Kaichiro Ishibashi

Radioimmunoassay for imidapril, a new angiotensin-converting enzyme inhibitor, and imidaprilat, its active metabolite, in human plasma and urine

A radioimmunoassay (RIA) was investigated for the determination of imidapril and its active metabolite, imidaprilat, in human plasma and urine. Imidapril is a new angiotensin-converting enzyme inhibitor and an oral prodrug of imidaprilat. Imidapril was determined after conversion to imidaprilat with esterase. Antiserum was raised in rabbits against the p-amino derivative of imidaprilat conjugated to bovine serum albumin. Radioligand was prepared by iodination (125I) of the p-hydroxybenzoylamino derivative of imidaprilat. Cross-reactivities of anti-imidaprilat antiserum for imidapril, its metabolites and several cardiovascular drugs were low. The calibration range was 0.1-100 ng ml-1 using a 100 microliters of human plasma of urine. Intra- and inter-day variations of imidaprilat assay in plasma were 2.0-7.9 and 4.1-6.2%, respectively, and intra- and inter-day variations of imidapril assay in plasma were 5.4-10.7 and 7.9-18.1%, respectively. The variations of the assay in urine were a little smaller than those in plasma. The recovery of imidaprilat and imidapril spiked in plasma or urine samples was approximately 100%. A good correlation between RIA and high-performance liquid chromatograpy was observed for both plasma and urine samples. Furthermore, this method was applied to the determination of imidaprilat and imidapril in human plasma and urine samples, for the evaluation of the pharmacokinetics of imidapril in humans. From the results, it was demonstrated that the developed RIA was useful for the determination of imidaprilat and imidapril in human plasma and urine, and was applicable to pharmacokinetic studies in humans.

Semiautomated Enzyme Immunoassay of Thyrotropin as a Mass Screening Test for Neonatal Hypothyroidism

ABSTRACT: A sensitive, simple, and rapid semiautomated sandwich enzyme immunoassay (EIA) was developed for measuring thyrotropin in dried blood samples on filter paper for use in screening for neonatal hypothyroidism. Good correlation was found between values for thyrotropin determined by this method and those determined by radioimmunoassay (RIA) (r = 0.94). In pilot tests on 17,160 newborn infants in the general population, five cases of primary hypothyroidism were detected by both EIA and RIA. The recall rate was slightly higher in EIA than in RIA.

A new radioimmunoassay of free thyroxine using 125I-labelled thyroxine—protein complex uninfluenced by albumin and thyroxine-binding globulin

We describe a double-antibody solid phase radioimmunoassay for free thyroxine (FT4) in serum with use of 125I-labelled thyroxine-human chorionic gonadotropin conjugate. Since the labelled conjugate does not bind to thyroxine binding globulin (TBG) and albumin because of its large molecular weight, the method is uninfluenced by TBG or albumin. The measurable range of FT4 in serum was 2.0 to 128 ng/l. The mean coefficients of variation within and between assays were 4.6-8.6% and 6.3-11.6%, respectively. The FT4 values determined by the proposed method correlated well with those determined by commercial radioimmunoassay in subjects with normal albumin concentration (r = 0.98). The FT4 concentrations in serum as determined by this method were 9 to 17 ng/l for healthy adult subjects; high for patients with hyperthyroidism; low for patients with hypothyroidism; and within normal limits for pregnant women, and patients with high or low concentrations of thyroxine-binding globulin.

Unforeseen effect of thyroxine binding globulin when using the microencapsulated antibody method to determine free thyroxine(FT4) : misleading results due to circulating unsaturated thyroxine binding globulin

The effect of varying concentrations (0-52 mg/l) of purified thyroxine binding globulin (TBG) on the microencapsulated antibody method for free thyroxine was investigated. The results demonstrated that the free thyroxine values were strongly influenced by the concentration of thyroxine binding globulin in the samples. The standard curve could no longer be distinguished at a concentration of purified thyroxine binding globulin of 52 mg/l. In the clinical application, we observed that the values obtained using the microencapsulated antibody method were significantly higher than the expected values in patients receiving triiodothyronine treatment after total thyroidectomy (theoretically nil) and in patients with untreated primary hypothyroidism with negligible thyroxine (less than 12.9 nmol/l). These false positive values are considered to be due to the methodological problem mentioned above, i.e. the microcapsule membrane is not efficient and therefore must be improved. Consequently, any data based on this method should be interpreted with caution.

Methodological aspects for free hormone estimation using microencapsulated antibody method—The effects of hormone binding protein on permeability of microcapsule membrane

The effect of T4 non-carrying thyroxine binding globulin (TBG) on free thyroxine determination using the microencapsulated antibody method was studied, to investigate the precise reliability of the membrane and to find possible applications for estimating other free steroid hormones. When increased amounts of purified TBG were added to a test tube containing microcapsule suspension, it affected the accuracy of the results. We found that with higher amounts,125I-T4 leaked through the membrane into the medium, thereby giving a falsely increased free T4 result. Our finding indicates that further improvements in the microcapsule membrane are necessary; or alternatively, it may also be possible to balance the binding affinity inside and outside the membrane by adding a suitable amount of carrier protein, to the contents of the capsule, so that both successful FT4 determination and other free steroid hormone assays may be undertaken.

Neonatal Hypothyroid Screening Using Enzymeimmunoassay of Thyrotropin — Comparison with Radioimmunoassay

In order to evaluate a new enzymeimmunoassay (EIA) of thyrotropin(TSH) for neonatal hypothyroidism, 6,100 blood samples on fdter paper, obtained through a neonatal hypothyroid screening program, were examined using a one‐step sandwich EI A and a radioimmunoassay(RIA). Measured TSH values, numbers of cases and false‐positives detected, and recall rates, were compared between the two methods. During this study two cases of congenital hypothyroidism were found by both EIA and RIA. However, a patient with hyperthyrotropinemia was detected only by the EIA. The reproducibility of the EIA was slightly better than that of the RIA. These results suggest that the EIA has same or higher sensitivity for detecting patients compared with the RIA. On the other hand, three cases with falsepositive elevation of TSH by EIA were found. High TSH values by the EIA were likely to be due to transplacental factors, probably immunoglobulin G. Because of high reproducibility of the EIA and false‐positive cases found with it, recall rates of the screening program were slightly higher for the EIA than for the RIA. The recall rate of the EIA could have been decreased by setting a cut‐off point at a higher percentile. Since the EIA is simple to use, this method is suitable for screening for congenital hypothyroidism.