (P>0.05) in cortisol concentrations in both periods, perinatal and weaning between male and female crias. Cortisol was elevated at day of parturition (125.8 ng/ml) and then decreased to 27.2 ng/ml at 3 days after birth (P<0.05). Conversely, cortisol was 32.5 ng/ml 2 days before weaning and then increased at day 3 to 64.7 ng/ml (P<0.05), but then decreased to 29.4 ng/ml at day 5 after weaning. Cortisol is elevated in new-born alpacas and then decreases at the third and fifth after birth. Concentrations of cortisol are also increased after weaning and then decreased 5 days after weaning.     

Determination of urinary cortisol with three commercial immunoassays.

Urinary cortisol determination was performed with three commercially available immunoassays: one enzyme-immunoassay (Cortisol Biotrol) (EIA) and two radioimmunoassays: Quanticoat Cortisol (Kallestad Diagnostics) (KD-RIA) and GammaCoat Cortisol (Clinical Assays) (CA-RIA). Four procedures were carried out. Procedure I (methylene chloride extraction) was applied to EIA and CA-RIA and procedure II (ethyl acetate extraction) to KD-RIA. Procedure III combining procedure I and column chromatography on Sephadex LH 20 in methylene chloride was applied to the three kits. Procedure IV consisting of carbon tetrachloride preextraction and extraction with cyclohexane-ethyl acetate (50:50, v/v) was applied to CA-RIA. The results obtained were compared with those of the reference technique, "on-line" HPLC with u.v. detection. Two groups of results were arbitrarily considered, those below (n = 28) and those above (n = 6) 270 nmol/l. In the first group, the results were markedly overestimated when the procedure was limited to solvent extraction. Conversely, the third procedure proved the efficiency of the chromatographic step since specificity was greatly improved in the three cases, the levels obtained with either kits being similar to those of the reference technique. The second group of results (above 270 nmol/l) yielded by the three kits were not always higher than those of HPLC when the procedure was limited to solvent extraction. When column chromatography was included in the procedure, the results were comparable to those of HPLC in three cases and lower in the three others. Since, the latter samples were collected after cortisol administration, and overestimated cortisol values obtained by HPLC might be due to the interference of some cortisol metabolites.     

Effects of naloxone and animal temperament on serum luteinizing hormone and cortisol concentrations in seasonally anestrous Brahman heifers

The effect of endogenous opioid peptides (EOP) and individual animal temperament on serum luteinizing hormone (LH) were investigated in seasonally anestrous Brahman heifers (n = 24). Animals that had shown behavioral estrus in previous months but that had not returned to estrus for at least 30 d were selected. The heifers were ranked by temperament (tame = 1, normal = 2, wild = 3) and randomly allotted into three groups. Blood was collected from one heifer of each group per day. Blood samples were taken via jugular cannula every 15 min for 6 h and every 30 min for another 4 h. After the first hour of sampling, the heifers received intravenous saline (SAL, n = 8); naloxone (LN, 0.5 mg/kg i.v., n = 8); or naloxone (HN, 1.0 mg/kg i.v., n = 8). Three hours after naloxone treatment, each heifer was given gonadotropin releasing hormone (GnRH, 100 mug i.m.). All samples were processed to yield serum and were assayed for LH by radioimmunoassay (RIA). Hourly samples were assayed for cortisol by RIA. The area under the LH curve 60 min postnaloxone treatment was higher in LN and HN than in SAL (57.0 and 40.8 vs 6.1 units; P<0.01); and the area under the 180 min postnaloxone curve remained higher in LN than in SAL (106.2 vs 35.1 units; P<0.05). Cortisol concentrations 60 min postnaloxone administration were above prenaloxone levels(38.2 vs 26.7 ng/ml; P<0.0002). Temperament scores of heifers were positively correlated with cortisol release. The area under the cortisol curve had a negative correlation with mean LH. Serum LH concentrations appear to be suppressed by EOP in seasonally anestrous Brahman heifers, and EOP appear to reduce serum cortisol concentrations. Excitable heifers had higher concentrations of serum cortisol, which negatively affected serum LH concentrations.     

Enzyme immunoassay for cortisol in serum using cortisol 21-amine

Cortisol 21-amine (21-amino-11β,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 μg/d1, using 10 μ1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme Immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.