A simple and sensitive enzyme-immunoassay technique for quantitation of human α1-microglobulin was established for the first time.The sandwich method, using anti-α1-microglobulin goat immunogiobulin G (lgG)-coated polystyrene beads and horseradish peroxidase-labeled antibody, was employed for immunoassay.Small polystyrene beads were loaded with goat antibody lgG using a simple adsorption technique.This method is highly sensitvie and is capable of detecting minimum concentration of α1-microglobulin in body fluids of 0.5ng/ml.The recovery rates of α1-microglobulin in sera and urine were 97.8% and 98.9%, respectively, and α1-microglobulin determinations on serial dilutions of both samples showed a satisfactory linearity.The coefficierlts of within-assay variation ranged between 2.8-6.2%in sera and 2.3-7.9%in urine, and those of between-assay variation 1.8-4.9%in sera and 1.3-6.0%in urine.α1-microglobulin leveis in the sera of 60normal adults quantitated in this study were found to be 8.82±2.9mg/l (mean±1SD), ranging from 5.89 to 11-75mg/l, While those in random Urines were 1.78±0.90mg/l, ranging from 0.69 to 3.92mg/l.This enzyme-immunoassay method should prove useful determining levels of α1-microglobulin in body fluids in which it might be present only in extremely low levels, and, moreover, is suitable for routine use in clinical laboratories.
This papar presents the existance of higher molecular form of intestinal aikaline phosphatase isozyme (ALP, EC 18.104.22.168) in serum of patients with liver cirrhosis. The ALP in the patient's serum was separated into two different molecuiar weight forms, 7S and 19S-7Sprotein fractions, by Sephadex G-200gel filtration.The apparent molecuiar weight of the 7S and 19S-7S ALP fractions was estimated to be about 200,000 and 350,000, respectively, from the elution voiume of the chromatography.The size of the 19S-7S ALP fraction was much smaller than that of so called high-molecular-weight liver isozyme in serum of patients with choiestatis. The 19S-7S ALP fraction was remarkably inhibited by L-phenylalanine, and the properties of amino acids inhibition were closely resemble to intestinal isozyme.The treatment of 19S-7S ALP fraction with Triton X-100 caused the partial decrease of its apparent molecular weight. The results suggest that the 19S-7S ALP fraction is a complex of intestinal isozyme and lipid.
The effects of two synthetic polyethyleneglycol flufenamates on plasmin and urokinase were investigated using fluorogenic peptide substrates. These flufenamates increased the plasmin activity about 1.5 times, and the urokinase activity about twice under optimum conditions. Moreover, they increased plasmin formation from plasminogen by urokinase 3-fold. They did not influence the antiplasmin activity of human plasma or the activation of plasminogen in plasma by urokinase or streptokinase. These findings show that the two flufenamates increase the activity of plasmin and urokinase, and the plasmin forming activity of urokinase in a purified system.
Ninety seven persons referred for assesment of thyroid function were studied in relation to their thyroid function and fasting serum lipids, especially high density lipoprotein cholesterol (HDL-C). In 42 hyperthyroid patients, HDL-C levels were significantly decreased as compared with those of normal subjects. When 7 patients were restudied after restoration of euthyroid state, HDL-C levels were increased from 32.5±6.1 (mean±S. D.) to 55.7±7.4mg/dl (P‹0.05). In addition, in 16 hypothyroid patients, HDL-C levels were also decreased as compared with those of normalsublects. Furthermore, HDL-C/LDL-C ratios were significantly decreased. When 3 patients were restudied after L-T4 replacement therapy, HDL-C/LDL-C ratios were increased from 1.92±0.18 to 3.11±0.23 (p‹0.05). From these results, it is suggested that especially in the patient with hypothyroid state, high level of total cholesterol and low level of HDL-C may become a risk factor of ischemic heart diseases.
A simplified colorimetric assay for blood levels of pyruvate kinase activity has been developed for the clinical use. After incubation of 0.1ml serum (or plasma) with substrates at pH 7.4 for 10min, pyruvate formed was detected as hydrazone formation by absorbance change at 510nm.Intraassay variance was 1.5% and the activity was stable for week on storage at-20°. In 36 cases with acute transmural myocardial infarction, plasma PK levels were all elevated from 3 hours to 4-5 days after the onset of the symptom. The mean peak value was 1761U/l, which was 5 fold over normal.Plasma levels of PK were not elevated in sublects with angina pectoris, acute hepatitis or hypothyroidism and seemed to be more specific to myocardial infarction compared with CPK, GOT and LDH. These results indicate that the present assay kit for blood level of pyruvate kinase is clinlcally useful for the diagnosis and follow up of myocardial infarction and other muscle diseases.
An enzymatic assay method using inulase and sorbitol dehydrogenase (SDH) for determination of inulin in human serum and urine was described. 0.2ml of serum or 100 times diluted urine was mixed with lml of O.2M acetate buffer of pH5.Oand 20μl of inulase solution.Following incubation of the mixture for 60min.at 37°, 0.2ml of 1.0M triethanolamine buffer of pH8.2, 20μl of NADH soution and 20μl of LDH solution were added. The mixture was then incubated further at 37° for ca.10min.lmmediately after addition of SDH solution (50μl), reduction of absorbance was measured at 340nm. The linear relationship was observed between the amount of inulin and the absorbance.The average recovery of inulin added to the serum and urine were 98.7%and 100.2% respectively.The reproducibility of the me thod was 3.4% (C.V.) with 50.1±1.70mg/dl (mean±S.D.).Giucose present in the sample was found to be inert in the present method. The results obtained by the present method corresponded with those obtained by the modified Heyrovsky's method.
Comparative study of Amberlite XAD7 and Amberlite XAD2 on the extraction of individual serum bile acids was observed.lndividual serum bile acids was determined by a highly sensitive determination of bile acids using high-performance liquid chromatography combined with enzymatic fluorometric measurement, which was previously reported by us (S.Baba et al.Kobe J.Med.Sci., 26: 89, 1980). The extraction rate of each bile acid using Amberlite XAD7 was higher than that of Amberlite XAD2.Therefore, the minimum detectable limits of each bile acid, especially taurine-conjugated bile acids by using Amberlite XAD7, was observed more sensitive and accurate than that of Amberlite XAD2.
Rapid microdetermination of D-galactose anomers with β-D-galactose dehydrogenase and mutarotase was devised.The reaction mixture (1ml) composed of 40μl (0.8unit) of purified β-D-galactose dehydrogenase solution (Pseudomonas fluorescens), 10μl of 50mM NAD+, and 930μl of 20mM Tris-HCI buffer (pH7.4) containing EDTA 1mM.By addition of 10μl of D-galactose solution (<25μg) at 25°, a rapid increase of absorption (NADH) at 340nm due to dehydrogenation of β-D-galactose was recorded, then 10μl (2 units) of hog kidney mutarotase solution was added in the reaction mixture to mutarotate the remaining α-D-galactose to β-D-galactose.The second increase of absorption (NADH) due to dehydrogenation of β-D-galactose changed from a-D-galactose was recorded.From these two absorptions, the ratio of β to α-D-galactose was calculated in 4.5min.