Radioimmunoassay of bleomycins

Rabbit antisera highly specific to the bleomycinic acid moiety of bleomycins were obtained by immunizing with a conjugate of copper-complex of bleomycin A5 and bovine serum albumin. These antisera not only reacted with bleomycin A5 but also with other bleomycins such as bleomycin A2, bleomycin B2 and peplomycin. The antisera showed little cross-reactivity with deamido-, depyruvamido- and decarbamoyl-bleomycins. Thus, these antisera were found to be highly specific for the intact bleomycinic acid moiety. One of the antisera was successfully applied to radioimmunoassay of bleomycin and peplomycin in mouse and human sera. The detection limit was 1 ng/ml. This radioimmunoassay is expected to be widely used for the determination of active bleomycins in biological and clinical samples.     

The pharmacokinetics of 2.5- to 10-mg oral doses of minoxidil in healthy volunteers

The dose proportionality of minoxidil was investigated by studying its pharmacokinetics after administration of single, oral doses of 2.5, 5.0, and 10.0 mg. The study, which was a Latin square cross-over design, was performed in 30 young, nonobese, normal subjects. Treatments were separated by a 4-day washout period. Serum and urine levels of minoxidil were determined by high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). Supine blood pressure and pulse were monitored during each study phase. Minoxidil concentrations determined by RIA were highly correlated with concentrations determined by HPLC; only the HPLC data was used in the pharmacokinetic analyses. No significant effects were observed for dose normalized Cmax, tmax, volume of distribution, minoxidil renal clearance, or the percentage of the dose excreted as either minoxidil or minoxidil glucuronide. Significant differences in apparent oral clearance, dose normalized AUC, and terminal elimination rate constant (beta) were observed between the 2.5-mg dose and the higher doses, but no differences in these parameters between the 5.0- and 10.0-mg doses were apparent. Thus, the available data support dose-independent pharmacokinetics for minoxidil over this range of doses. Repeated measures analysis of variance detected significant time and treatment effects on supine blood pressure and pulse rate, but the effects were generally small and of little clinical significance. The results support the hypothesis that minoxidil has little effect on blood pressure in normotensive subjects.     

Effect of incubation time and temperature on the interference of digoxin-like immunoreactive substances in digoxin immunoassays

We have tested the cross-reactivity of digoxin-like immunoreactive substances (DLIS) of cord serum in three commercially available radioimmunoassay systems. We found that there is a marked difference in the specificity of the three antibodies. We were able to eliminate or greatly reduce the interference of DLIS by incubating for 1 h at 37 degrees C. The degree of interference for various cord blood sera is given at three temperature levels and at incubation times ranging from 15 min to 48 h.     

Development of a radioimmunoassay for SR 31747A,a new sigma ligand,in human plasma

A specific and sensitive radioimmunoassay was developed for SR 31747A, a new sigma ligand, using a monoclonal antibody. This antibody was produced from spleen lymphocytes of a mouse immunized with SR 31747A coupled to bovine serum albumin via a peptide bond using SR 120684A, a succinamic acid derivative of SR 31747A. Negligible binding occurred when metabolites, obtained by chemical synthesis or by “indashvitro” incubation with hepatic microsomal fraction, were tested for cross-reactivity. A quantitative recovery in serum of the exogenous analyte was obtained for all the concentrations tested and the quantification limit was found to be 0.25 ng ml⁻¹ of SR 31747 (the nondashsalified derivative). Intra- and inter-assay relative standard deviations ranged from 6.3–10.9 and from 5.3% to 15.4%, respectively. Furthermore, comparison of results from samples which were assayed by radioimmunoassay and gas chromatography demonstrated an excellent correlation (r = 0.984).     

The role of minoxidil on endogenous opioid peptides in the spinal cord: a putative co-agonist relationship between K-ATP openers and opioids

ATP-gated K(+) channel openers produce antinociception that is attenuated by opioid receptor antagonists, indicating K-ATP openers produce antinociception, in part, via the release of endogenous opioid peptides. Utilizing the spinal perfusion method, male Sprague-Dawley rats were administered minoxidil intrathecally (i.t.) at doses ranging from 12.5 to 200 microg/rat for 3 min, tested for antinociception using the tail-flick test, and perfused with artificial cerebrospinal fluid (aCSF) to collect endogenous opioid peptides. Endogenous opioid peptide levels were measured by radioimmunoassay. Naltrindole, a delta-opioid receptor antagonist, at 4 mg/kg, subcutaneously (s.c.), blocked minoxidil-induced antinociception. beta-Funaltrexamine, a mu-opioid receptor antagonist, at 100 microg/rat, partially blocked minoxidil, whereas the kappa-opioid receptor antagonist nor-binaltorphimine, at a dose of 100 microg/rat, did not attenuate minoxidil. Although antagonists of the mu- and delta-opioid receptor attenuated minoxidil-induced antinociception, there was no increase in beta-endorphin, an endogenous ligand with affinity for both micro- and delta-opioid receptors or [Leu(5)]enkephalin, an endogenous ligand with affinity for delta-opioid receptors.     

