The laser nepherometric quantitation of α-thiol proteinase inhibitor (α-TPI) in human serum was established by using a monospecific rabbit antiserum against human α₂-TPI.The reaction was performed by incubating 100μl of 30-fold diluted serum and 200μl of 5-fold diluted antiserum in a cell at room temperature for 60 min.The accuracy of the method was determined to be 4.3% for the within-run and 8.7% for the betweenrun. The average α-TPI level in sera from normal healthy individuals was 425±97 mg/l (mean±S.D., n=67). There was no statistical correlation between sex and age.
We observed a rough correlation between the α-TPI level determined by laser nepherometry and by a single radial immunodiffusion (r=0.53, p<0.05), and a strict correlation between laser nepherometry and rocket immunodiffusion (r=0.75, p<0.01). We observedthe remarkable changes of α-TPI in sera from the patients with chronic renal failure, between before and after receiving hemodialysis. The patients with inflammatory diseases tended to show a lower level of serum α-TPI within the normal range. We concluded that the laser nepherometry provides a more simple, rapid and sensitve procedure to determine serum α-TPI, comparing with other immunochemical methods.

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Mouse monoclonal antibodies and rabbit sntisera against human prostate specific antigen (PA) were produced. A double antibody sandwich enzyme immunoassay system was developed for serum PA determination using these monoclonal and polyclonal antibodies. The assay sensitivity is I ng per ml of serum. An initial clinical study indicates that serum PA levels were specifically high in patients with progressive prostate cancer. The new EIA method offers great potential for clinical management of prostate cancer.

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An attempt to characterize the amino acid residue of the terminal and internal bond in uremic peptide chains by enzymatic digestion and chemical hydrolysis after dansylation was made. Thirteen uremic peptides chosen from two groups of 36 uremic peptids which had been obtained from hydrophobic middle molecule fraction in hemofiltrates of previously reported two dialysis patients, were reacted with 8 kinds of proteases. The result of enzymatic digestion revealed that these peptide chains were hardly cleaved with the following enzymes; CPase B (EC 3.4.12.3), pyroglutamate aminopeptidase (EC 3.4.11.8), trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.2) and papain (EC 3.4.22.2). Only one residue of each terminal amino acid of 3 uremic peptide chains was slightly released by the action of CPase A (EC 3.4.12.2) or Y and LAP (EC 3.4.11.2), whereas the sequential release of amino acids was not observed. Although only α-amino terminal residue of 10 uremic peptides as dansylamino acids was produced by chemical hydrolysis after dansylation and ε-amino-dansyl lysine was undetectable in the hydrolysate of the peptide chain including lysine residue, results indicate possibility of the modification of theε-amino group of lysine in the chain. On the other hand, it was also clarified these uremic peptides have no inhibitory effect against protease. These results suggest that there are possible modifications on the amino acid residues in uremic peptide chains which lower hydrophobicity and hinder the conjugation with plasma components or enzyme proteins. This undigestable character in itself may lead to the retention of uremic peptides in blood circulation.

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The new chemiluminescence assay of glucose oxidase (GOD) has been developed and applied to the enzyme immunoassay using GOD as the label enzyme. GOD activity can be assayed by two reaction steps in which at the first step non-fluorescent leuco-2', 7'- dichlorofluorescein is oxidized to fluorescent 2', 7'-dichlorofluorescein (DCF) by H₂O₂ generated from glucose with the catalytic reaction of GOD (to be assayed), and then at the second step DCF gives chemiluminescence by reaction with bis (2, 4, 6-trichlorophenyl) oxalate-H₂O₂ in acetonitrile. Detection limits of H₂O₂ and GOD were 10^-7 mol/l and 1.0μU/ml, respectively. This method was applied to the EIA of thyroxin (T₄) using GOD as the label. Results for T₄ by the EIA correlated well with those for serum determined by radioimmunoassay (r=0.947).

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in the determination of lipid peroxide (LPo) by the method using 2-thiobarbituric acid (TBA) the addition of various metal ions to the reaction mixture containing authentic unsaturated fatty acids as sample led to a marked increase in LPO value. Metal ions showing a stimulation of LPO production were Fe²⁺, Fe³⁺, V³⁺ and Cu²⁺ and the orders of their stimulating activities were Fe²⁺>Fe³⁺>V³⁺>Cu²⁺.ln contrast, the metal ions such as Zn²⁺, Mg²⁺, Ca²⁺ and Mn²⁺ had no effect on the LPO production.ln the determination of serum LPO by the same method, however, Fe³⁺specifically caused an increase in LPO Value unlike the case on the authentic fatty acids.
With respect to the correlation among the TAB method, the Fe³⁺-TBA method and the Hb-MB method, there was a significant correlation between the TBA method and the Fe³⁺- TBA method (p<0.001), while the Hb-MB method had no correlation with the remaining two methods.
We further exammed the correlation between each determined value of serum components and serum LPO values estimated by the thre emethods. LPO values estimated by the TBA method were positively correlated with contents of total choiesterol, phospholipid and uric acid. LPO values estimated by the Fe³⁺-TBA method were positively correlated with contents of totalcho-esterol, phospholipid, triglyceride and albumin, and negatively correlated with LDH activity and ZTT level. LPO values estimated by the Hb-MB method showed a positive correlation with total protein content and uric acid values

