The levels of putrescine, cadaverine, spermidine and spermine in uremic erythrocytes were determined by high-performance liquid chromatography-Spermidine and cadaverine were significantly (p<0.05) higher in 10 patients with chronic renal failure than in 8 normal subjects.Putrescine and spermine values were only slightly elevated.The erythrocyte polyamines were also measured in the patients before and after hemodialysis.The spermidine content was lowered but those of the other polyamlnes Were not.
A rapid and simple method for the determination of Lecithin cholesterol Acyltransferase (LCAT) activity in serum was investigated by using immobilized cholesterol oxidase (COD)-peroxidase (POD)-voltammetry system and by applying to flow injection analygis. In this system, suitable protdn donor (Resorcinol) was oxidized by immobilized POD and by hydrogen peroxide (H2O2) which had been produced from the reaction of free cholesterol and immobilized COD.Then, oxidized proton donor was reduced voltammetrically and the reduced current was proportional to the amount of free cholesterol as well as H2O2. Detection limit of free cholesterol in serum was 5 ng and calibration curve was inear to 200ng of free cholesterol.This system was much more sensitive than conventional colorimetry using phenol 4-aminoantipyrine-COD-POD system.Accordingly, it was realized that incubation time could be shorten from 120 minutes of the conventional method to 30 minutes and sample volume could be decreased from 500μl to 50μl. Furthermore, this system was free from influence of other compounds in serum at the voltage of-150mV vs.Ag-AgCl and was comparatively simple to use. The correlation coefficient of the present method to colorimetry was 0.911 (n=20). Therefore, the present method was found to be suitable for the routine assays of serum LCAT activity in the clinical laboratories.
A rapid and sensitive technique for colorimetric determination of iron in hemoglobin which requires no deproteinization is described. Iron (about 17 nmoles) in hemoglobin of 200μl of 100 times diluted blood is oxidized to F3+and released by adding 200μl of sodium hypochlorite (active chlorine: 0.28 mol/l).This released Fe3+is reduced to Fe2+by adding 300μl of 0.17 mol/l ascorbic acid.ln order to completely release and reduce iron, after standing for 5 minutes at a room temperature, the released Fe2+is determined by colorimetry with 300μl of 1.05 mmol/l bathophenanthroline at 535 nm.Simultaneousy, denatured protein should be dissolved by adding 500μl of 0.2mol/l tris-maleate buffer (pH 7.0).Thus this method (C.V.=2.15%) requires no deproteinization procedure.This method correlates enough with mineralization-bathophenanthroline method (y=1.005x-0.01, r=0.995, n=30).
It was Observed that the values of HDL-cholesterol (HDL-TC) in plasma by ultracentrifugation method were occationally higher levels than the values of HDL-TC by phosophotungs-MgCl2 (ph.Tg-MgCl2) precipitation method.We studied on the mechanism of this discrepancy between the values of HDL-TC by the two methods.ln the plasma which were shown the discrepancy in values of HDL-TC, sinking pre β band by electropholesis and immunoprecipitation of Lp [a] with HDL fraction were observed.Sinking pre β lipoprotain (Lp. [a]) was separated into HDL fraction by ultracentrifugation. On the other hand, the supernatant after ph.Tg-MgCl2 precipitation did not contain sinking Pre β lipoprotain (Lp [a]). These results show that in the plasma which contains sinking pre β lipoprotain, the values of HDL-TC by ultracentrifugation method were higher than the values of HDL-TC by ph.Tg-MgCl2 precipitation method.
A simple fluoroenzymatic method is presented for the microdetermination of tissue cholesterol.The method is based on the same principle as a conventional enzymatic cholesterol determination except that homovanillic acid, a fluorogenic reagent, was used as the substrate for the hydrogen peroxide-peroxidase system, and Triton X-100 was added to the enzymatic reagent solution for solubilization of cholesterol and activation of cholesteryl ester hydrolase and cholesterol oxidase.Rat liver, aorta, serum and high-density lipoprotein fraction samples were used to compare this fluoroenzymatic method with the standard colorimetric method.Results of the two methods were Similar.The advantages of the fluoroenzymatic assay for free and esterified cholesterol include speed, easy procedure and high sensitivity.Using this method, as little as 0.25 μg of cholesterol can be determined.This. method should prove very useful for the quantification of cholesterol in limited amounts of tissue such as in biopsies.