Immunochemical studies on thymosin: radioimmunoassay of thymosin beta 4

Thymosin beta 4, a peptide with hormonal-like properties first isolated from the thymus gland, can be measured in serum using a newly described radioimmunoassay. The radioimmunoassay utilizes an antibody raised in rabbits against synthetic thymosin beta 4 conjugated by glutaraldehyde to keyhole limpet hemocyanin. A 125I-tyrosine-C13 analogue of the biologically active C-terminal fragment is used as the radioactive tracer. The radioimmunoassay is sensitive in the nanogram range and no cross-reactivity with common serum proteins is demonstrable. High performance liquid chromatography of serum samples indicates that two thymosin beta 4 cross-reactive species are present in human serum. Levels in serum range from 450 to 1100 ng/ml and decline with age.     

A radioimmunoassay for the angiotensin converting enzyme inhibitor ramipril and its active metabolite

A radioimmunoassay (RIA) has been developed for the measurement of 2-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-(1S,3S,5S)- 2-azabicyclo[3.3.0]octane-3-carboxylic acid (ramipril, Hoe 498) and its active metabolite (diacid: hydrolysis product of ramipril) in serum or plasma. Antibodies to the diacid were raised in rabbits against a lysine analogue conjugated to bovine serum albumin. A 125I-ramipril-diacid derivative was used as radioligand. Separation of the antibody-bound and free radiolabeled ligand was achieved by employing a polyethylene glycol solution. The RIA is specific for the active metabolite (diacid). Studies on specificity showed less than 0.6% cross-reactivity with intact ramipril or with dioxopiperazine metabolites. The levels for intact ramipril were obtained by prior enzymatic hydrolysis to the diacid. The sensitivity of the assay was 0.1 and 0.5 ng/ml for diacid and ramipril, respectively. Intra- and inter-assay coefficients of variation ranged from 3 to 14% at 1 to 30 ng diacid per ml. A recovery experiment yielded an average of 103%. The assay has been used to investigate the pharmacokinetic profile of both ramipril and its pharmacologically active metabolite.     

Simultaneous determination of glucocorticoid alcohols, their succinates and hydrocortisone in plasma

A reversed-phase high-performance liquid chromatographic (HPLC) assay is described for the simultaneous determination of methylprednisolone, methylprednisolone-21-hemisuccinate and endogenous hydrocortisone in biological fluids. This assay is also applicable to the determination of prednisolone-21-hemisuccinate in the presence of prednisolone and hydrocortisone in biological fluids. Prednisolone and hydrocortisone are determined by normal-phase HPLC. The stability of hemisuccinate esters in ampoules, in saline and in plasma has been studied. Whereas the esters were stable in saline at 37°C, they were considerably hydrolysed in plasma at the same temperature. Comparison of hydrocortisone levels obtained by HPLC and radioimmunoassay (RIA) showed the large influence of the cross-reactivity of methylprednisolone and hydrocortisone in the presence of high serum concentrations of methylprednisolone.     

Properties of novel anti-digoxin antisera in radioimmunoassay using homologous and site heterologous tritium-labeled antigens involving a [3H]-leucine moiety

The specificities of antisera against digoxin C-3' or C-3' hemisuccinate-bovine serum albumin (BSA) conjugate were assessed by cross-reactivity studies with digoxin metabolites by radioimmunoassay (RIA) using the homologous and the site heterologous tritium-labeled antigens. One of the tracers used was digoxin 3'-hemisuccinyl-[3H]-leucine; the other was digoxin 3'-hemisuccinyl-[3H]-leucine, which had been prepared from digoxin 3'-hemisuccinate. When the tracer with [3H]-leucine at the C-3' position was used, antisera (I-1, I-3) elicited by digoxin 3'-hemisuccinate-BSA conjugate showed the following cross-reactivity: digoxigenin bisdigitoxoside (0.34%, 76%), digoxigenin monodigitoxoside (0.11%, 65%), digoxigenin (0.02%, 26%) and dihydrodigoxin (9.4%, 1.2%). However, when using the homologous antigen, antiserum (I-1) was highly specific against the digitoxose chain. When the site heterologous antigen, digoxin 3'-hemisuccinyl-[3H]-leucine was combined, this antiserum showed high cross-reactivity to digoxin degradation products. This digoxin RIA using antiserum (I-1) with the homologous antigen measures unmetabolized digoxin. On the other hand, the RIA system using antiserum (I-3) with the homologous antigen had cross-reactivity with the metabolites in accordance with their relative cardio-activities, so this system would be useful in therapeutic drug monitoring of digoxin.     

