Radioimmunoassay for prednisolone

A radioimmunoassay method for the measurement of prednisolone and its 20β-OH metabolite in peripheral serum is presented. The method is capable of detecting 10.0 picograms of prednisolone in 0.1 mls of diluted serum or 1 nanogram in 0. 1 ml of undiluted serum. Hydrocortisone was the only endogenous steroid tested that cross-reacted significantly with the prednisolone antibody. The specificity of the primary antibody is strongly influenced by the double bond at the 1-position and the hydroxyl group at the 11-position on the steroid nucleus.Measurement of serum concentrations of prednisolone was accomplished in man and dog following oral dosages of Delta-Cortef^® (prednisolone) and Deltasone^® (prednisone). It was possible to use prednisolone as an index of the latter compound because of its rapid metabolic conversion to prednisolone.The number of serum samples which may be processed each day is limited only by the capacity of the liquid scintillation counter used. As many as 200 samples per day may easily be processed by one skilled technician. This capability, aside from the greater sensitivity, precision and accuracy offered by this method, provides a distinct advantage in the analysis of large numbers of samples which are required in bioavailability studies.     

Radioimmunoassay for fluoxymesterone (halotestin®

A specific, sensitive, precise and accurate radioimmunoassay has been developed for fluoxymesterone, 9α-fluoro-11β, 17β-dihydroxy-17-methyl-4-androsten-3-one (Halotestin^®). The method is capable of detecting 25 picograms of drug in 0. 1 ml of unextracted serum. The primary antibody was prepared against fluoxymesterone 3-[0-(carboxymethoxime)] (CMO) bovine serum albumin. The specificity of the assay is greatly influenced by the hydroxyl group at position 11 and the methyl group at position 17. Physiological levels of endogenous steroids did not cross-react significantly with the primary antibody. Blood levels of fluoxymesterone were determined in both human subjects and male beagle dogs after oral administration of Halotestin.     

Radioimmunoassay for the detection and quantitation of bruceantin

A radioimmunoassay for a new anticancer drug, bruceantin, has been developed using [³H]acetylbruceantin and antibody induced by immunizing rabbits with succinylbruceantin-bovine serum albumin conjugates. [³H]Acetylbruceantin was synthesized by reacting bruceantin with [³H]acetyl anhydride. The assay is simple and reproducible. The standard curve was linear on a logit-log plot, and the lower limit of sensitivity of the assay was 1 ng/ml. Using this assay, drug levels were easily determined in tissues of experimental animals following bruceantin administration. The assay procedure does not require sample extraction for plasma, urine, and bile. Bruceantin in other tissues can be extracted quantitatively with ethanol before being measured by the radioimmunoassay.     

A radioimmunoassay for 11-oxotestosterone: Its application in the measurement of levels in blood sebum of rainbow trout (S. Gairdneri).

17β-Hyd.roxyandrost-4-ene-3,11-dione was linked via its 3-(O-carboxymethyl) oxime to bovine serum albumin to give a conjugate which was used to generate antiserum in rabbits. The antiserum, at an overall dilution of 1 in 16,000, together with [1,2-³H] 17β-hydroxyandrost-4-ene-3,11-dione synthesized from [1,2-³H] cortisone have been used to develop a radioimmunoassay for the parent steroid. The assay incorporates a purification step in which serum or plasma extracts are chromatographed on silica gel layers bound to plastic or aluminium sheets and the steroid, containing zones cut out and eluted directly with assay buffer. The cross-reactivities of several steroids with the antiserum and the specificity, sensitivity, accuracy and precision of the assay are described. Blood sera from Immature male rainbow trout contain $\text{ca}$ 0.2–0.4 μg/100 ml of 17β-hydroxyandrost-4-ene-3,11-dione. As male fish mature, serum levels rise sharply to reach values of 2 to >9 μg/100 ml. Levels in immature females rarely exceeded the assay sensitivity but serum from three ripe females showed low but detectable levels ($\text{ca}$ 0.2 μg/100 ml) of steroid. The assay has found application in sexing live fish for experimental purposes.     

