Aldosterone plasma radioimmunoassay interference by a spirolactone metabolite

The plasma aldosterone radioimmunoassay developed by Ito et al. was found to be non-specific for aldosterone following administration of the spirolactones, spironolactone and canrenoate-K, in rabbits, dogs and humans. The assay interfering principle was identified as a hydroxylated derivative (M_B) of canrenone, which itself is a metabolite common to both spironolactone and canrenoate-K. The metabolite M_B possessed a high cross-reactivity to the 21-hemisuccinate aldosterone antibody relative to other spirolactones. A modified procedure was developed specific for plasma aldosterone in the presence of MB. Following single doses of spironolactone and canrenoate-K, aldosterone plasma levels were unchanged in humans and in dogs and decreased in rabbits.     

Modulation of prostaglandin of the renal vascular action of arginne vasopressin

To determine whether the renal vascular effect of arginine vasopressin (AVP) is modulated by renal prostaglandin E₂ (PGE₂) were determined during the infusion of AVP in dogs during control conditions and after the administration of the inhibitor of prostaglandin synthesis, indomethacin. During control conditions, intrarenal administration for 10 min of a dose of AVP calculated to increase arterial renal plasma AVP concentration by 75 pg/ml produced a slight renal vasodilation (p<0.01) and an increase in renal venous plasma concentration of PGE₂. Renal venous PGE₂ concentration during control and AVP infusion averaged 33 ± 7 and 52 ± 12 pg/ml (p<0.05), respectively. After administration of indomethacin, the same dose of AVP induced renal vasoconstriction (p<0.05) and failed to enhance renal venous PGE₂ concentration (9 ± 1 to 8 ± 1 pg/ml). Intrarenal administration of 20 ng/kg. min of AVP for 10 min induced a marked renal vasoconstriction (p<0.01) and increased renal venous plasma PGE₂. Renal venous PGE₂ during control and AVP infusion averaged 31 ± 10 and 121 ± 31 pg/ml (p<0.01), respectively. Administration of the same dose of AVP following indomethacin produced a significantly greater and longer lasting renal vasoconstriction (p<0.01) and failed to increase renal venous plasma PGE₂ (10 ± 1 to 9 ± 1 pg/ml). These results indicate that a concentration of AVP comparable to that observed in several pathophysiological conditions induces a slight renal vasodilation which is mediated by renal prostaglandins. The results also indicate that higher doses of AVP induce renal vasoconstriction and that prostaglandin synthesis induced by AVP attenautes the renal vasoconstriction produced by this peptide.     

Radioimmunoassay of PGE and an approach to the specific measurement of PGE1

A specific radioimmunoassay for prostaglandins of the E series is presented. Several refinements of the conventional procedures used for unknown sample preparation prior to assay are described. Validation of the method through recovery of labeled and unlabeled prostaglandin E from plasma indicates: 74% extraction efficiency, 80% chromatographic separation efficiency, and a 67% overall recovery figure for the complete assay. The development of an “absorbed system” approach for measuring one E prostaglandin (PGE₁) without interference from the other E prostaglandin (PGE₂) is also described. A preliminary study of rat tissue and male plasma samples measured by these assay systems is included. Alternate approaches to the development of a specific PGE₁ assay, although less feasible in these authors' hands, are discussed.     

A convenient lactic dehydrogenase-coupled assay for determining pyridoxal 5′-phosphate in plasma

An assay for determining the concentration of pyridoxal 5′-phosphate in plasma from 0.4 ml whole blood is reported. The assay consists of incubating deproteinized plasma with d-serine apodehydratase from Escherichia coli in 0.5 mN-2-hydroxyethyl-piperazine-N′-3-propanesulfonic acid, pH 7.8, at 37°C for 15 min, and then determining the d-serine dehydratase activity of an aliquot of the incubation mixture. A lactic dehydrogenase-coupled assay (at 25°C) was used to measure the rate of enzymically catalyzed conversion of D-serine to pyruvate, wherein depletion of NADH was followed continuously at 340 nm. The concentration of pyridoxal 5′-phosphate in the plasma sample was estimated from the enzymic activity which is a linear function of the amount of pyridoxal 5′-phosphate present in the assay.     