Development of a radioimmunoassay for RX77368 (pGlu-His-3,3-dimethyl proline amide)--a stable analogue of thyrotrophin releasing hormone (TRH)

The recent interest in RX77368 for the treatment of Motor Neurone Disease (MND) has led to the requirement for an assay (RIA) capable of detecting the peptide at low levels in plasma. Several drug conjugates were prepared in which RX77368 was covalently linked to larger proteins, e.g. bovine serum albumin, keyhole limpet haemocyanin or bovine thyroglobulin, the best yield being obtained with the bis-diazotized benzidine reaction (BDB) linking RX77368 to KLH. The latter conjugate was injected into sheep and ultimately produced an antibody of sufficiently high titre to be used. This combined with an iodinated radiolabel formed the basis of the radioimmunoassay. Cross-reactivity studies using similar analogues and RX77368 metabolites showed that the antibody was specific for RX77368. The greatest cross-reactivity was exhibited by the pGlu-His-monomethylProNH2 peptide (RX74355), but, not being a natural metabolite, this did not interfere with the assay. The RIA was used to measure RX77368 in MND patients in a recent clinical study, where RX77368 was administered both by the intravenous and oral routes. High plasma concentrations of RX77368 were found in the patients given intravenous drug by infusion. The oral route exhibited much lower levels, but had a sustained duration of action of up to 12 h.     

Quantification of thyroid hormones and analogs by liquid chromatography coupled to mass spectrometry. Preliminary results in athletes and non-athletes serum samples

This paper aimed to assess a method to measure eight thyroid-related compounds in serum by liquid chromatography-mass spectrometry (LC-MS/MS), to verify the correlation with radioimmunoassay (RIA), to evaluate the possible cross-reactivity, and to observe differences between athletes declaring the consumption of sodium levothyroxine and nonathletes serum samples. Validation was carried out to assess carryover, working range and linearity, limit of detection and limit of quantification, precision, matrix influence, recovery, accuracy, and uncertainty. Comparison between RIA and LC-MS/MS results was done. The assay was applied to serum samples, and comparison with RIA was done for T3 and T4 levels supported by RIA Thyroid-stimulating hormone (TSH) measurements. Validation parameters showed satisfactory results. Correlation between RIA and LC-MS/MS for T3 and T4 showed good results, but a cross-reactivity between T3 and T3AA was observed. Although no significant differences were proved, preliminary comparison between athletes and nonathletes serum samples showed a shift towards high values of TSH and lower for T4 values in the athletes' group. Differences between thyronine and T4AA concentrations and ratios were observed. The trend of T4 values supported by TSH measures might indicate subclinical hypothyroidism in athletes. This represents one of the most controversial thyroid statuses as different criteria about its treatment are described, especially since one of the exogenous causes is inadequate levothyroxine therapy. Because the variation of thyroid hormones and TSH has been extensively studied in high-performance sports, it is worth considering the need to set an adequate reference interval to accurately assess the thyroid status in athletes.     

Effects of minoxidil on ischemia-induced mechanical and metabolic dysfunction in dog myocardium

Effects of minoxidil on ischemia-induced myocardial mechanical and metabolic dysfunction were examined in anesthetized open-chest dogs. A regional portion of the left ventricle was made ischemic for 20 min by ligating the left anterior descending coronary artery, and then reperfused for 120 min. Dimethylsulfoxide or minoxidil (0.3, or 1.0 mg/kg) was injected intravenously 10 min before ligation. Ischemia decreased regional myocardial contraction, and reperfusion recovered it but incompletely. Myocardial metabolic derangement was observed during ischemia, such as decreases in the myocardial levels of ATP and creatine phosphate. These metabolic changes caused by ischemia were restored by reperfusion. Minoxidil injection at 0.3 and 1.0 mg/kg significantly decreased blood pressures but increased coronary flow. Pretreatment with minoxidil significantly enhanced the recovery of myocardial contraction during reperfusion after ischemia. The levels of ATP and creatine phosphate in the ischemic myocardium were significantly preserved by minoxidil at 0.3 mg/kg. No significant effect of minoxidil on the metabolism was observed in the 120 min reperfused myocardium. In conclusion, minoxidil improved the mechanical dysfunction in the reperfused heart and the drug at low dose preserved high-energy phosphates during ischemia.     

