Plasma protein binding interaction of prednisone and prednisolone

The plasma protein binding interaction of prednisone and prednisolone were characterized by equilibrium dialysis. Prednisone and prednisolone are bound equally but weakly to human albumin (affinity constant, K approximately equal to 1 X 10(3) M-1). Transcortin affinity for prednisolone is 10-fold greater (51.3 X 10(6) M-1) than that for prednisone (4.3 X 10(6) M-1). In competition under pharmacologic conditions, prednisolone inhibits prednisone binding to transcortin producing linear binding averaging 55%. Prednisone does not affect prednisolone binding and does not complicate pharmacokinetic studies of the latter.     

Pharmacokinetics of prednisone and prednisolone in a case of hypothyroidism: effect of replacement therapy

We found this particular case during the course of a clinical trial designed to assess the pharmacokinetics of oral prednisone in normal and diseased children. The plasma concentrations of prednisone, its main metabolite prednisolone, and endogenous cortisol were measured by HPLC at selected times during 8-h periods starting at 7:30 a.m. One 9.9-year-old administered prednisone 0.5mg/kg p.o. was found to be hypothyroid (TSH: 351microIU/mL; fT4: <2pg/mL; fT3: <1pg/mL); four age-matched normal boys (aged 6.6+/-4.9 years) served as a control group. In comparison with the controls, the hypothyroid boy showed a marked increase in the total AUC of prednisone (3360microg h/L versus 215+/-83microg h/L) and prednisolone (4040microg h/L versus 724+/-77microg h/L), and an altered pattern of endogenous cortisol, which is known to be impaired in hypothyroid subjects. After 6 months of thyroxine replacement therapy (75microg/day), the AUCs of prednisone and prednisolone returned to normal values (prednisone: 248microg h/L; prednisolone: 528microg h/L), as did the pattern of circadian cortisol secretion. In conclusion, our data indicate that the pharmacokinetics of prednisone and prednisolone can be profoundly altered by hypothyroidism, and subsequently restored by thyroxine replacement therapy.     

Effect of cucurbitacins on bilirubin-albumin binding in human plasma

The aim of this study is to investigate the effect of three cucurbitacins (Cuc) E, D and I on the bilirubin-albumin binding, both in human serum albumin (HSA) and in plasma. Bilirubin-HSA solution and plasma free of cucurbitacins were prepared as well as others containing serial concentrations of cucurbitacins. The concentration of unbound bilirubin was determined in bilirubin-HSA solution and the direct and total bilirubin concentrations were measured in plasma (with normal or elevated bilirubinemia) by Jendrassik and Grof method. In the conditions we adopted Cuc E and D (to a lesser extent), decreased the levels of unbound bilirubin in bilirubin-HSA solution and decreased direct bilirubin concentration and total bilirubin concentration in plasma in a dose-dependent manner while Cuc I had no effect. The effect of Cuc is related to the presence of native HSA. Thus, when albumin was absent or has been denatured by heating or by urea, Cuc E did not modify bilirubin levels, suggesting that the native structure of albumin is essential for such activity. The interaction of HSA with Cuc E was investigated by fluorescence spectroscopy. Cuc E increased the intrinsic fluorescence of the protein and the magnitude of fluorescence intensity of bilirubin-albumin complex. We concluded that Cuc E and D produced a rearrangement in the structure of albumin, particularly in the domain-II, resulting in an increase in the binding of bilirubin to albumin regardless to whether it's conjugated to glucuronic acid or unconjugated.     

Kinetics of bilirubin oxidase and modeling of an immobilized bilirubin oxidase reactor for bilirubin detoxification

The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin-serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin-serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K(1) = 6 x 10(7)M(-1) and K(2) = 4.5 x 10(6)M(-1), respectively. The rate of total bilirubin degradation in bilirubin-serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8-mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350muM and a serum albumin concentration of 500muM.     

Experimental determination of net protein charge, [A]tot, and Ka of nonvolatile buffers in bird plasma.