Plasma prolactin in the periparturient sow

A homologous radioimmunoassay was used for measurement of porcine prolactin in blood plasma collected from sows during the periparturient period. The assay was able to detect prolactin over a range of 0.5 to 7.0 ng/assay tube. There was no significant cross reaction with growth hormone, luteinizing hormone, or follicle stimulating hormone at amounts up to 10⁵ ng/assay tube while porcine ACTH gave 30% binding at 10⁴ ng. Prolactin was not detected in plamsa from a hypophysectomized pig or 2 ergocryptine-treated sows when 100 μ l plasma were assayed. Prolactin concentration in plasma was then measured in 14 periparturient sows within a period extending from 7 days before farrowing to 7 days after farrowing. Samples were collected at 15 min intervals between 1330 and 1630 h each day. However, prolactin assays were done only on the even-numbered samples (30 min interval). Plasma prolactin concentrations (ng/ml, $\text{X}\text{± SEM}$) were 23.7 ± 2.0 on days ?7 to ?5 prepartum, began to rise by day ?3 prepartum (42.5 ± 5.9), and peaked at 127.5 ± 17.6 on day 1 prepartum. By day 3 postpartum, prolactin concentrations in plasma had decreased to 80.5 ± 12.6 and further declined to 51.6 ± 4.6 on day 7 postpartum. The mean prolactin concentration in plasma for all pigs on days ?1 to +2 was 116.8 ± 13.8. This mean concentration for days ?1 to +2 was different (P < 0.025) from the mean prolactin concentration for the period both prior and subsequent to these days (?8 to ?2 and +3 to +8 days).     

A Radioassay for GM1 Ganglioside Concentration in Cerebrospinal Fluid

A radioassay for the rapid determination of G_M1, ganglioside concentration in small volumes of CSF from individual patients is described. The assay utilizes the high-affinity interaction between cholera enterotoxin and G_M1 ganglioside. The lower detection limit of G_M1 ganglioside by this radioassay under the described incubation conditions is 2.5 ng/ml. The radioassay-determined lumbar CSF G_M1 ganglioside concentrations in a small group of patients with diverse neurologic disorders are presented. The radioassay G_M1 ganglioside concentration is in good agreement with the G_M1 ganglioside concentration determined, in one patient, by the tlc-densitometry technique.     

A sensitive, precise, and convenient method for determination of 1,25-dihydroxyvitamin D in human plasma.

A new, highly sensitive and relatively convenient method has been developed for the determination of 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ in blood plasma. The method involves a simplified and more specific extraction procedure, new rapid and effective methods of purification, and a competitive binding assay using intestinal cytosol from rachitic chicks. The method also includes a procedure for stabilizing the cytosol binding protein and a convenient procedure for the separation of bound from free 1,25-dihydroxyvitamin D₃ with the use of polyethylene glycol. The recovery of 1,25-dihydroxyvitamin D₃ during extraction and purification is 68% and triplicate determinations can be made on a 5-ml plasma sample. With this method, rachitic chick plasma, plasma from anephric patients, and plasma from patients suffering severe endstage renal failure show no detectable 1,25-dihydroxyvitamin D, while normal human values have been found to be 29 ± 2 pg/ml.     