小于胎龄儿血清IGF-1、IGFBP-3检测的临床意义（附26例分析）

目的探讨小于胎龄儿血清胰岛素样生长因子-1（IGF-1）、胰岛素样生长因子结合蛋白-3（IGFBP-3）检测的临床意义。方法46例新生儿分成小于胎龄（SGA）儿26例（A组）;适于胎龄（AGA）儿20例（B组）,新生儿均于生后3天内抽取股静脉血,采用放射免疫法检测血清IGF-1、IGFBP-3。结果A组血清IGF-1、IGFBP-3浓度明显低于B组,差异有统计学意义（分别P〈0．01和P〈0．05）。结论小于胎龄儿生后3天内IGF-1、IGFBP-3仍呈低水平。     

Antibody specificity studies for reserpine, its metabolites, and synthetic reserpine congeners: radioimmunoassay

Progress in the development of radioimmunoassay techniques for reserpine and related compounds is reported. A conjugate of reserpine with human serum albumin was prepared, involving linkage at the indole nitrogen atom of reserpine. Injection of the purified conjugate into sheep elicited antibodies of high titer, which bound reserpine selectively. Tritiated reserpine was employed in the procedure, and dextran-coated charcoal was utilized to separate free and bound forms of the drug. Antibodies exhibited a selectivity for reserpine and did not cross-react significantly with major human metabolites. Cross-reactivity of antibodies with other reserpine derivatives (i.e., syrosingopine, deserpidine, and rescinnamine) also was investigated. A stable tritiated or radioiodinated reserpine derivative of high specific activity is being sought to improve assay sensitivity for use in bioequivalence and bioavailability studies. In the absence of any extraction or concentration procedures, at least a 10-fold increase in immunoassay sensitivity would be required to follow reserpine levels in humans given normal doses of the drug. The methods show promise also for the assay of reserpine derivatives such as deserpidine, which exhibits cross-reactivity to reserpine antibodies.     

米诺地尔联合地塞米松对肾缺血再灌注损伤大鼠肾功能指标和氧化应激指标的影响

目的探究米诺地尔联合地塞米松对肾缺血再灌注损伤大鼠肾功能指标和氧化应激指标的影响。方法 40只SD大鼠通过ip 1%戊巴比妥钠溶液4 mg/kg建立肾缺血再灌注损伤模型,分为假手术组、模型组、地塞米松组、米诺地尔组、米诺地尔联合地塞米松组,每组8只。假手术组不进行肾缺血再灌注处理;模型组术前30 min ip生理盐水1 mL;地塞米松组术前30 min ip 4 mg/kg地塞米松磷酸钠注射液;米诺地尔组术前30 min ip 0.6 mg/kg米诺地尔;米诺地尔联合地塞米松组术前30 min ip 0.3 mg/kg米诺地尔和2 mg/kg地塞米松磷酸钠注射液。考察米诺地尔联合地塞米松对肾缺血再灌注损伤大鼠肾功能指标和氧化应激指标的影响。结果地塞米松组、米诺地尔组、米诺地尔联合地塞米松组的血清尿素氮(BUN)、肌酐(Cr)值和肾组织丙二醛(MDA)值均显著低于模型组(P0.05、0.01),以米诺地尔联合地塞米松组最为显著(P0.01);肾组织匀浆总超氧化物歧化酶(T-SOD)活性均显著高于模型组(P0.01),以米诺地尔联合地塞米松组最为显著(P0.01)。结论地塞米松和米诺地尔预处理均可以显著减轻急性肾缺血再灌注的损伤,且两者对急性肾缺血再灌注损伤的保护作用总体相近,联合用药预处理的保护作用则更加显著。     

Radioimmunoassay of carteolol in human plasma

A radioimmunoassay for the direct measurement of carteolol, a new beta-adrenoreceptor blocker, in human plasma was developed. Carteolol was acylated to form O-glutarylcarteolol, which was conjugated to bovine serum albumin to provide the immunogen. Antibody to carteolol was raised in New Zealand albino rabbits. The tracer was the radioiodinated derivative of O-glutarylcarteolol-tyrosine methyl ester conjugate. The method is highly sensitive, with a lower quantifiable concentration of approximately 0.4 ng of carteolol/ml using 0.1 ml of plasma, and has good specificity, with the major metabolite (8-hydroxycarteolol) showing only 0.2% cross-reactivity. It is reproducible, with relative standard deviations from triplicate standard curves being mostly within +/- 8%. The method is currently being used to monitor carteolol levels in clinical samples.     

