A study on the extraction method of plasma nicotine and cotinine for FTD gas chromatography is described. Ether anhydrous for nicotine and chloroform for cotinine are suitable organic solvent, respectively. A internal standard method using quinoline or 5-aminoquinoline is profitable to obtain a good result. Two kinds of extraction method were adopted, which were the method l for single determination of nicotine and the method II for simulataneous determination of nicotine and cotinine. Reproduciblility of the methods was good and the recovery of nicotine and cotinine was approximately 90-100% of added ones to plasma. The method was sufficiently sensitive to detect nicotine of 0.1ng·ml^-1and cotinine of0.2ng·ml^-1.
The concentration of nicotine in plasma of 10 non-smokers was2.5±2.93ng·ml^-1 and cotinine could not be detected. Plasma nicotine and cotinine concentration of 20 smokers before smoking were14.5±8.68ng·ml^-1and145.3±138.23ng·ml^-1respectively. Reagent blank value in these methods was about2-3ng·ml^-1as nicotine and none as cotinine, it may be due to contaminations in reagent and during the procedure. Plasma nicotine rised to maximum value at just behind or 15minutes after voluntary smoking of one cigarette and falled to the former value after 1hour. In case of plasma cotinine, its rise and fall was more gradual in comparison with nicotine and the retention-time in plasma was longer than nicotine consequently.

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The spots of the corresponding trigryceride, digryceride and monogryceride on thin-layer chromatography for the quantitative analysis of the lipid in human serum have been investigated.
The human seram was treated to separate into individual components by means of column arld thin-layer chromatography. The separated grycerides were reduced to the corresponding alcohols with lithium alumirlum hydride in diethyl ether. The structure deter. mination of ester components was performed on the ground of chemical and spectral evidence.
The liverated acids from glycerides were C_14:0, C_14:1, C_16:0C_16:1, C_18:0, C_18:1, C_18:2and C_20:4.

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A simple fluorometric method has been developed for determining urinary free cortisol or corticosteroids. The procedure is as follows: Extract 2ml of urine with 2ml of dichloroethane, wash the organic layer once with 0.5ml of water and twice with 0.5ml of 0.1N NaOH, mix 0.2ml of the organic layer with ethanol-conc. H₂SO₄ (3:7, v/v), stand it for 40min, and then perform fluorometry (λ_ex=468nm, λ_em=520nm).
This method was compared with an isotope dilution method and other methods for the determination of urinary free cortisol or corticosteroids. The values obtained by the present method, dichloroethane extraction-fluorometry, showed good correlation with those by the isotope dilution method and dichloromethane extraction-RIA, and were coincident well with those by the latter two methods. A common method, dichloromethane extraction-fluorometry, showed good correlation with the present method but gave apparently high values. Application of RIA to unextracted urine gave three times the values assayed by the present method.
Thus, this method is suitable for a routine laboratory test for urinary free cortisol from the view point of simplicity, accuracy and precision.

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A new continuous monitoring method for the determination of leucine aminopeptidase (LAP) was presented. This method is based on the principle which consists of the measurement of ammonia liberated from the substrate leucineamide by the action of LAP with glutamate dehydrogenase as a coupling enzyme. Kinetical properties of glutamate dehydrogenase from Proteus inconstans used for the coupling system were studied and the optimum condition for the measuring of LAP was settled on the basis of the theory described by Horio et al.⁹
L-Leucine-β-naphthylamide and L-leucine-p-nitroanilide have been used as substrate for the determination of LAP in clinical laboratories. The activities of LAP which used these coventional chromogenic substrates (Arylamidase, A. A) and that of LAP presented in this study were compared in various diseases. Good correlation was obtained from both LAP and A. A activities in almost all cases of normal and hepatobiliary disorders, however, remarkable discrepancy was observed in both activities in the cases of hepatitis, malignant lymphoma and leukemias. It was suggested that the method presented in this study would be useful for the detection of these lymphomatosis and leukemic diseases.

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A noble system for estimating HDL-cholesterol by a continuous ultrafiltration method is described. Immunocomplexes, produced after reaction between Apo β-associated lipoprotein in sample serum and anti β-lipoprotein goat serum, form gradually large precipitates in a mixing coil and are removed by a membrane filter attached to a dialyzer.
The immunocomplex-free HDL fraction is continuously mixed with enzymic reagent to start cholesterol determination. The day-to-day precision of this system is fairly good (±CV% of 2.75 to 4.36%). Correlation of this system with the airfuge ultracentrifugal procedure was studied by the use of least squares regression analysis, and we obtained the following values: n=55, y=0.89x+3.7, r=0.978.
Our data indicates that continuous flow system for the estimation of HDL-cholesterol can be used efficiently for routine tests.

