A new HPLC (High Performance Liquid Chromatography) method for the determination of HbAlc (Hemoglobin Alc) is proposed. This method uses the hydrophobic stationary phase, which can separate minor components of hemoglobin more clearly than the previous method.
As this stationary phase is hard enough to bear high pressure, precise data has been achieved for many loadings.

抄録全体を表示

The Km value (0.ssmM) of the brush border-bound maltase from the rat small intestine for a-maltose was lower than that (2.3mM) for β-maltose, whereas the Vmax values for both anomers were the same. Thus, at finite concentrations, α-maltose was better hydrolyzed than β-maltose by the enzyme. This α-anomeric preference seemed to be purposive in starch digestion because pancreatic α-amylase is known to produce maltose in the a form from starch. A modification, utilizing fluorometry of NADH, of a spectrophotometric method for the assay of glucose anomers with β-D-glucose dehydrogenase and mutarotase was developed and applied to the anomer analysis of the glucose released by hydrolysis of maltose by the brush border-bound maltase. The glucose of the non-reducing end was released exclusively in the a form: i.e., almost entirely pure a-Dglucose was produced from a-maltose. The data obtained in this study were discussed in relation to the transport mechanism of the glucose liberated from maltose.

抄録全体を表示

Adenosine deaminase, purine nucleoside phosphorylase and guanase activities in sera of patients with various diseases were determined by the new colorimetric methods.
It was observed that the serum guanase activity increased specifically in liver disorders and correlated with serum transaminase activity. Although the serum adenosine deaminase activity also increased in liver diseases, the clinical value of adenosine deaminase may be different from that of guanase.
It was found that the serum purine nucleoside phosphorylase activity, which increased in patients with malignant lymphoma and systemic lupus erythematosus, correlated with the amount of sensitized lymphocyte.

抄録全体を表示

Transferrin (Tf) and ceruloplasmin (Cp) in human serum are found to strongly inhibit lipid peroxidation by rat liver microsomes, and a study was made of this inhibition. Apo-Tf strongly inhibited NADPH dependent and ascorbic acid dependent lipid peroxidations of microsome. However, the inhibition of apo-Tf almost disappeared by adding iron ion to the system of the reaction. Inhibition of holo-Tf formed by adding Fe³⁺ to apo-Tf was mar kedly decreased. As a result of this, it was inferred that inhibition of apo-Tf is due to bon ding with iron ion necessary for lipid peroxidation in microsome. Cp showed a stronger in hibition than apo-Tf. It was confirmed that inhibition of Cp is due to oxidation of Fe²⁺, which is necessary for lipid peroxidation of microsome, into Fe³⁺ by ferroxidase action of Cp.

抄録全体を表示

A new and simple method for protein assay in strongly acidic solution was developed and applied to the assay of perchloric acid soluble glycoproteins (ASP) in serum. Serum ASP levels were determined directly with the supernatants deproteinized by 0.6M perchloric acid using Coomassie Brilliant Blue G-250. This method required neither pH neutralization nor further precipitation of ASP by phosphotungstic acid.
The comparative studies of ASP and seromucoid fraction have shown that the composi tion of both protein fractions was almost identical. Also the serum levels of ASP was highly correlated with seromucoid levels by Winzler method. The results suggest that ASP determi nation by C.B.B.-G-250 method is thought to be identical with serum mucoprotein assay.

抄録全体を表示

A new radioimmunoassay method (Amerlex® Free T₄) for measurement of the serum concentration of free thyroxine (FT₄) was evaluated with the following results.
1. The estimated values were almost constant during incubation for 30 to 2₄0 min at37°C.
2. The coefficients of variation, which were 2.5-3.5% (within assay) and 5.2-16.5% (between assays), were acceptable for clinical tests.
3. The FT₄ values of sera with added T₄ determined by this method were identical to those estimated by computational analysis.
4. The FT₄ concentration determined by this method in normal sera was 1.₄2±0.2₄ng/dl (normal range: 0.90-1.90ng/dl). The serum FT₄ concentration was increased in hyperthyroidism, decreased in hypothyroidism, at the lower limit of normal in pregnancy (18-26 weeks) and liver cirrhosis, and within normal limits in familial TBG excess and TBG deftciency.
5. The FT₄ values determined by this method correlated well with those determined by computational analysis, and equilibrium dialysis and with the FT₄ index.