Quantitative determination of 9-deoxo-16,16-dimethyl-9-methylene-PGE2 in human plasma by GC-MS,RIA and HPLC

Comparisons were made of 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ levels in plasma determined by three assay methods. Plasma samples from Rhesus monkeys treated with 200 μg/kg 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ intravenously were analyzed by radioimmunoassay (RIA) and by high pressure liquid chromatography (HPLC). In a second experiment known amounts of 11β-³H-9-deoxo-16,16-dimethyl-9-methylene-PGE₂ were added to human plasma at several concentration levels. The samples were analyzed by RIA, HPLC and gas chromatography-mass spectrometry (GC-MS). A limited number of comparisons have been made between RIA and GC-MS analysis of plasma samples from human subjects treated with 9-deoxo-16, 16-dimethyl-9-methylene-PGE₂. The results indicated that the three assay methods generally give comparable estimations of 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ content in plasma.     

Determination of mivacurium in plasma by high-performance liquid chromatography

An assay has been developed and validated for the routine monitoring of mivacurium in plasma. It consists of liquid-liquid extraction with dichloromethane and high-performance liquid chromatography with fluorometric detection (excitation and emission wavelengths 220 nm and 320 nm, respectively). A Spherisorb C₁ 5 μm column and a mobile phase containing acetonitrile, KH₂PO₄ and methanol are used. At a flow-rate of 1 ml/min, a concentration gradient is applied. The detection limit is approximately 1 ng/ml in plasma. For the separation of stereoisomeres, the Spherisorb SCX 10 μm column and acetonitrile-Na₂SO₄ as a mobile phase can be used. The assay shows good linearity over the range 1–1000 ng/ml. The accuracy and precision allows the utilisation in clinical pharmacokinetic studies.     

High-performance capillary electrophoresis measurement of dolastatin-10

A high-performance capillary electrophoresis (HPCE) assay was used to determine the concentration of a potent cytotoxic agent, dolastatin-10, in human plasma. Following extraction from plasma, using a solid-phase C₁₈ cartridge, capillary zone electrophoresis was used to separate, detect and quantitate dolastatin-10 using the structurally related compound dolastatin-15 as the internal standard. Migration times for both dolastatins are less than 20 min. The recovery of the drug was approximately 90% and was quantified over the assay range of 39 to 5000 ng/ml with good precision and accuracy. The method is linear up to 5000 ng/ml with a lower limit of detection of 25 ng/ml. Data resulting from the use of the assay for the in vitro metabolism of the drug are presented. This is the first report of a validated HPCE assay for determining dolastatin-10 levels in human plasma.     

Determination of a novel indolylpiperazine anti-migraine agent in rat, monkey, mouse and rabbit plasma by high-performance liquid chromatography with electrochemical detection

A specific, accurate, precise and reproducible assay for the quantitation of a novel indolylpiperazine anti-migraine agent (I) in plasma from various animal species is described. The method involves addition of internal standard (I.S.) and 1.0 M sodium carbonate to the plasma sample, vortex-mixing and extraction with ethylene dichloride. The organic layer is then back-extracted in a buffer consisting of 0.1 M tetramethylammonium hydroxide (TMAH), pH 3.0 and 0.1 M (NH₄)₂HPO₄, pH 3.0, in water. The aqueous layer is injected on to a Zorbax cyano analytical column with a mobile phase consisting of acetonitrile, methanol and water (15:5:80, v/v/v) with 0.01 M TMAH, pH 3.0 and 0.01 M (NH₄)₂HPO₄, pH 3.0. The eluate is monitored by electrochemical detection at 0.9 V (guard cell), 0.5 V (detector 1) and 0.8 V (detector 2). The retention times of I and I.S. were 7 and 10 min, respectively. In drug-free control plasma, there were no interfering peaks seen at the retention times of I or I.S. The standard curve was linear over the concentration range of 5–500 ng/ml in rat, monkey, mouse and rabbit plasma. The lower limit of quantitation in all four matrices was 5.0 ng/ml. Within- and between-assay variability of quality control samples was less than 9% relative standard deviation and the predicted concentration of the quality control samples deviated by less than 15% from the nominal concentration. The stability of I was established for up to 36 h in the autosampler tray, up to 10 months in plasma at −20°C and up to 2 h in plasma at room temperature. The assay is validated for determination of I in plasma.     