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High-dose methotrexate and citrovorum factor (HD-MTX-CF) treatment was performed for the following patients suffering from osteosarcoma: case 1 (7-year-old female), case 2 (9-year-old male), case 3 (19-year-old female), case 4 (45-year-old female). In this treatment, it is important keeping urine alkaline to prevent renal failure caused by crystallization of MTX in acid urine. For this purpose, meylon (sodium bicarbonate solution) was injected intra-venously. Though the importancy of keeping urine alkaline has been emphasized, there are few reports describing urine pH profile during HD-MTX-CF.
We studied urine pH profile during HD-MTX-CF for the patients described above. Becouse urinary pH decreases from midnight to morning, then the additional meylon should be injected in the early morning.
We also studied plasma and urine amylase and N-acetyl-β-D-glucosaminidase, plasma immunosuppressive acidic protein (IAP) and Sialic scid. This HD-MTX-CF for patients described above was performed successfully, these data and general liver and kidney function tests showed no significant changes during the treatment.

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The assay of furosine, which is acid-hydrolyzed from fructose-lysine, glucose bound to lysine residues of protein, was used as a specific method for determining nonenzymatic glycosylation of tissue protein.
In order to determine furosine derived from fructose-lysine of hair as a tissue sample which is easily taken from living body, fundamental studies were performed. The diabetic patients showed significantly higher furosine levels than healthy subjects. These resultssuggest that the hair furosine may become an indicator of past blood glucose control at any time by selecting the portion of hair in diabetic patients.

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A simple and rapid method for the determination of uric acid in human serum by high-performance liquid chromatography is described.Serum (0.1ml) was mixed with 1.0 ml of 0.4 mol/l perch-oric acid and the mixture was centrifuged at 2000×g for 10 min. The supernatant diluted twofold with 0.25mol/l disodium hydrogen phosphate was injected onto a reversed-phase ODS column.The mobile phase was 40 mmol/l sodium phosphate buffer, pH2.2, containing 2%methanol.The eluent was monitored at 284 nm.The lower detection limit for uric acid was 1μmol/l.The analytical recovery of uric acid was found to be almost 100%.The within-run precision (CV) was0.52% and the day-to-day CV was 1.1% for a serum uric acid concentration of 238 μmol/l.No interferences from ascorbic acid, hypoxanthine and xanthine were observed.The correlation between the value by the present method (y) and that by the uricase-catalase method of Kageyama (x) was good (y=0.997x -4.57, r=0.996).

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Effect of dissolved oxygen on the measurement of the plasma g-ucose by theglucose analyzer using oxygen electrode, New Glucoroder, was examined.
The value of plasma glucose measured by this method was higher than the enzyme method using hexokinase, and the increased value of glucose were observed in the case of low levels of dissolved oxygen in plasma.This plus errors were related to concentration of dissolved oxygen and were independent of the concentration of plasma glucose, and significant effect was observed in the venous blood and the blood drown by evacuate tube with holder.This errors were disappeared by shaking the test tube to increase oxygen level in the plasma.
These errors were minimized by compensating the influence of dissolved oxygen using double cathodes of oxygen electrode.

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We investigated the effect of pH on the formation of glycosylated hemoglobin. Erythrocytes collected from 5 healthy subjects were incubated in the 30 mM triethanolamine HCI buffer contaning 2000mg/dl glucose at 37° for 3 days. Glycosylated hemoglobin was determined by high-performance liquid chromatography (HPLC) using a ion-exchange column chromatography (Auto A_1c) and affinity column chromatography (Glyc-Affin system).
Furthermore, as glycosylated hemoglobin (GHb-f) we determined furosine which is acidhydrolyzed from fructose-lysine, glucose bound to a lysine residue of hemoglobin, by HPLC using a reversed phase column. Both stable and labile component levels of glycosylated hemoglobin were positively correlated with pH levels. Therefore, it is suggestedthat pH level is important in considering the changes of glycosylated hemoglobin levels in the clinical state such as uremic acidosis or diabetic ketoacidosis.

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ln order to investigate the possible involvement of the plasma kallikrein-kinin system (K-K system) in Crow-Fukase syndrome, high molecular weight kininogen (HMWK), low mclecular weight kininogen (LMW-K), plasma prekallikrein (P.KaI), kininaseI (KI), kininaseII (KII) andFactor XII levels were measured in the plasma of 7patients.
Factor XI Ievels in the plasma of all patients assayed were significantly Iower than that of normal control (between20%and70% of normal control).A downward value in HMW-K level was also observed, while no significant change in the levelsofLMW-K, P.KalandKI was observed. On the other hand, markedly decreasedKII level was obsen/ed in the plasma of 6 patients (between 30% and 70% of normal control).
Judging from these results, a following possible hypothesis which suggests the involvement of the K-K system in this syndrome could be proposed. Namely, the activation of Factor XII by some reasons would occure and the generated Factor XIIa activated P. Kal. Then, the generated kallikrein selectively acted on HMW-K and liberated bradykinin from HMW-K molecule. Kallikrein also acted on Factor XII and the consumption of Factor XII wouldl be accelerated.therefore kinin would play important roles in this syndrome, such as edema formatlon etc. Decreased KII level would facilitate the kinin action.
ln our present studies no significant change ofP. Kal level and not so much altera- tion of HMW-K level were observed, so further details are now being studied to make sure the involvement of the K.K system in this syndrome.

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