Liquid chromatographic analysis of prednisolone, prednisone and their 20-reduced metabolites in perfusion media

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20α-dihydro and 20β-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6α-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0–1 μg/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20α-dihydroprednisone) to 8.8% (20β-dihydroprednisolone) and 4.1% (20β-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20α-dihydroprednisone) to 9.1% (20β-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C₁₈ columns which were linked together and maintained at 40°C.     

Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.     

Analysis of prednisone,prednisolone and their 20β-hydroxylated metabolites by high-performance liquid chromatography

A high-performance liquid chromatographic technique primarily developed for use on samples from kidney perfusion studies is presented for simultaneous determination of prednisone, prednisolone and their 20β-hydroxylated metabolites. The technique employs 6β-hydroxycortisol as the internal standard. Samples are extracted with ethyl acetate, washed with sodium hydroxide and water and injected onto a silica gel column with UV detection at 254 nm. Inter- and intraday variability of the assay was determined at two concentrations of each steroid and was less than 10%. Assay steroid recovery ranged from 54.1% for prednisone to 63.2% for 20β-hydroxyprednisone. Sensitivity is 4–10 ng/ml for the steroids measured. The chromatographic conditions may be modified to permit quantitation of these steroids from plasma samples. This method may alternatively be used for quantitation of 6β-hydroxycortisol, an endogenous indicator of enzyme induction. A perfusate concentration-time profile is presented from a kidney perfusion study using prednisolone.     

EFFECTIVENESS OF ANTACIDS IN REDUCING DIGESTIVE DISTURBANCES IN PATIENTS TREATED WITH PREDNISONE AND PREDNISOLONE

To appraise the efficiency of complemental antacid administration in preventing and reducing digestive disturbances during prolonged treatment with prednisone and prednisolone, 100 patients with active rheumatoid arthritis who were maintained on combined antacid and prednisone or prednisolone therapy for periods of one year or longer, were studied clinically and roentgenographically. Antacid therapy consisted of 300 mg. of dried aluminum hydroxide gel and 50 mg. of magnesium trisilicate taken with each 2.5 mg. dose of the steroids.Digestive symptoms, such as indigestion, heartburn, sour eructations, gnawing epigastric distress and the like, were experienced by 18 per cent of patients during treatment with prednisone or prednisolone combined with antacids. Among patients who had been maintained on the steroids without antacids beforehand, the incidence of digestive complaints was reduced from 38 per cent to 17 per cent by the addition of alkali therapy, and the severity of the distress decreased in others.Active peptic ulcers were detected roentgenographically in three of the 100 patients. In two instances the ulcers were asymptomatic and in two instances they were considered as reactivations of previously healed lesions. The incidence of active ulcers in this series was substantially lower than that reported by several investigators among patients treated with prednisone and prednisolone without the concomitant administration of alkalis. The size of dosage and individual susceptibility appeared to be important factors in the development of digestive disturbances from steroids.Results of the study indicated that the complemental use of antacids with each divided dose of steroid is highly effective in reducing the frequency and severity of digestive symptoms during prednisone and prednisolone administration. The low incidence (3 per cent) for roentgenographically demonstrable active lesions in the series suggests that the addition of acid-neutralizing agents during prolonged treatment with these steroids may afford at least partial protection against the development and reactivation of peptic ulcers.     

Corticosteroid analysis in biological fluids by high-performance liquid chromatography

A sensitive, specific, and reproducible high-performance liquid chromatographic assay for the simultaneous determination of prednisone, prednisolone and cortisol in biological fluids was developed with dexamethasone as the internal standard. Samples are extracted with methylene chloride, washed with sodium hydroxide and then water, and chromatographed on a microparticulate silica gel column with UV detection at 254 nm. Sensitivity was greater than 15 ng for all four steroids. Specificity was supported by use of dual wavelength UV detection and/or radioimmunoassay. The assay has been applied in pharmacokinetic studies and a typical plasma concentration—time profile for the three steroids is presented for one subject who received 50 mg of prednisone.     

Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics

Steroid resistance is a significant problem in management of chronic inflammatory diseases, including asthma. Accessible biomarkers are needed to identify steroid resistant patients to optimize their treatment. This study examined corticosteroid resistance in severe asthma. 24 asthmatics with forced expiratory volume in one second of less then 80% predicted were classified as steroid resistant or steroid sensitive based on changes in their lung function following a week of treatment with oral prednisone. Heparinised blood was collected from patients prior to oral prednisone administration. Phosphorylated mitogen activated kinases (MAPK) (extracellular regulated kinase (ERK), p38 and jun kinase (JNK)) were analyzed in whole blood samples using flow cytometry. Activation of phospho-p38 MAPK and phospho-mitogen- and stress-activated protein kinase 1 (MSK1) in asthmatics’ peripheral blood mononuclear cells (PBMC) were confirmed by Western blot. Dexamethasone suppression of the LPS-induced IL-8 mRNA production by steroid resistant asthmatics PBMC in the presence of p38 and ERK inhibitors was evaluated by real time PCR. Flow cytometry analysis identified significantly stronger p38 phosphorylation in CD14⁺ monocytes from steroid resistant than steroid sensitive asthmatics (p = 0.014), whereas no difference was found in phosphorylation of ERK or JNK in CD14⁺ cells from these two groups of asthmatics. No difference in phosphorylated p38, ERK, JNK was detected in CD4⁺, CD8⁺ T cells, B cells and NK cells from steroid resistant vs. steroid sensitive asthmatics. P38 MAPK pathway activation was confirmed by Western blot, as significantly higher phospho-p38 and phospho-MSK1 levels were detected in the PBMC lysates from steroid resistant asthmatics. P38 inhibitor significantly enhanced DEX suppression of LPS-induced IL-8 mRNA by PBMC of steroid resistant asthmatics. This is the first report demonstrating selective p38 MAPK pathway activation in blood monocytes of steroid resistant asthmatics, suggesting that p38 and MSK1 phosphorylation can serve as blood biomarkers of steroid resistance.     

Hypothalamic-pituitary-adrenal axis following glucocorticoid prophylaxis against acute mountain sickness.

The pituitary-adrenocortical and adrenomedullary response to high altitude (HA) stress was studied following daily single dose administration of prednisolone as a prophylaxis against altitude-induced acute mountain sickness (AMS). Forty healthy men, randomly divided into two groups of twenty, received placebo or prednisolone 20 mg once a day at 08.00 h for two days prior to induction to HA and during an initial three days stay at an altitude of 3450 m. The AMS score and circulatory levels of ACTH, cortisol, epinephrine and norepinephrine were measured at sea level (SL) and during residency at HA. The sensitivity of the hypothalamic-pituitary-adrenal axis in subjects receiving prednisolone therapy was evaluated at SL and on day four of stay at HA. Administration of prednisolone significantly (p < 0.01) decreased the severity of AMS in all the subjects. The steroid dose used did not inhibit endogenous secretion of ACTH, cortisol, epinephrine or norepinephrine, as HA response to adrenocortical and adrenomedullary hormones was identical in placebo and prednisolone treated subjects. The integrity of the hypothalamic-pituitary-adrenal axis was maintained well in subjects receiving low dose prednisolone therapy. These observations suggest that short-term administration of prednisolone is able to curtail AMS without causing suppression of the hypothalamic-pituitary-adrenal axis.     