Interaction of Mg+2 with beef heart mitochondrial ATPase (F1).

The soluble mitochondrial ATPase, F₁, can be slowly inactivated by incubation with Mg⁺² in a manner consistent with the observations of Moyle and Mitchell $\text{(}\text{FEBS}\text{Lett}\text{.}\text{56}$, 55 (1975)). This inhibition results in a low initial rate of ATP hydrolysis upon addition to an ATPase assay medium of F₁ which has been incubated with Mg⁺². This inhibition, however, is completely reversible by Mg·ATP in a time dependent process and results in the rate of ATP hydrolysis increasing during the ATPase assay to reach control levels after 30 sec. The length of the lag is independent of the F₁ concentration in the ATPase assay and the lag is also completely reversed by subsequent incubation with excess EDTA before assay.F₁ is unstable if incubated with EDTA in the absence of free nucleotides or Mg⁺². The rate of inactivation increases with decreasing protein concentration until a limiting rate is reached at high dilution. Mg⁺² in excess of the EDTA or 50 μM ADP stabilize the F₁ against the inactivation but cannot reverse prior denaturation.     

A new reliable chemiluminescence immunoassay (CLIA) for prostaglandin E2 using enhanced luminol as substrate

A sensitive and reliable chemiluminescence immunoassay suitable for the quantitative determination of prostaglandin E₂ (PGE₂) has been developed using 96 well microtiter plates (MTP). The assay is based on a competitive reaction between a highly specific monoclonal anti-PGE₂ antibody (mouse), free antigen and solid phase bound antigen. The MTP was first coated with a bovine serum albumin (BSA)-PGE₂ conjugate. Then, after preincubating, the anti-PGE₂ antibody (Ab) and the analyte were added. The remaining amount of free antibody was captured by the solid phase bound BSA-PGE₂ conjugate. The monoclonal antibody captured on the MTP was determined using biotinylated antimouse-Ab and a complex of avidin and biotin-labelled horseradish peroxidase (HRP). Substrate for HRP was the cyclic diacyl hydrazide compound luminol, enhanced by p-iodophenol. Photons emitted during the reaction were measured using a photomultiplier tube. The assay has been validated with assay buffer and human plasma over a concentration range of 10–50,000 pglml. The lower limit of quantification is 100 pglml (2 pglwell) and 150 pglml (3 pglwell) for buffer and plasma, respectively. The intea-day coefficients of variation (CV) for the range of 100–50,000 pglml are 3.2–8.9% (buffer) and 4.2–17.7% (plasma) and inter-day CV are 2.9–19.8% (buffer) and 3.6–21.2% (plasma). The method can be used for quantification of PGE2 in biological fluids like plasma and suction blister fluid.     

Application of a direct spectrophotometric assay employing a chromogenic substrate for tryptophanase to the determination of pyridoxal and pyridoxamine 5′-phosphates

Improved procedures for the isolation of apotryptophanase and its use in estimation of the vitamin B-6 coenzymes are presented. An excess of the apoenzyme is allowed to react with limiting amounts of pyridoxal-P. Estimation of the holotryptophanase thus formed by use of the chromogenic substrate. S-o-nitrophenyl-l-cysteine, provides a sensitive (1–400 pmol) and conveniently direct spectrophotometric assay for pyridoxal-P. For the specific estimation of pyridoxamine 5′-phosphate, samples are first reduced with NaBH₄ to convert pyridoxal-P to pyridoxine-P (inactive). By nonenzymatic transamination with glyoxylate, pyridoxamine-P is then converted quantitatively to pyridoxal-P and estimated with apotryptophanase. The method gives excellent recoveries of the added coenzymes and indicates that in many tissue extracts pyridoxamine-P surpasses pyridoxal-P in concentration.     

