A sensitive and simple method for a color · producing determirlation of tropic acid hydrolyzed from atropine by atropinesterase is described.This method is based on the coupling reaction of carboxylic acid with 2-nitrophenylhydrazine.Tropiohydrazide has a purple color wlth an absorption peak at 535 nm.This method is useful for determing atropinesterase activity in microliter samples or extracts of biopsied tissues.lt can be used also to determine other specific carboxylester hydrolyzing enzyme activities.
A radioreceptor assay (RRA) system for a simple and rapid determination of human serum chorionic gonadotropin (hCG) 1evel was developed by using a receptor preparation from porcine ovaries.The system mainly consists of a hCG-receptor-activity containing fraction, which was obtained from porcine ovary homogenate through the differential centrifugation, and a  -labeled hCG.A serum hCG level more than 0.025 lU/ml was able to be determined by this method within a 30 min-incubation and the coefficient of variation was at 8.6%.Serum hCG levels from individuals of different gestational periods were estimated by the method, showing a peak around 10 gestational weeks, gradually decreased and again representing a second peak around 30-35 weeks.The hCG levels thus obtained were compared with those obtained by the radioimmunoassay (RIA) with a NlH kit.Both values were significantly correlated (r=0.501, P<0.001) with each others.
We investigated CPK.BB activity in serum of various individuals at Ueno Civic Hospital.CPK-BB activity was determined by Stream Switching System containing micro colum packed DEAE-sepharose CL 6B, six-port valve, injector and fluorescence detector (Ex 360nm, Em 460nm).The system was useful for the correction of background caused serum and reagents. The CPK-BB activities of healthy individuals were0.86mU/ml (CPK-BB/total CPK=0.0098) for female (n=35) and 0.92 mU/ml (CPK-BB/total CPK=0.0060) for male (n=35) respectively.We found abnormaiities of CPK-BB activity and relative activity (CPK-BB/total CPK) in patient's serum of renal failure, prostatic cancer, gastric cancer and so on.Especially, in the case of renal failure, CPK-BB activity was determined sensitively without the effects of artifact like vitamines and atbumin in serum.
We studied the optimum conditions for measurement of creatine kinase (EC 22.214.171.124, CK) activity in serum at 30°. Imidazole acetate buffer containing EDTA was superior for both CK and glucose-6-phosphate dehydrogenase (G6PD) activities to Bis-Tris acetate with and without EDTA. The optimum pH of partially purified CK isoenzymes CK1, CK2, CK3 and serum CK (abundant of CK3) were 6.2-6.6, 6.2-6.6, 6.0-6.4, and 6.4-6.8, respectively. The affinities for creatine phosphate were significantly different not only among the purified CK isoenzymes but also between the purified CK3 and serum CK (abundant of CK3). Since approximately the same activities were obtained by several thiol compounds at the concentrations of 10 to 30 mmol/I, N-acetylcysteine was selected because of its easy handling. The combined additions of AMP (4-5 mmol/l) and diadenosine pentaphosphate (10μmol/l) resulted in remaining of less than 5% of adenylate kinase (AK) activities and 1 to 4% suppression of CK activities. As a coupling enzyme, G6PD from leuconostoc mescenteroides was recommended because of the low concomitant activity of glucose dehydrogenase and the stability in the CK assay reagents except for its large Km-value for glucose-6-phosphate.
Basic conditions of discriminative assay of arylsulfatase (AS)-A and -B activities was examined. AS inhibitors, such as NaCI, Ag+ and thimerosal, were found to be useful for inhibiting AS-A and -B. However, the inhibitors were only applicable to discriminate AS-A from AS-B in human lung, but not in human leukocytes, and brain and serum of rat. So, a chromatographic separation of AS-A from AS-B was attempted by using mini-column, 0.6×1.0 cm, of DEAE-cellulose. The present separating method by mini-column was found to be applicable for the separating determination of AS-A and AS-B activities in human lung, human leukocytes, rat brain and rat serum.
A sensitive radioimmunoassay was developed for the determination of diazepam. Antisera capable of binding [3H] diazepam were obtained by repeated immunization of rabbits with temazepam (oxydiazepam)-3-hemisuccinate conjugated to bovine serum albumin. The level at least as low as 1 pg of diazepam could be detected by this procedure.The lack of either a methyl group or a keto group at theposition-1 and-2 of a benzodiazepine skeleton markedly decreased the binding affinity for theantibody.
we have established the double antibody radioimmunoassay for α1-microgloblin (α1-m).The range of reproducibilities for three various α1-m levels was 3.6 to 4.5% for within-assay variation and 4-0 to 8.0% for between-assay variation. The mean analytical recovery was 94.8% for serum α1-m and 97.0%for urinary α1-m, and reliable lirlearities were obtained in dilution tests.Correlation coefficients between the results by RIA and SRID for serum α1-m and those by RIA and EIA were 0.953 and 0.935, respectively.Using our method.the normal levels of serum α1-m were 18.8±4-4mg/l (mean±SD) in healthy males and 15.5±3.9mg/l in healthy females. Measurement of serum α1-m might be useful as a screening marker for diagnosis in early renal dysfunction and probably as a diagnostic tool for liver failure.
Highly sensitive fluorimetric methods for the enzymatic assays of glucose, uric acid and free and total cholesterol in biological samples are described.Glucose.uric acid and cholesterol are oxidized by glucose oxidase-, uricase-and cholesterol oxidase-catalyzed reactions to give hydrogen peroxide, respectlvely, which is measured by horseradish peroxidase-mediated reaction with a highly sensitive fluorogenic substrate 3-(p-hydroxyphenyl) propionic acid.Cholesterol ester is hydrolyzed in the presence of cholesterol ester hydrolase.The methods are readily performed with human serum of 1μl or less for glucose, 2μl for uric acid and 1μl or less for total cholesterol and with rat liver of 0.5mg (wet weight) for free cholesterol.The lower limits of determination for glucose, uric acid, total cholesteroi in human serum and free cholesterol in rat liver are 50ng (275 pmol), 60 ng (354 pmol), 110 ng (286 pmo-) and 0.6μg (1.55 nmol), respectively.
The distribution of three peptidases that may cleave collagen-derived peptides, PZ-peptidase (PZ-Pro-Leux Giy-Pro-D-Arg as substrate).SU-peptidase (Succinyl-Gly-Pro-Leu-Gly-Pro-4-methy-coumarinamide assub strate).and dipeptidyl-aminopeptidase I0V (DAP-IV-Giy-Pro-p-nitroanilide as substrate).were measured in human tissues.The activity of PZpeptidase was about 1/20 of that of SU-peptldase, but the activities of two enzymes were similarly distributed various tissues.whereas the activity of DAP-IV was predomlnant in kidney and submandibular gland and relatively high in the digestive system such as pan-Creas.liver, and small intestine.The activity of PZ-peptidase and that of SU-peptidase had weak correlation, but the activity of DAP-LV had no correlation with that of PZ-peptidase or SU-peptidase.The results indicate that PZ-peptidase and SU-peptidase may be similar enzymes and that DAP-IV may have different functions from those of PZ-peptidase and SUpeptidase. Since the correlation between PZ-peptidase and SU-peptidase was weak.clinical data obtained by SU-peptide should not be assumed to be identical with those obtained by PZ-peptide.