Investigation on possible transformations of cortisol,cortisone and cortisol glucuronide in bovine faecal matter using liquid chromatography–mass spectrometry

Given the close resemblance of the ring A structure of prednisolone and prednisone on the one hand, and of androstadienedione on the other, the transformation of cortisol and cortisone into prednisolone and prednisone in cattle faeces was evaluated. A simple method that does not involve extraction but only the 1:100 dilution of cattle faeces, spiking with 400 ng/mL cortisol, cortisone or cortisol glucuronide and incubation of the suspension, was used. The analyses were performed by HPLC–MS³ to detect the supposed Δ¹ dehydrogenation of the glucocorticoids. The decision limits (CCα) and detection capabilities (CCβ) were 2.0 and 3.0 ng/mL for cortisol, cortisone and prednisolone, 3.0 and 4.0 ng/mL for cortisol glucuronide and 7.0 and 10.0 ng/mL for prednisone, respectively. Intra-day and inter-day coefficients of variation (CV%), were 5.6–6.2 and 5.2–6.6 for cortisol glucuronide, cortisol, cortisone and prednisolone, and 16.0 and 16.2 for prednisone, respectively. The recoveries were in the range 110–143% for all analytes. Regression coefficients (R2) were in the range 0.996–0.999 for all analytes. The results show the hydrolysis of the conjugated form and the dehydrogenation in ring A in diluted faeces. It is therefore predicted that urine contaminated with faeces may be positive for prednisone and prednisolone in the same way as they are positive for boldenone, i.e. as a result of microbiological dehydrogenase activity on cortisol and cortisone.     

A competitive enzyme-linked immunosorbent assay for plasma 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione)

An enzyme-linked immunosorbent assay (ELISA) for 11-deoxycortisol is described for the first time. 11-Deoxycortisol-thyroglobulin conjugate is adsorbed onto the wells of a 96-well ELISA plate and competes with 11-deoxycortisol in the standards or plasma extract for antibody binding sites. After washing, immobilized primary antibody is probed with peroxidase-labeled goat anti-rabbit IgG. The ELISA plate is further washed and o-phenylenediamine added, color developed and the absorbance read at 492 nm. The ELISA shows good agreement with our existing 11-deoxycortisol radioimmunoassay (RIA) and has similar specificity and performance which allow its use in the routine steroid laboratory for assessing pituitary adrenal function by the metyrapone test.     

Statistical survey of “saturation analysis” calibration curve data for prednisolone,prednisone and digoxin

An extensive survey of radioimmunoassay calibration data for prednisolone, prednisone and digoxin indicated that the common practice of preparing calibration curves with individual subject's pre-dose plasma or serum, and using this to estimate unknown concentrations for the same subject, is not supported by statistical considerations. Preparation of calibration plots from pooled data is better because this introduces less bias in estimated concentrations. Such a method also saves a great deal of time, since it is not necessary to repeat the calibration procedure each time “unknowns” are being assayed. The data suggest that there is no optimum calibration plot for all radioimmunoassays. Rather, each antibody-drug combination should be investigated thoroughly to determine the best calibration plot for the particular combination. We found that the best calibration plots are; the logistic-logarithmic plot for prednisolone; nonlinear least squares fit to a polyexponential equation for prednisone; and a weighted least squares regression of normalized % bound $\text{versus}$ concentration for digoxin. The error in the radioimmunoassay is usually concentration-dependent, and, in certain regions of the standard curve, is larger than the literature indicates, since, frequently, the error has been gauged from % bound values, but should be gauged from inversely-estimated concentrations.     

Determination of prednisolone,prednisone, and cortisol in human plasma by high-performance liquid chromatography

A method is described for the measurement of prednisone and prednisolone in human plasma by high-performance liquid chromatography (hplc). An ether/dichloromethane extract (60:40, $\text{v}\text{v}$) of plasma is evaporated to dryness and chromatographed on a 250 × 4.5-mm Whatman Partisil column with uv detection at 239 nm. Prednisone, prednisolone cortisol, and the internal standard dexamethasone are satisfactorily resolved with the elution system of water-saturated dichloromethane/ethanol (95:4, $\text{v}\text{v}$). The hplc method can be used for plasma prednisolone concentrations as low as 25 ng/ml. The values correlate well with those obtained by a radioimmunoassay procedure for prednisolone.     