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Using high performance liquid chromatography, a quantitative, specific and direct determination method for urinary catecholamines [Adrenaline (Adr.), Noradrenaline (NA) and Dopamine (DA)] was established. The principle of our present method is that with high-performance liquid chromatograph, catecholamines were selectively separated from other urinary components by gradient elution method. For continuous detection of catecholamine fractions, trihydroxyindol procedure was employed. Each catecholamine was separated definitely from other urinary components. Only 60 minutes was required for analysis of one sample. As little as 10-20μl urine specimens were found to be enough for the analysis. The sensitivity was 1.0ng/ml for Adr., 1.5ng/ml for NA and 150ng/ml for DA. The coefficient of variation byintra-assay (n=5) was ±2.9% for Adr.,±3.0% for NA and ±5.0% for DA. The coefficient of variation by inter-assay (n=7) was±1.7% for Adr.,±2.3% for NA and ±7.2% for DA. The average of recovery was 101.4% for Adr., 98.7% for NA and 97.7% for DA. Thus, the present methodhas proved to be feasible for the specific assay of urinary catecholamines in monitoring patients with hypertention.

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The Cyclization of N-carbomyl-α-alanine, and N-carbomylglycine by the treatment with hydrochloric acid was Investigated by means of the Jaffé reaction and Fearon reaction. Effect of the corresponding hydantoin compounds produced on Velocity and Coloration in the Jaffé reaction were compared. The results thus were as follows:
1. The Jaffe reaaction proceeded more rapidly and the color from 1-methyl hydantoin developed more storongly than that from hydantoin.
2. The Jaffe reaction of 1-methyl hydantoin proceededs more sluggishly than that of creatinine.
3. 5-methylhydantoin, the product from N-carbamyl-α-alanine, hardly reacts with the Jaffe reagent.
4. There is no differance in absorptionspectra in the visible range among the complexs of the Jaffe reagent with creatinine, hydantoin and 1-methylhydantoin.
5. N-carbamyl-glycine was very difficult to cyclization product when heated under the acidic condition.

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A duplex preliminary treatment procedure was studied for the determination of catecholamines (CA) in human urine using high-performance reversed-phase chromatography with electrochemical detector system (HPLC-ED). Three mobile phases were applied to the chromatography; A (citrate/phosphate), B (sodium phosphate) and C (citrate/acetate). Preliminary purification of the specimen was carried out with a boric acid gel and alumina seccessively.
Urinary CA levels thus determined revealed to be stable in each mobile phase as compared to those determined afterinitial treatment with alumina or boric acid gel alone. The duplex purification system also eliminated many unkown peaks which appeared in the chromatogram of urinary CA's and interfered with CA determination. A elongated elution time which was often the case with single purification system could be shortened by the successively purified procedure.
Those results have proved that the procedure should be available in urine CA determination.

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Rapid spectrophotometric assay of mutarotase activity with β-D-glucose dehydrogenase for 3 min was deviced. The reaction system composed of 960μl of 50mM Tris-HCI buffer (pH 7.4), 10μl of β-D-glucose dehydrogenase solution (2,500 units/ml), and 15μl of 200mM NAD⁺. When 15μpl of 100mM α-D-glucose was added, a slow increase of absorption (A) of NADH at 340nm due to dehydrogenation of β-D-glucose formed from α-D-glucose nonenzymatically was recorded. Mutarotase was then added to record a rapid absorption (B) of NADH due to dehydrogenation of β-D-glucose formed from α-D-glucose nonenzymatically and enzymatically. The difference (B-A) of two rates of NADH formations corresponds to the activity of mutarotase.

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A simple high-performance liquid chromatographic method for determination of the psychotropic drug, clocapramine, in plasma is described. The method permits the accurate determination of the drug in plasma as low as 5ng/ml and is suited for monitoring the drug in the therapeutic dose range and for the investigation of the bioavailabilities of the drug preparations. Simultaneous determination of clocapramine and carpipramine, another psychotropic drug, is possible.

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We evaluate E₃ kit which is commercially available, and have found some problems. Effect of protein on adsorption of estrogen to Amberlite XAD-2 is observed and the effect is increased by increased concentration of protein.
Abnormal absorption curve is also observed with samples from pregnant woman given some antibiotics. The interference of these drugs is corrected by three points wavelength measurement, but the effects of protein could not be avoided.

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For the test of curve fitting, an indicator g was proposed, which is calculated_??_, where f_i is the observed frequency, e_i is the theoretical frequency calculated from the model distribution, and k is the number of classes within the normal range. Decisions are made as follows;“good” for g less than 1.18,“acceptable” for g between 1.18 and 2.00, and “dissociate” for g larger than 2.00. The fittness of Usui-Kawai's distribution model to the patients' data disribution of 22 laboratory tests were tested using this indicator, many of them showed good or acceptable fitting except very skewed distribution such as GPT,γ-GTP and TTT tests.

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