The quantitative mechanistic acid-base approach to clinical assessment of acid-base status requires species-specific values for [A]tot (the total concentration of nonvolatile buffers in plasma) and Ka (the effective dissociation constant for weak acids in plasma). The aim of this study was to determine [A]tot and Ka values for plasma in domestic pigeons. Plasma from 12 healthy commercial domestic pigeons was tonometered with 20% CO2 at 37 degrees C. Plasma pH, Pco2, and plasma concentrations of strong cations (Na, K, Ca), strong anions (Cl, L-lactate), and nonvolatile buffer ions (total protein, albumin, phosphate) were measured over a pH range of 6.8-7.7. Strong ion difference (SID) (SID5=Na+K+Ca-Cl-lactate) was used to calculate [A]tot and Ka from the measured pH and Pco2 and SID5. Mean (+/-SD) values for bird plasma were as follows: [A]tot=7.76+/-2.15 mmol/l (equivalent to 0.32 mmol/g of total protein, 0.51 mmol/g of albumin, 0.23 mmol/g of total solids); Ka=2.15+/-1.15x10(-7); and pKa=6.67. The net protein charge at normal pH (7.43) was estimated to be 6 meq/l; this value indicates that pigeon plasma has a much lower anion gap value than mammals after adjusting for high mean L-lactate concentrations induced by restraint during blood sampling. This finding indicates that plasma proteins in pigeons have a much lower net anion charge than mammalian plasma protein. An incidental finding was that total protein concentration measured by a multianalyzer system was consistently lower than the value for total solids measured by refractometer.     

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.     

Measurement of plasma unbound unconjugated bilirubin

A method is described for measuring the unconjugated fraction of the unbound bilirubin concentration in plasma by combining the peroxidase method for determining unbound bilirubin with a diazo method for measuring conjugated and unconjugated bilirubin. The accuracy of the unbound bilirubin determination is improved by decreasing sample dilution, eliminating interference by conjugated bilirubin, monitoring changes in bilirubin concentration using diazo derivatives, and correcting for rate-limiting dissociation of bilirubin from albumin. The unbound unconjugated bilirubin concentration by the combined method in plasma from 20 jaundiced newborns was significantly greater than and poorly correlated with the unbound bilirubin determined by the existing peroxidase method (r = 0.7), possibly due to differences in sample dilution between the methods. The unbound unconjugated bilirubin was an unpredictable fraction of the unbound bilirubin in plasma samples from patients with similar total bilirubin concentrations but varying levels of conjugated bilirubin. A bilirubin-binding competitor was readily detected at a sample dilution typically used for the combined test but not at the dilution used for the existing peroxidase method. The combined method is ideally suited to measuring unbound unconjugated bilirubin in jaundiced human newborns or animal models of kernicterus.     

Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

Paclitaxel is pharmaceutically formulated in a mixture of Cremophor EL and ethanol (1:1, v/v). The unbound fraction of the anticancer drug paclitaxel in plasma is dependent on both plasma protein binding and entrapment in Cremophor EL micelles. We have developed a simple and reproducible method for the quantification of the unbound paclitaxel fraction in human plasma. Human plasma was spiked with [3H]paclitaxel and [14C]glucose (unbound reference) and incubated at 37 degrees C for 30 min. Plasma ultrafiltrate was prepared by a micropartition system (MPS-1) and collected in a sample cup containing 100 microl of plasma to prevent the loss of paclitaxel due to adsorption. The radionuclides were separated after combustion of the biological samples using a sample oxidizer and the radioactivity was determined by liquid scintillation counting. The unbound fraction of paclitaxel was calculated by dividing the ratios of 3H and 14C in plasma ultrafiltrate and in plasma. The method was thoroughly validated using human plasma spiked with pharmacologically relevant concentrations of paclitaxel (10-1000 ng/ml) and Cremophor EL (0.25-2.0%). The method was precise, with a within-day precision ranging from 3.9 to 11.0% and a between-day precision ranging from 5.8 to 13.1%. In patient plasma with low serum albumin values containing 1% of Cremophor EL, the unbound fraction appeared to be significantly higher than that in plasma with normal albumin values. The determination of the unbound fraction of paclitaxel proved to be stable during a 10-week storage at -20 degrees C. Furthermore, the assay was applicable in patient samples. This assay can be used to determine the unbound fraction of paclitaxel in plasma. Moreover, its design should allow the determination of the unbound concentrations of other hydrophobic drugs.     

