Polyamine levels in blood cells were suggested as a sensitive and specific indicator for cancer. We report here a simple and sensitive method to determine free polyamine levels in blood cells. Using 0.2ml of hemolyzed blood, free polyamines in blood were measured by a putrescine oxidase chemiluminescent method. The analytical specificity and sensitivity of this method were good enough for clinical use. The coefficient of variation (CV) of within-run (n=9) was 2.1% for a blood sample with 20.4μmol/L of free polyamine, and the CV of between-run (n=12) was 4.0% for a sample with 20.3μmol/L of polyamine. Analytical recovery was about 100% for all free polyamines except spermine. The linearity of measurement by this method was observed in the range of spermidine from 0 to 70μmol/L. The results determined by this method were correlated well with those determined by an HPLC method (r=0.967). Since the concentrations of free polyamine in plasma are negligibly low as compared to those in blood cells, the free polyamine concentrations in blood as measured by this method can represent the free polyamine concentrations in blood cells. The free polyamine levels in blood cells (PBC), when devided by the corresponding hematocrit values,were 41.0±10.3uM/Ht for healthy subjects (12 males and 27 females). High PBC were observed in some patients with cancer. The usefulness of PBC for screening cancer, however, remaines to be established.
A study was undertaken to establish interrelationship between oxygen affinity of whole blood and level of glycated hemoglobin and intracellular concentration of 2, 3-diphosphoglyceride (2, 3-DPG), the major organic phosphate affecting the oxygen affinity of hemoglobin, in patients with diabetes mellitus. 74 non-acidotic patients, with or without microangiopathic complications were studied. Our findings obtained in this study can be concluded as follows: 1) The major factor contributing to the increased oxygen affinity of red-cells in non-acidotic patients is the increase in HbA1 concentration. 2) The increased affinity is partially or fully compensated by an increase in red-cell 2, 3-DPG. 3) In patients under good or fair metabolic control, the compensation is adequate. In poorly controlled patients, however, it is disorderly inadequate resulting in increased oxygen affinity of various degrees. Possible role played by the augmented affinity for development of microangiopathic complications in diabetic patients is discussed.
This paper reports a simple and sensitive method for the determination of ceruloplasmin in human serum based on its ferroxidase activity. In acetate buffer solution (pH 6.5), iron (Fe2+) was rapidly oxidated to Fe3+ by ceruloplasmin. The addition of trichloroacetic acid solution stopped the reaction from deproteinization, but the remaining Fe2+ in the reaction mixture was stable for about 2h. The remaining Fe2+ was chelated with 2-nitroso-5-(N-propyl-N-sulfopropylamino)phenol as a specific reagent for Fe2+. The chelated compound developed a color that has a peak absorbance at 745nm upon addition of ammonium acetate solution. The method was suitable for the determination of ceruloplasmin at concentrations up to 100mg/dl. The within-run precision, or coefficient variation, was 3.25%. Close correlation was found between this method and other methods currently available.
A simple and rapid nonradiochemical method for the simultaneous determination of adenine phosphoribosyltransferase (APRT, EC 2. 4. 2. 7) and hypoxanthine phosphoribosyltransferase (HPRT, EC 2. 4. 2. 8) activities in human erythrocytes by high-performance liquid chromatography is described. APRT and HPRT activities were simultaneously assayed with adenine and hypoxanthine as substrates. adenosine-5'-monophosphate (AMP) and inosine-5'-monophosphate (IMP) produced by both enzyme reactions were converted to adenosine and inosine with alkaline phosphatase (EC 3. 1. 3. 1), respectively, and the adenosine and inosine separated from adenine and hypoxanthinelby a reversedphase silica column were determined by the absorbance at 270nm. The activities of APRT in erythrocytes of 36 healthy subjects were 0.42±0.17μmol/min/gHb and 2.00±0.39μmol/min/gHb (mean±2SD), respectively The method is suitable for screening erythrocytes for APRT and HPRT deficiencies.
A simple and sensitive method for the colorimetric determination of hemoglobin in plasma is described. It involves a color reaction with leuco crystal violet, which is oxidized to a colored form by the pseudoperoxidase effect of hemoglobin in the presence of hydrogen peroxide. This assay system is sensitive enough to determine 2mg/dl of hemoglobin and is linear up to 100mg/dl. The precision, that is the coefficient of variation, was 2.75%, and recovery was 99.9%. Close correlation was found between this and the other two methods.
We developed a new device for rapid and convenient amylase assay, by combining the rapid quenching and the continuous flow techniques, with a novel apparatus which can measure the amylase activity in a trice (iodine coloration). The instrument was successfully applied for determining glucoamylase, pancreatic α-amylase and amylase in human serum samples. All the procedures are operated automatically under the regulation of a microcomputer program.
The present work was undertaken to develop a method for the determination of testosterone (T) and epitestosterone (ET) in human urine using the technique of gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring (SIM). Urinary steroids were extracted by means of SEP-PAK C18 cartridge, and were hydrolyzed with β-glucuronidase. The hydrolyzate was treated by running it through a SEP-PAK silica cartridge and a thin layer chromatography, to remove interfering materials. Furthermore, proper amounts of internal standard, 1-dehydrotestosterone (1-dehydro T) were added. Urinary steroids in extract were converted into 3-enol, 17-ditrifluoroacetate derivatives and were quantitated with a GC/SIM using each molecular ion (T, ET: m/z=480, 1-dehydro T: m/z=478). As a result, T and ET were completely separated by using a 3% SE-30 as an isolation column for GC and it became feasible to finish the analysis in less than 3min without disturbing peaks. The T values in urine measured by this method correlated well with those by radioimmunoassay (r=0.928). This method is logical and was found to be specifically suitable for the clinical laboratory test.
This report describes the serum-dependent nonspecific oxidation of hydrogenperoxide, which strongly interferes with the determination of hydrogen-peroxide by use of continuous flow hydrogen-peroxide electrode. Properties and characteristics of the H2O2-oxidizing factors in the serum were studied. Dialysis experiments showed that a nondialyzable, high molecular weight fraction of the pooled serum (named“A” fraction) is mostly responsible for H2O2-oxidation, while a dialyzable, low molecular weight fraction (named“B” fraction) containing various chemical reducing substances such as ascorbic acid, glutathione, cysteine, bilirubin and uric acid is playing only minor role. The heat-labile and sodium azide-sensitive properties strongly suggests“A” fraction to be a catalase.
A calculation algorithm for the probability chart method of normal range estimation is proposed. In order to get a similar normal range to the result of the eye fit regression line on the probability chart, the regression line is calculated with weighted least square method between upper limit of the class mark and the probability scale of the proportional cumulative sum using the frequency table. Normal ranges are calculated before and after logarithmic variable transformation, and they correspond to the normal ranges of normal distribution and of log-normal distribution respectively. Correlation coefficients (r) calculated with the weighted regression method can be used for selection of well fit distribution type. The result which shows larger r is selected as the well fit distribution. The normal range thus obtained is coincident well with that calculated with iterative truncation parametric method.