Measurement of beta-methyldigoxin level in serum from patients by enzyme immunoassay using novel specific antiserum with a phenyl boric acid column

The authors compared serum beta-methyldigoxin (MDx) levels in digitalized patients by enzyme immunoassay (EIA) using anti-MDx 3'-hemisuccinate BSA antiserum (antiserum-I) with commercial antidigoxin antiserum (antiserum-II). The usefulness of a phenyl boric acid (PBA) column for pretreatment of the serum samples was also investigated. The assay using antiserum-I demonstrated good accuracy and precision in the concentration range of 0.5 to 5 ng/mL. When the specificities of antiserum-I and antiserum-II were assessed by cross-reactivity studies with various related compounds, antiserum-I was much more specific for MDx antiserum-II. Using a phenyl boric acid (PBA) column, MDx, and digoxigenin, which exhibits a negligible cross-reactivity, were separated from serum, including MDx and its metabolites. The recovery tests of MDx using antiserum-I with a PBA column in human serum were satisfactory and no interference of metabolites of MDx was observed. Mean MDx concentrations in serum samples (n = 30) from digitalized patients by EIA using antiserum-I with PBA column, antiserum-I, and antiserum-II were 1.06, 1.30, and 1.74 ng/mL, respectively. These results indicate that our EIA system using antiserum-I with a PBA column for pretreatment of serum samples is useful to more precisely measure the unchanged type of MDx in patients.     

1-Methyl-4-phenylpyridinium (MPP+) does not exhibit paraquat-like immunoreactivity

The structural analogy of paraquat with 1-methyl-4-phenylpyridinium (MPP+) has been implied in the aetiology of Parkinson's disease. The cross-reactivity of MPP+ to a specific antibody to paraquat was assessed by radioimmunoassay and was found to be very low. The results suggest that this polyclonal paraquat antibody does not mimic the MPP+ receptor.     

Pharmacokinetics of minoxidil in patients with cirrhosis and healthy volunteers

Objectives: To determine the effect of reduced hepatic function on the pharmacokinetics of minoxidil. The pharmacokinetics of antipyrine, lorazepam, and indocyanine green were included as indicators of hepatic function. Methods: Eight mild cirrhotics and eight healthy subjects received antipyrine (po), lorazepam (IV), indocyanine green (IV) and minoxidil 5 mg (po). Blood and urine were sampled for up to 72 h after each drug, and drug concentrations were measured by validated HPLC methods. Blood pressure and heart rate were measured for safety. Results: For unchanged minoxidil, the serum elimination rate constant was significantly smaller and mean residence time was significantly longer in cirrhotic patients. Urinary elimination rate constant for minoxidil glucuronide was significantly smaller and fraction of dose excreted in urine was significantly higher in cirrhotic patients. Antipyrine elimination was significantly slower for cirrhotic patients. No differences were observed in lorazepam pharmacokinetic parameters. Conclusion: Pharmacokinetic analysis suggests a longer dosage interval may be appropriate in patients with hepatic impairment. In the absence of multiple-dose minoxidil pharmacodynamic studies in this population, minoxidil should be used as in other populations: begin with a modest dose, and adjust the dose based on clinical response. Copyright © 1998 John Wiley & Sons, Ltd.     

Cyclosporine blood concentrations determined by different assay methods in heart transplant patients

Cyclosporine (CsA) through blood concentrations were determined by polyclonal nonspecific radioimmunoassay (RIA) (Sandoz), monoclonal specific and nonspecific RIA (Sandimmun, Sandoz), monoclonal specific and polyclonal nonspecific RIA, (Cyclo-Trac, INCSTAR) and fluorescence polarization immunoassay (TDx, Abbott), at multiple points in time, in two patients receiving CsA for immunosuppression after heart transplantation. Results obtained by the different nonspecific methods have a correlation (r) from 0.76 to 0.94. Concentrations determined by Sandimmun nonspecific RIA, Cyclo-Trac nonspecific RIA and TDx were consistently higher than those by Sandoz nonspecific RIA, owing to different cross-reactivity with CsA metabolites, giving ratios of 1:8, 1:4, and 1:6, respectively. In the first patient, the ratios between nonspecific and specific results were higher during days 1-20 than later on, which coincides with the high serum bilirubin level observed (r = 0.72, n = 31). This observation was not made in the second patient, whose liver function was within normal limits. Because CsA and its metabolites are eliminated primarily in the bile, the high values obtained with the nonspecific assay indicate that CsA metabolites accumulate in blood when liver function is impaired.