抄録全体を表示

A reverse phase high performance liquid chromatography (HPLC) method was developed for the determination of Hypoxanthine, Xanthine, Uric acid, Allopurinol (4-hydroxypyrazolo-(3, 4-d) pyrimidine) and Oxypurinol (4, 6-dihydroxypyrazolo-(3, 4-d) pyrimidine) in human serum and urine.
This technique is as follows: Speciments were deproteinized with Amicon centriflo membrane cone type CF25 (Amicon Corp.) and were injected into the HPLC column directly. 0.04mol/l phosphate buffer, pH 2.2, contained 2% methanol (V/V) was used as the mobilphase and the eluent were monitored at 254nm.
Proposed method, which has good sensitivity (the lower limites of determination 1.0 μmol/l for the compounds), precision (CV, 2-3% for 100 μmol/l compounds) and accuracy (almost 100% recovery), were observed.
This method should be useful in the clinical studies for abnormal purine metabolism.

抄録全体を表示

Methods for a determination of 3-methoxy-4-hydroxy-mandelic acid (VMA) based on the Pisanos' method are complicated and time consuming, so the convenal method is modified to simplify and increase speed of an analysis. The procedure can eliminate interfering substances (for example, vanillin) from urine by the pre-extraction using ethyl acetate, and the treatment for oxidation with periodic acid to convert VMA to vanillin is performed at the room tempareture. Vanillin which formed is extracted with toluene, followed by the extraction with 1M potassium carbonate solution, and then absorbance of the potassium carbonate layer is determined spectrophotometrically.
The coefficient of variation between the Pisano's method and the proposed method was 0.95, and mean recovery of standerd added to urine was 99.8 per cent (n=21).
The determination of VMA, the end-product of catecholamine metabolism, is important for diagnosis of patients with pheochromocytoma, ganglioneuroma, neuroblastoma, metastatic carcinoid, and familial dysautonomia, etc, so this modified simple method is useful for the clinical test.

抄録全体を表示

A semi-automated method for arginase activity in a dried blood spot and hemolysate is described. Urea produced by arginase action was measured with the enzymes of urease and glutamate dehydrogenase, 2-oxoglutarate and NADH. The decrease in absorbance at 340nm at 30°C was measured by a centrifugal analyzer, the Centrifichem 400. The conditions of activation with Mn²⁺, incubation and urea determination are discussed. In dried blood spots, elution of erythrocyte and activation of arginase were performed by incubating discs at 37°C for 30min. We used 25mmol/L MnCI₂ to activate and 150mmol/L arginine as substrate to obtain the maximum velocity of arginase activity. As HCIO₄ was used for the stopper of the enzyme reaction, pH adjustment for the following enzymatic urea determination could be ommited. Linearity and reproducibility were acceptable. The correlation between arginase activities from dried blood disc and hemolysate in the same subjects was r=0.953. The reference values were 41.7±7.75U/g·Hb in dried blood disc and 39.3±9.44 U/g·Hb (mean±SD, n=20) in hemolysate. The activity from dried blood spot is stable at room temperature for one week, which allows to be mailed.

抄録全体を表示

A cation-exchange high-performance liquid chromatographic method for the sensitive determination of dityrosine in hydrochloric acid-hydrolysate of insoluble protein is described. The method is based on the clean-up of the acid hydrolysate by Dowex 1×8 column chromatography and the separation of dityrosine in the eluate from the column by high-performance liquid chromatography with fluorescence detection on Hitachi Gell 3011-S. The limit of detection for dityrosine is 0.6 pmol per 10μl (the injecton volume) of the sample solution for the chromatography. This method permited the determination of dityrosine in collagen from cow skins and tendons, and should be useful for the determination of dityrosine in various tissues in biological samples.

抄録全体を表示