Optimum reaction conditions were evaluated for assay of serum copper by use of guanidin hydrochloride as a decomposing agent for high order structure of protein and TAMSMB as a copper chelating and color deveroping agent.These reaction condition were judged to be optimum: (a) concentration of guanidin hydrochloride should be more than2.5mol/l, pH4.2with acetate buffer 0.5mol/l, the mixture of potassium iodate4.5mmol/l and potassium iodide 0.4mmol/l as a oxidizing agent for various reductants in serum.(b) sample pretreatment with the solution of buffered guanidin hydrochioride and oxidizing agent for 5min period is absolutely effective to cause copper release from protein and combert it to oxidized form, After this treatment, the TAMSMB-copper coupling reaction are compieted within one min.Using these reaction conditions, we devised an improved direct method for measuring serum copper without a deproteinizing process.Results compare well with those by atomic absorption spectrometry.The within day and day to day reproducibility with pooled serum (copper concentration of 110μg/dl) by this method were c.v. 0.65% and 1.62%, respectively.

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A highly sensitive fluorescent determination for CPK isoenzyme with continuous flow injection system is described.
In the system, -iquid chromatography with stepwise gradient and stream switching technique with six-port valve are utilized. CPK isoenzyme is separated on a micro column packed with DEAE-sepharose and NADH is produced with a coupled enzymaticreactions.Serum and reagents backgrounds are separated from fluorescence NADH by stream switching technique. A major advantage of the detection system is the sensitive monitoring of the isoenzyme activities.
In the measurement of control serum, coefficient of variation was2.5%for CPK-MM, 6.6%for CPK-MB and 9.3%for CPK-BB (n=10).
We have applied this technique to the patient serum to study about clinical investigation. especially CPK MB and BB activities in the case of myocardial infarction for recent ll months.

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The measurement of free γ-Carboxyglutamic acid (Gla) in urine was examined. After separation of Gla from whole urine by anion exchange chromatography, the eluent was concentrated by evaporation and subsequently physio-ogical fluid analysis was performed by Hitachi high performance amino acid analyzer (type835-50) using lithium citrate buffer (Li 0-15N, pH 2.3) as eluant. Gla peak was eluted at 18 minutes. The precision with 10 nmole /50μl of Gla by this method was 0.86-2-52%in CV. Recovery in the anion exchange concentration step 85.9-93.5% and recovery of Gla added to urine was 97.3%. The values obtained by this amino acid analysis correlated well with those by high performance liquid chromatography in 17patients with urolithiasis (r=0.95).
In 11 male calcium stone formers. urinary free Gla excretion ranged from 25-2 to 43.8μmole/24h (37.3±5.4, Mean±SD) and 19 normal male adults excreted from 35.6to62.4μmole Of Gla per day (46.9±7.4, Mean±SD). AS Urinary free Gla excretion is SignifiCantly decreased in calcium stone formers compared to normal adults, the urinary free Gla seems to be an inhibitory factor in calcium stone formation. However, further studies are needed to draw a definite conclusion.

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Colorimetric methods for the determination of serum guanase, based on the formation of H2O2 by the coupling of xanthine oxidase (XOD) followed by color development with peroxidase-MBTH-aniline derivative system, were deveKoped.
As the aniline derivatives, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-aniline (ALOS) andN-ethyl-N-(3-sulfopropy1)-m-anisidine (ADPS) were used for fixed time assay and continuous monitoring assay, respectiveiy.
The addition of superoxide dismutase (SOD) to the coupling step of XOD markedly improved the linearity of reaction, and the formation of H2O2 was stoichio-metrically measured.
The coefficient variance of normal serum material was less than3.3%both on fixed time and Continuous monitoring assay.
The proposed methods, which were with only two reagents system and relatively higher reproducibility, would be simply applied to commercial automated analyzer and useful for the diagnosis of liver cell damage.

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Direct injection of plasma samples on to high-performance liquid chromatographic system for the determination of procainamide and N-acetylprocainamide is proposed. After injection of plasma sample on to protein-coated ODS column, the initiaalq ueous solution eluted plasma proteins and hydrophilic compounds, and then the second solvent containing3.5% acetonitrile eluted procainamide and N-acetylprocainamide, which were adsorbed in sma-l pores of ODS resins from an aqueous solution, by reverse phase mode.
The recoveries of drugs spiked in human plasma were almost quantitative (101.8% for procainamide and99.3%for N-acety-procainamide) with good reproducibility (c.v.;1.1% and 1.4%, respectively, n=6, within-run), and the analyzed drugs were total amount in plasma regardiess of free or bound to plasmaproteins.The procedure was superior, in view of simplicity and accuracy, to the conventional method, which included deproteinizingorsolvent extractionstepwhich should have been done prior to injection of samples.

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The serum apoproteins A-I and A-II concentrations were determined in124 patients with hepatobiliary diseases and30normal subjects.The determination of these apoproteins by single radial immunodiffusion was highly corre-ated with that by electroimmunoassay.
The mass of apoproteins A-I and A-II, the major proteins of high-density lipoprotein (HDL), decreased in many liver parenChymal diseases and obstructive jaundice. But, the ratio of apoprotein A-I to A-II increased only in the decompensated stage of liver cirrhosis and obstructive jaundice-The amounts of apoproteins A-I and A-II in hepatobi-iary diseases correlated with HDL3-cholesterol (d=1.063-1.125).The clinical course of apoproteins A-I and A-II in one patient with subacute hepatitis showed the discrepancy with HDL-cholesterol, and the histological findings of biopsy specimen were identifiable with the amounts of the former than that of the latter.The amounts of apoproteins A-I and A-II correlated strognly with conventional liver functional tests (e.9., albumin, choline-esterase, total protein, lecithin-cholesterol acyltransferase).
These results indicate that apoproteins A-I and A-II are metabo-ized somewhat independently of each other and the lipid components in HDL.We must, therefore, measUre apoproteins as well as lipid moieties when -ooking at patlents with hepatobiliary diseases.

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A sensitive method for the simu-taneous determination of dexamethasone and cortisol in serum is described.The procedure involves pre-column derivatization of corticosteroids through their 21-hydroxyl groups with9-anthroyl nitrile followed by high-performance liquid chromatography of the fluorescent esters.These two were efficiently separated on a Cosmosi-5SL column with hexane/ethyl acetate (7: 5) and determined with a detection limit of20fmol employing the21-(9-arlthroyl) derivative of triamcinolone acetonide as an internal standard.The clean-up of corticosteroids hbiologicalfluidswasattainedbythe use of a Sep. pak C18cartridge.

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