we have applied an enzyme immunoassay for the determination of 17α-hydroxyprogesterone (17-OHP) in serum and dried blood sample on filfer paper to the“Multistat MCA III F/LS”Centrifugal Analyzer. Glucose oxidase (GOD) was used as the label enzyme and conjugated with 17-OHP-3-(O-carboxymethyl) oxime.The immune reaction and separation of free from bound fractions were carried out in the sample cap coated with second antibody.The enzyme activity was measured by fluoresence reaction using Horseradish peroxidase and 3-(P-hydroxyphenyl) propionic acid after incubaticn with glucose. The detection limit of H2O2, activity for GOD and 17-OHP are 5×10-6M, 2×10-6unit and 1 pg/tube, respectively. The results were compared with those obtained by radioimmunoassay. Multistat MCA III F/LS is useful for the routine assay of enzyme immunoassay of 17-OHP.
Fosfomycin (FOM) is an antibiotic drug of low molecular weight, exhibiting antibacterial activity against gram-negative and positive bacteria.FOM has no distinct absorption in the UV spectrum. This paper describes a highly sensitive and speedy method for the determination of FOM in human serum. This measurement was achieved by highperformance liquid chromatography with refractive index detection. Only a 50μl of senum was enough for this method.lt took 8 minutes to analyze FOM in the sample, using a silica-NH column and 0.08M NaC1-0.1M phosphate buffer (pH 2.0) as mobile phase. The detection limit of this method was 100ng FOM. The results demonstrated that the proposed method is suitable for the detrmination of FOM in human serum.
A rapid and simple method for determination of both total and direct bilirubin is reported.The reagent consists of 1, 5-naphthalene disulfonate of diazosulfanilic acid and dimethylsulfoxide as an accelerator of the reaction.The reagent develops an endpoint of reaction within 15 and 10min at room temperature and 37°C, respectively.The developed color of reaction mixture, which was found to be stable at least for 1h, has its maximum absorption at560nm.The influence of hemoglobin on the bilirubin determination was demonstrated to be smaller for the present method than other two conventional methods employing diazotization reagents.The results were well correlated with those obtained by both manual (Jendrassik-Grof and Malloy-Evelyn) and automated (SMA 12/60) methods. In addition, this new method can be applied to automated analysis with either discrete type or centrifugal analyzers.
A simple method for the determination of titer of total hemolytic complement activity is described.The principle of the method is based on the original CH 50 hemolysis method of Mayer.Modifications employed are;a reduced reaction volume, only a single colorimetric determination and addition of undiluted test serum to the reaction mixture.A table is constructed for easy calculation of the sample's titer.The assay is simple, rapid, economic and quantitative.The complement titers in sera measured by this new method correlate very well with those measured by the original method, r=0.998 (least squares regression), n=80.
A simple and sensitive assay for serum superoxide dismutase (SOD) was deviced by using a xanthine-xanthine oxidase system in suitable pH condition and with Triton X-100. Respective coefficients of variation for within-run and day-to-day precision of 3.8% and 8, 0% were obtained. The analytical recovery for sera supplemented with SOD was 96% and the normal range of serum SOD was 8.08±1.76 (mean±SD) U/ml. Serum SOD activity were determined in adult patients of diabetes mellitus, hypertensive diseases, hepatitis, malignant tumors and lver-cirrhosis which seem to be attributable to oxigenic toxicity. Higher level of SOD was shown in several of these patients.
A new method for the determination of β-glucuronidase activity using steroidglucuronide as substrate has been described Principle of the reaction is as follows;steroid-glucuronide as substrate is hydrolyzed by the catalysis of β-glucuronidase and steroid released is determined by the enzymatic method using 3α-hydroxysteroid dehydrogenase or 3β, 17β, -hydroxysteroid dehydrogenase. The specifisity of β-glucuronidase from E.coli and H.pomatia was compared with the conventional method using phenolphthalein glucuronide. The results have shown that the measured activity of the enzyme was directly proportional to the enzyme concentrations and to the incubation time.The specificity of β-glucuronidase was the most for testosterone-glucuronate.