Radioimmunoassay for reserpine

Antisera against reserpine have been developed in rabbits immunized with reserpine conjugated to bovine serum albumin. These antisera were used in a radioimmunoassay procedure for reserpine. No appreciable cross-reactivity was observed either with reserpine metabolites or with other drugs commonly used in conjunction with reserpine for the treatment of hypertension. The assay was able to detect as little as 150 pg of reserpine in serum (15 ng/ml), without the need for an extraction procedure. Serum concentrations were measured in rats following the oral and intravenous administration of 1 mg/kg of reserpine.     

The use of immobilized estradiol antiserum in the study of receptors and other estradiol-binding proteins.

Antiestradiol antibody immobilized on a polymer film is set in competition for tritium-labeled estradiol with estradiol receptor or antiestradiol antibody in solution. Measurement of equilibrium quantity of radiolabel in test solutions in the presence and in the absence of dissolved antibody or receptor gives a data base for evaluating the association constant and the quantity of the estradiol-complexing species in solution. The immobilized antibody method avoids the problems and uncertainties arising in the step of separating the bound from the free hormone which is the pivotal step in all currently used procedures for the determination and characterization of steroid receptors. With the exception of equilibrium dialysis, it is the only procedure presently available to study steroid antibodies and receptors in solution at unperturbed equilibrium. Using the immobilized antibody method at 4°C, 0.86 ± 0.12 × 10¹¹m^?1 was the association constant found for estradiol receptor in rat uterine cytosol, and 2.6 ± 0.5 × 10¹¹m^?1 was the association constant found for antiestradiol antibody raised in a rabbit to estradiol linked to bovine serum albumin via a C-6 carboxymethyloxime.     

Antigen-antibody complexes produced in chickens tolerant to bovine serum albumin.

A radioimmunoassay for circulating soluble antigen-antibody complexes is described. The assay detects complexes either in antigen or antibody excess. Soluble complexes were found in the sera of chickens tolerant to bovine serum albumin (BSA). The complexes appeared in the serum as soon as 7 days following neonatal induction of tolerance. The amount of complexes reached a peak between 1 to 2 weeks of age and disappeared by 6 weeks when responsiveness returned. The complexes were found in the bottom third of a 10 to 40% sucrose density gradient and by analytical ultracentrifugation indicated a size of 22.8S. If tolerant chickens were challenged with BSA at 2, 4 or 6 weeks, the disappearance of complexes was not accelerated, and a proportion of the previously tolerant chickens exhibited a heightened antibody response.     

Maximizing the precision of the mathematically ideal radioimmunoassay at a specified dose

Mathematical analysis of the equation for the radioimmunoassay, R²+R(1+Kp+Kp^*–Kq)–Kq=0, gives a general solution for the dose of maximum precision, ²R/p², of the assay. This solution, (R–c₁+Kp)(R–c₂), and the radioimmunoassay equation can be used to determine theoretical conditions for maximum precision of a radioimmunoassay at any dose p. A theoretically optimal antibody concentration, q, can be calculated for precision at a critical concentration of interest, p. This is dependent on the affinity constant, K, of the particular antibody being used and, potentially, the amount of labeled tracer, p^*. This solution idealizes the radioimmunoassay to a one affinity constant system. Though this may be achievable, particularly with monoclonal antibodies or receptors, the applicability needs to be explored experimentally.     

Development, validation and application of an enzyme immunoassay (EIA) of atriopeptin

A rapid, convenient, and sensitive enzyme immunoassay (EIA) for atriopeptin (AP) has been developed. The tracer-ligand for the assay is the 24-amino acid peptide, AP24, which has been covalently coupled to the tetrameric form of acetylcholinesterase (AChE) (EC 3.1.1.7). Tracer, unknown, and primary antibody are incubated in a 96-well microtiter plate precoated with secondary antibody. After washing, a colorimetric reaction is used to measure acetylcholinesterase activity. A direct linear correlation was obtained when comparing the conventional radioimmunoassay and the EIA by using the same primary antibody to assay: plasma samples (rat or human), HPLC column fractions, or atrial extracts. Besides being technically much less demanding and not requiring the use of the radioisotopes, the EIA is more sensitive than the radioimmunoassay and thereby lends itself to a "flash" same-day assay of samples.