High-performance liquid chromatographic assay with ultraviolet detection for the determination of etoperidone and two active metabolites, 5-(1-hydroxyethyl)etoperidone and 1-(3-chlorophenyl)piperazine, in plasma

A selective and sensitive high-performance liquid chromatographic assay with ultraviolet detection for the determination of the antidepressant drug etoperidone and two active metabolites in plasma is described. The drug, metabolites and internal standard are isolated from plasma using a two-step liquid—liquid extraction procedure. The resulting sample is chromatographed on a C₁₈ column (10 cm × 2.1 mm I.D.) with ultraviolet detection at 254 nm. Standard curves are linear for each compound over the concentration range 2–1000 ng/ml. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from the true value and the relative standard deviation (inter-run), are ≤ 10% at all concentrations except the minimum quantification limit. Using an automated injector and computerized data acquisition, eighty samples can be routinely processed in one day. The assay has been successfully used for the analysis of plasma samples from pharmacokinetic studies in mice, rats, dogs and humans.     

Prostaglandin F2α (PGF2α) and prolactin secretion in rats

Plasma prolactin and F-prostaglandins (PGF) were measured in anesthetized male Sprague-Dawley rats before and at 15, 30, 45 and 60 minutes following i.v. injection of either PGF_2α (4 mg/kg), chlorpromazine, 1 mg/kg or chlorpromazine (1 mg/kg) after pretreatment with i.p. indomethacin (2 mg/kg). Following PGF_2α administration, plasma prolactin levels increased significantly only at 15 and 30 minutes in spite of extremely high PGF levels throughout 60 minutes. Besides the expected rise in plasma prolactin, chlorpromazine caused a transient but statistically significant increase in PGF. Indomethacin blocked the chlorpromazine-induced PGF rise but not prolactin increase. Animals stressed with ether anesthesia showed elevation of plasma prolactin, which was not blocked by indomethacin although PGF concentration fell. These results indicate that PGF_2α can stimulate prolactin release. This effect does not appear to be physiologic since very high PGF levels are required. Furthermore, blockade of prostaglandin synthesis by indomethacin does not prevent the release of prolactin in response to chlorpromazine or stress. Our findings do not support a possible role of PGFs as intermediaries in prolactin release. However, it is possible that PGFs may work through other mechanisms not investigated in our study.     

Thromboxane and pulmonary hypertension following E. coli endotoxin infusion in sheep: Effect of an imidazole derivative

We assessed the effect of a specific thromboxane synthetase inhibitor (an imidazole derivative) on pulmonary hemodynamics and the concentrations of TxB₂ (TxA₂), 6-keto-PGF_1α (PGI₂), and PGF_2α in pulmonary lymph and transpulmonary blood samples following intravenous administration of E. coli endotoxin (1 μg/kg) in sheep. In control animals the rise in pulmonary artery pressure correlated with increases in plasma and lymph TxB₂ concentrations and large transpulmonary concentration gradients of this metabolite were measured. In imidazle treated animals both pulmonary hypertension as well as increases in plasma and lymph TxB₂ concentrations were substantially reduced. In contrast, peak concentrations of 6-keto-PGF_1α (PGI₂) and PGF_2α were severalfold higher than those measured in control animals. This suggests a shunting of endoperoxide metabolism towards prostacyclin and primary prostaglandins and documents the specificity of the thromboxane synthetase inhibitor. Out study provides evidence that endotoxin-induced pulmonary hypertension is mediated by pulmonary synthesis of TxA₂.     

