A microextraction method for the determination of plasma nicotine, which required only 100μl of sample, was described. Gas chromatographic technique was in accordance with the condition of previous reports. Recovery of nicotine in plasma was about 100% and reproducibility of the method was sufficient for practical use. Plasma nicotine level of 20 non-smokers was 4.6±2.08ng ml-1 in average and its of 8 smokers before smoking was 14.8±10.74ng ml-1. After smoking of one cigarette (nicotine content in smoke is 0.8mg a cigarette), nicotine level in plasma increased to maximum value at just behind after smoking (30.4±21.49ng ml-1) and decreased rapidly to former value until 60 minutes after. Reagent blank value in this method was about 2-3ng ml-1 as nicotine. This method is a profitable technique for a rapid deternination of plasma nicotine.
In order to determine the B-cell function in the patients with chronic pancreatitis who had glucose intolerance, we measured C-peptide immunoreactivity (CPR) in plasma. In patients with chronic pancreatitis who are insulin requiring diabetics, concentrations of fasting plasma CPR were low as compared with those never treated with insulin. Concerning the CPR response to glucose ingestion, no significant peak was observed in the patients with chronic pancreatitis as a whole, and the CPR response is smaller than that of adult type diabetics in spite of the equal glucose changes. As for the patients with chronic pancreatitis, ΣCPR/ΣBS showed a higher score in those treated by diet than in those who had been treated by insulin. On the other hand, the group with normal or low values in one factor of the pancreozyminsecretin test showed higher CPR responses than the group with low values in two or three factors of the pancreozymin-secretin test.
1) With use of high performance Liquid chromatography (HPLC) and continuous flow fluorometric monitoring of LDH, LDH isoenzymes were assayed within 7min in 10μl of human serum. 2) HPLC method was more rapidly and easily performed than electrophoretic method, and its reproducibility and quantitativity were sufficient for utilization in clinical chemistry. This method was favorable for LDH assay, because LDH reaction was conducted in a short time such as 8min at 37°C, and in a homogeneous solution. 3) A good correlation was obtained between HPLC method and elecrophoretic method. However, some problems of this method are remained to be solved such as an appearance of a minor LDH peak usually eluted before LDH-3, and the effect of coexistent substance in serum on the fluorometric LDH assay.
A simple and sensitive assay method for kallikrein-like arginine esterases using N-α-carbobenzoxy-L-lysine thiobenzyl ester (ZLSB) as substrate was established. The present method was sixteen thims more sensitive than the spectrophotometric method using N-α-benzoyl-L-arginine ethyl ester (BAEE) as substrate. Minimum identification limit of the enzyme activity in the present method was recognized as 0.95 n mole of substrate hydrolyzed per minute as enzyme activity judging from the minimum value in the present expreiment.
The modified urease-indophenol method was applied to determine salivary urea nitrogen concentration. This method enables easy sampling and yields good correlation between blood urea nitrogen and salivary urea nitrogen. From these results, it has been determined that this method is especially suitable for mass-screening, and for patients whose blood sampling is difficult.
Heparin-Ca precipitation method for measurement of cholesterol in high-density lipoprotein (HDL-C) has been improved for a good separation of HDL from low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) in normo-and hyperlipemic sera by adding NiCl2 (1mM) to heparin-Ca reagent. We proved that this method can be used for the separation of HDL fraction from the other lipoproteins in a specimen of serum triglycerides (TG) concentration of about 1g/dl. The correlation between the results of dextran sulfate-Mg precipitation method and present method is very high (n=46, r=0.980) with regression line, y=1.05x+0.4. Precision (CV) % was found to be 2.3% (n=10) within run and 3.5% (n=20) run-to-run. Present method was applied to measure HDL-C in normo-and hyperlipemic sera in the routine clinical measurement.
In the determination of uric acid we investigated the interference of ascorbic acid on the uricase-peroxidase reaction, and studied the elimination of its interference with use of ascorbate oxidase. The investigation was conducted in comparison with the uricase-catalase method described by Kageyama. 1) Interference exerted by one mole of ascorbic acid on the uricase-peroxidase (MBTH-DMA) reaction was correspondent to about one mole reduction of uric acid value. 2) 0.5U/ml of ascorbate oxidase in the uricase solution (pH 7.2) was enough to break down ascorbic acid up to 17mg/dl in the samples within 2.5min at 37°C. 3) In the case of urine samples including patients, the addition of ascorbate oxidase gave the almost concordant values of uric acid with those obtained by the catalase method. 4) In the case of serum samples, there remained significant discrepancies in uric acid values from those of the catalase method, although ascorbic acid was removed by ascorbate oxidase. The discrepancies were revealed as the kind of proportional bias both in normal adults and patients, and in the latter there were some non-icteric sera indicating furthermore considerable discrepancies.
The 3-methyl-2-benzothiazolone hydrazone hydrochloride (MBTH) method for the assay of glucose was improved to facilitate simple, rapid and sensitive estimation of this compound in serum. In this procedure, serum can be incubated with the enzyme system in the presence of MBTH. The incubation time was remarkably shortened by the elevation of incubation temperature. The present method is suitable for the assay of glucose using diluted serum.
Sex difference in urinary excretion levels of β-phenylethylamine, tyramine and tryptamine in healthy male and female adults was investigated. These amines were determined by the method of Watanabe et al. In female subjects, changes of urinary excretion levels of these amines during menstrual cycle were also examined. Tyramine and tryptamine levels in urinary excretion were not different between male and female. But, the amount of β-phenylethylamine excretion in the premenstrual period was increased significantly when compared with that of male subjects. Effects of administration of precursors on the urinary excretion of these three amines were examined. A load of 1g tryptophan produced an increase of tryptamine excretion in four subjects out of five.