Determination of unbound ticagrelor and its active metabolite (AR-C124910XX) in human plasma by equilibrium dialysis and LC-MS/MS

Ticagrelor is the first direct acting reversibly binding oral platelet P2Y(12) receptor antagonist. The parent molecule and the main metabolite (AR-C124910XX) are both able to block adenosine diphosphate-induced receptor signaling with similar potency. Drug binding to plasma proteins reduces free drug available for pharmacologic activity. Therefore, assessing unbound drug is important for interpretation of pharmacokinetic/pharmacodynamic findings. This paper describes the development and validation of an equilibrium dialysis/LC-MS/MS method for measuring unbound ticagrelor and AR-C124910XX in human plasma. Plasma samples (200μl) were dialysed against phosphate buffered saline (37 °C, 24h) in 96-well dialysis plates to separate unbound analytes. Drug-protein binding alterations during dialysis were minimized by maintaining physiologic conditions (pH 7.4, 37 °C). Ticagrelor and AR-C124910XX were quantified in dialysates (unbound fraction), retentates and plasma (total concentration) using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods. Calibration curves were established for the retentate and plasma (total concentration) in the ranges 5-5000 ng/ml (ticagrelor) and 2.5-2500 ng/ml (AR-C124910XX), and for the dialysate in the range 0.25-100 ng/ml (both analytes). Both ticagrelor and AR-C124910XX were highly protein bound (>99.8%), i.e. unbound fraction <0.2%. Yet, the methodology was successfully applied to determine unbound concentrations of ticagrelor and AR-C124910XX in clinical samples.     

Continuous measurement of protein osmotic pressure in blood and lymph of sheep

We have continuously measured protein osmotic pressure of blood and lymph in sheep to compare two kinds of needle osmometers (rigid and flexible) with a membrane osmometer (Wescor). We also compared the averaged values of the continuous measurement with osmotic pressure calculated from total protein and albumin fraction, using the Yamada equation. The rigid-needle and membrane osmometers showed excellent correlation (y = 1.00x + 0.06; r greater than 0.99). The flexible-needle osmometer tended to overestimate osmotic pressure (avg 16%). We used the rigid-needle osmometer for continuous measurements of protein osmotic pressure of blood and lymph in anesthetized or unanesthetized sheep to observe changes in protein osmotic pressure of blood and lymph through the three different interventions. The relationship between the theoretical values (x) and the continuous measurements (y) of osmotic pressure was good (y = 0.99x + 0.16, r = 0.97), but after various interventions, the continuously measured protein osmotic pressure tended to exceed the calculated measurements. The continuous measurement should be monitored with spot samples measured in a stationary osmometer or by calculation of osmotic pressure from total protein concentration and albumin fraction.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

Serious renal transplant rejection and adrenal hypofunction after gradual withdrawal of prednisolone two years after transplantation.

Ten patients with stable renal function two years after transplantation had their sole immunosuppressive treatment (oral prednisolone 10 mg daily) withdrawn by reducing the daily dose by 1 mg at monthly intervals. Plasma prednisolone concentration, cortisol concentration, creatinine clearance, and serum creatinine concentration were measured in all patients, and the adrenal response to corticotrophin was determined in five by measuring plasma cortisol concentrations before and after tetracosactrin injection. No episodes of rejection occurred in patients taking over 7 mg prednisolone daily. Although three patients apparently required only minimal immunosuppressive treatment (less than 5 mg daily) the remainder suffered episodes of rejection at daily doses below 7 mg. There was a tenuous association between rejection and low plasma cortisol concentration, but neither the pattern of plasma prednisolone concentrations nor the response to tetracosactrin were related to episodes of rejection. Reducing the daily dose of oral prednisolone to under 7 mg should not be attempted in patients with renal transplants unless there are extenuating circumstances.     

Development of a sensitive and selective method for the quantitative analysis of cortisol,cortisone, prednisolone and prednisone in human plasma

A highly selective, sensitive and robust LC–MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M + 2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 μm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.     