The treatment of the hepatorenal syndrome with intra-renal administration of prostaglandin E1

Three patients with the hepatorenal syndrome were treated with prostaglandin E₁ administered through a selective renal arterial catheter. Prostaglandin E₁ was given in progressively increasing doses (2 to 100 ng/kg/min) over a 60-minute period. Control plasma prostaglandin E levels were elevated in all three patients, 0.98, 0.91, and 0.83 ng/ml, respectively. At the end of the infusion, plasma prostaglandin E levels had risen to 10.4, 2.63, and 10.3 ng/ml in the three patients respectively. Plasma renin activity increased during the course of the infusion in two of the patients. The plasma aldosterone concentration did not change during the prostaglandin E₁ infusion. Intrarenal prostaglandin E₁ failed to increase urine volume or urinary sodium concentration in three patients with the hepatorenal syndrome.     

Pharmacokinetics Study of Recombinant Hirudin in the Plasma of Rats Using Chromogenic Substrate,ELISA, and Radioisotope Assays

Aim

To compare the analytical methods used to study the pharmacokinetics of recombinant hirudin in the plasma of rats that had been injected with ¹²⁵I-recombinant hirudin.

Methods

2.0 mg/kg ¹²⁵I-recombinant hirudin were injected intravenously into rats. The recombinant hirudins in the plasma was analyzed by chromogenic substrate assay, enzyme-linked immunosorbent assay (ELISA), total radioisotope assay (RA) and trichloroacetic acid pre-treated total radioisotope assay (TCA-RA).

Results

The chromogenic substrate assay standard curve was linear over the concentration range from 3.12 to 40.00 ng/ml for the recombinant hirudin in plasma. The relative standard deviations (RSD) for the intra- and inter-day variation were 5.0 to 6.3% and 11.9 to 12.6%, respectively. The recoveries of recombinant hirudin was 89.8% to 100.7%. The limit of quantification (LOQ) was 3.12 ng/ml. The concentration-time curve of the recombinant hirudin in the plasma could be explained as a two-compartment model. Pharmacokinetic parameters, including the half-life of distribution phase (t_1/2 α), the half-life of elimination phase (t_1/2 β), volume of apparent distribution (Vd), and area under the concentration-time curve from zero to infinite time (AUC_0–t) were 7.59 min, 46.99 min, 0.17 L/kg, and 204.5 mg/L/min, respectively, as determined by chromogenic substrate assay; 6.41 min, 47.28 min, 1.24 L/kg, and 575.18 mg/L/min, respectively, as determined by ELISA; 3.69 min, 701.90 min, 0.04 L/kg, and 4189 mg/L/min, respectively as determined by RA; and 4.57 min, 724.9 min, 0.09 L/kg, and 2329 mg/L/min, respectively, as determined by TCA-RA.

Conclusions

The chromogenic substrate assay on the concentration dynamics of the recombinant hirudin in the plasma is a specific, sensitive, and accurate analytical method for pharmacokinetic studies. Moreover, the pharmacokinetic parameters determined by the chromogenic substrate assay and ELISA are congruent except for AUC.     

Simultaneous rapid high-performance liquid chromatographic determination of phenytoin and its prodrug, fosphenytoin in human plasma and ultrafiltrate

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of phenytoin and fosphenytoin, a prodrug for phenytoin, in human plasma and plasma ultrafiltrate is described. For plasma, the method involves simple extraction of drugs with diethyl ether and evaporation of solvent, followed by injection of the reconstituted sample onto a reversed-phase C₁₈ column. Plasma ultrafiltrate is injected directly into the HPLC column. Compounds are eluted using an ion-pair mobile phase containing 20% acetonitrile. The eluent is monitored by UV absorbance at 210 nm. The fosphenytoin standard curves are linear in the concentration range 0.4 to 400 μg/ml for plasma and 0.03 to 80 μg/ml for ultrafiltrate. Phenytoin standard curves are linear from 0.08 to 40 μg/ml for plasma and from 0.02 to 5.0 μg/ml for ultrafiltrate. No interferences with the assay procedure were found in drug-free blank plasma or plasma ultrafiltrate. Relative standard deviation for replicate plasma or ultrafiltrate samples was less than 5% at concentrations above the limit of quantitation for both within- and between-run calculations.