Effect of glycyrrhizin on the pharmacokinetics of prednisolone following low dosage of prednisolone hemisuccinate

We investigated the pharmacokinetics of prednisolone (PSL) in six healthy men, with or without glycyrrhizin (GL), to confirm whether GL influences the metabolism of PSL in humans. Each subject received an intravenous administration of 0.096 mg/kg of prednisolone hemisuccinate (PSL-HS, equivalent to 0.075 mg/kg of PSL), with or without 200 mg of GL. Blood samples were taken from a peripheral vein at 5, 10, 15, 30 and 45 min, and 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h after PSL-HS infusion. The concentration of total PSL in the plasma was analyzed by high-performance liquid chromatography, and the free PSL was measured by an isocolloidosmolar equilibrium dialysis method. The pharmacokinetic parameters of PSL were determined, using noncompartmental analysis. GL was found to increase significantly the concentration of total PSL at 6, 8 h, and of free PSL at 4, 6, and 8 h after PSL-HS infusion. GL was also found to modify the pharmacokinetics of PSL. After the administration of GL, the area under the curve (AUC) increased, total plasma clearance (CL) decreased, and the mean residence time (MRT) was prolonged. However, only those of AUC, CL, and MRT of free PSL were significantly different. The volume of distribution at a steady-state (Vdss) of both total and free PSL showed no evident change. This suggests that GL increases the plasma PSL concentrations by inhibiting the metabolism of PSL and that it potentiates pharmacological effects of PSL.     

Acid-base effects of altering plasma protein concentration in human blood in vitro

We altered the concentration of plasma proteins in human blood in vitro by adding solutions with [Na+], [K+], and [Cl-] resembling those in normal blood plasma, either protein-free or with a high concentration of human albumin. After equilibrating the samples with a gas containing 5% CO2-12% O2-83% N2 at 37 degrees C, we measured pH, PCO2, and PO2; in separated plasma, we determined the concentrations of total plasma proteins and albumin and of the completely dissociated electrolytes (strong cations Na+, K+, Mg2+ and anions Cl-, citrate3-). With PCO2 nearly constant (mean = 35.5 Torr; coefficient of variation = 0.02), lowering plasma protein concentration produced a metabolic alkalosis, whereas increasing plasma albumin concentration gave rise to a metabolic acidosis. These acid-base disturbances occurred independently of a minor variation in the balance between the sums of strong cations and anions. We quantified the dependence of several acid-base variables in plasma on albumin (or total protein) concentration. Normal plasma proteins are weak nonvolatile acids. Although their concentration is not regulated as part of acid-base homeostasis, hypoproteinemia and hyperalbuminemia per se produce alkalosis and acidosis, respectively.     

Plasma binding of oestradiol in progesterone-treated rats

Progesterone treatment of female rats causes an increase in body weight possibly via suppression of oestradiol secretion. This study was carried out to investigate the effect of progesterone on the non-protein bound and hence presumably biologically active fraction of oestradiol. Oestradiol binding to plasma proteins was studied in female Wistar rats during the oestrous cycle and after 12 days of treatment with progesterone (5 mg/day). There was no change in either the unbound fraction of oestradiol or plasma albumin concentrations during the oestrous cycle. Plasma oestradiol concentrations in progesterone-treated rats were similar to those seen during dioestrus, as were the degree of oestrogen binding and the plasma albumin concentrations. Although it was not feasible to calculate unbound concentrations, these results suggest that the increased body weight seen in progesterone-treated rats, and also during pregnancy, may be a result of suppression of unbound oestradiol concentrations to levels similar to those occurring during dioestrus.     

Enhanced prednisolone elimination: a possible cause for failure of glucocorticoid therapy in Graves' ophthalmopathy

This study was undertaken to determine the pharmacokinetics of intravenous prednisolone in patients with Graves' eye disease. 6 women with Graves' ophthalmopathy treated with prednisolone for severe endocrine exophthalmos were compared with 6 healthy female volunteers. All subjects with Graves' disease had been taking carbimazole and I-thyroxine as concurrent drugs for at least 4 months prior to study day. All subjects were euthyroid. Each subject received .54 mg/kg prednisolone as an i.v. bolus. Plasma concentrations for total and unbound prednisolone were determined by HPLC and equilibrium dialysis. Significant increase (p less than .01) in clearance values and significant decreases in half-life times (p less than .01) were found for both total and unbound prednisolone in women with Graves' disease compared with the control subjects. Volumes of distribution at steady-state were unchanged in both groups. The data suggest that patients with Graves' ophthalmopathy show an enhanced elimination for prednisolone and that is why they may need higher doses of corticoid although the function of the thyroid gland is euthyroid.     

Hormonal determinants of apolipoprotein B,E receptor expression in human liver. Positive association of receptor expression with plasma estrone concentration in middle-aged/elderly women

The relationships of the expression of hepatic low-density lipoprotein (LDL) receptors (apo B,E receptors) to several plasma hormone concentrations were examined in 15 fasted women aged 37-75 years (mean, 57 years), who were undergoing laparotomy for non-neoplastic disease. No subject had clinical or biochemical evidence of familial hypercholesterolemia, renal disease, hepatic disease, or endocrine disease. Hepatic apo B,E receptor expression was quantified in vitro as the EDTA-suppressible binding of 125I-labeled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C; values were 23-75 ng LDL protein/mg cell protein (mean, 47 ng/mg). Receptor expression was strongly correlated with plasma estrone concentration (rs = +0.70, P = 0.035), but was unrelated to the concentrations of testosterone, thyroxine, free triiodothyronine, cortisol, sex hormone-binding globulin (SHBG) or cortisol-binding globulin. Insulin and estradiol concentrations were mostly very low. The correlation of receptor expression with plasma total estrone concentration reflected associations with both the albumin-bound (rs = +0.78, P = 0.014) and unbound (rs = +0.80, P = 0.009) fractions, but not with the SHBG-bound fraction (rs = -0.22, P = 0.574), of this hormone. As the non-SHBG-bound fractions of gonadal steroids are considered to be the biologically active components, these results are consistent with experimental evidence that the synthesis of apo B,E receptors in hepatocytes is stimulated by estrogens, and suggest that circulating estrone may be the major hormonal determinant of receptor expression in fasted middle-aged/elderly women.     

Effect of low-calorie diets on plasma retinol-binding protein concentrations in overweight women

The concentrations of total protein, albumin and retinol-binding protein, a major transport protein for vitamin A, are significantly decreased by protein-calorie malnutrition. Weight-loss diets, sometimes involving severe energy deficits over prolonged periods of time, are common in the United States. The effect, if any, of prolonged low calorie weight-loss diets with normal intakes of protein on albumin, total protein and retinol-binding protein concentrations (and potentially on vitamin A metabolism) had not been extensively studied. We measured total protein, albumin, apo + holo retinol-binding protein and holo-free- and holo-transthyretin-bound retinol-binding protein concentrations during the course of a nutritionally adequate weight-loss diet (50% calorie restriction). We found that this type of dieting did not affect total protein, albumin or apo + holo, holo-free or holo-transthretin-bound retinol-binding protein concentrations significantly. This suggests that protein intake is more critical than caloric intake for retinol-binding protein status.     

Radioimmunoassay for prednisone

A radioimmunoassay for the measurement of prednisone in serum has been developed. The method is accurate, precise, specific, and very sensitive. Using a primary antibody elicited against prednisone 21-hemisuccinate-bovine serum albumin and a chemical precipitation step, the assay is capable of detecting 6.25 picograms of prednisone in 0.1 ml of unextracted diluted serum or plasma.The specificity of the assay is influenced by the carbonyl groups at position 11 and 20, and the double bond at the 1-position of the steroid nucleus. Physiological levels of endogenous steroid interfered only slightly with the primary antibody.Measurement of serum concentrations of prednisone in man and dog were accomplished following the administration of prednisone (Deltasone^®). This assay, used in conjunction with a published radioimmunoassay for prednisolone (1), can be used to determine the interconversion of these two drug products following prednisone or prednisolone administration.