We developed and tested a simple, practical procedure for the isolation of bilirubin covalently bound to albumin (delta bilirubin: Bd), employing ammonium sulfate precipitation and ultrafiltration. A sample isolated using the procedure had properties consistent with those previously demonstrated for Bd. We were able to isolate 99-100% purified Bd from a sample of serum from jaundiced patients at a recovery rate of 78%.

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When high density lipoproteins (HDL) containing serum amyloid protein A (SAA) were incubated for 3 hr with granulocyte elastase in vitro, SAA were degraded rapidly and almost completely disappeared, but no significant effects were noted to occur for other HDL apoproteins. A high concentration of elastase could also degrade apo C-III protein, though the rate of degradation was lower than that for SAA. SAA in HDL were also catabolized rapidly by polymorphonuclear leucocytes (PMNL) in a manner similar to that by elastase. Elastase, however, degraded SAA more specifically than did PMNL. Aprotinin inhibited the degradation of SAA apoproteins in HDL by elastase and PMNL. This inhibitory effect on SAA degradation was weaker than that on apo C-III degradation.

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A high-performance liquid chromatographic method involving a postcolumn photochemical reaction and fluorometric detection has been developed for the determination of nalidixic acid concentration in human urine. The mobile phase consists of 0.1M potassium dihydrogenphosphate-sodium tetraborate buffer (pH8.6)-methanol (75: 25, v/v) containing 20mM hydrogen peroxide. The compound in the column effluent is irradiated with ultraviolet light to produce fluorescence. This fluorescence is monitored with excitation at 380 nm and emission at 440nm. The calibration curve for nalidixic acid is linear over the range of 0.2-300ng/injection. The mean recovery from urine is nearly 100%.

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Pyridinoline (PYR) and deoxypyridinoline (DP YR) promiss to be more excellent bone resorption markers than hydroxyproline, because they are only present in mature collagen, their major sources are bone and cartilage, and their excretion in urine is not influenced by the degradation of newly synthesized collagen molecules or noncollagenous protein. Therefore, a quantitative method was developed for the determination of PYR and DPYR cross-links of collagen in urine. Using this method, the inherent fluorescence of PYR and DPYR was detected using a high-performance liquid chromatography. The values of PYR and DPYR are given as equivalent amounts of pyridoxamine because there are no authorized standards, and are expressed as pmol/μmol creatinine. The mean values±SD of PYR and DPYR in urine from 26 healthy adults (13 males, 24-56 years old; 13 females, 23-31 years old) were 4.50±1.03 and 1.09±0.28, respectively. The PYR and DPYR excretion levels of a patient with hypothyroidism were similar to those of healthy adults, but those of two patients with hyperthyroidism were significantly higher. In cases of patients with bone metastases of malignant tumors, significantly higher levels of PYR and DPYR were detected in the urine.

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Urinary estradiol 17-sulfate (ES) concentration during a pregnancy was measured by radioimmunoassay using a highly specific antiserum to ES. The ES levels rose gradually from the 6th gestational month, and such levels in late pregnancy became over four-times higher than those in non-pregnant women. The ES concentration at the 7, 8, 9 and 10th gestational months were 15.0±2.3, 17.0±2.2, 23.5±2.4, and 41.1±4.7ng/ml (mean±SE), respectively. After delivery, the urinary ES rapidly decreased; the values at 8 and 24 hours after delivery were 7.0±0.9 and 5.8±0.6ng/ml, respectively, or almost at the same levels in non-pregnant women. The physiological role of the ES production during a period of pregnancy is described herein.

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We studied the mechanism of the diazo coupling reaction of bilirubin using various diazonium salts.
It was found that an intermediate, which is stable and can be extracted easily with waterchloroform, was produced in this reaction. This substance turned into a yellowish color with a maximum absorption of about 420 nm, which was similar but not identical to that of bilirubin itself. However, it did not produce the green colored product called biliverdin following its oxidation with a nitrous acid solution unlike bilirubin.
The intermediate reacted comparatively gradually with various diazonium salts and produced an azo dye like the diazo coupling reaction of bilirubin itself. Also, aldehyde was detected in this reaction, and its amount was approximately proportional to that of bilirubin used. Based on the result of H¹-NMR spectroscopy, and the reaction with the diazonium salts it was assumed that the intermediate is a dipyrrole, a part of the bilk ubin molecule.
From these results, it is assumed that in the diazo coupling reaction of bilirubin the azo dye and the intermediate are produced by a cleavage of the bilirubin molecule with an electrophilic attack by the diazonium salts, and then the intermediate reacts with the diazonium salt and produces the azo dye and the aldehyde.

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We have found that the Lana AG® kit used for determination of 1, 5-anhydroglucitol in plasma or serum interfered with the intravenous infusion of maltose, which rarely exists in normal plasma. The principle of the kit consists of the removal of interfering substances with a mini-column, an oxidation of the recovered 1, 5-anhydroglucitol by pyranose oxidase and a detection of the hydrogen peroxide with a color development reaction. At the detection system, glucose is a major interfering sugar but it is completely trapped in the mini-column. Because of the lower affinity of the mini-column for maltose, infused maltose leaks out of the mini-column slightly. As the pyranose oxidase does not react with maltose, it is stipulated that pyranose oxidase is contaminated with maltase. The leaked maltose is hydrolyzed into glucose with maltase and the formed glucose is oxidised with pyranose oxidase and changes color.

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A simple and specific enzyme immunoassay for betamethasone was developed. In order to improve the specificity and affinity of antibodies for betamethasone, the antiserum was prepared by immunizing rabbits with a 4-(carboxymethylthio) betamethasone-bovine serum albumin conjugate. Alkaline phosphatase was used as a labeling enzyme. Cross reactivity for cortisol which is considered as a candidate for the interfering substance in serum with the antibody was 0.03%. Intra-and inter-assay coefficient of variations were 4.1-8.3% (n=6) and 3.1-13.7% (n=6), respectively. The minimum amount of betamethasone detected was 1 pg/tube and the measurable range was from 0.1 to 100ng/ml, using 10μl of serum sample without extraction. The chronological changes of serum betamethasone and cortisol concentration in five normal subjects after an oral administration of 1 mg of betamethasone were also reported.

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We have developed a sensitive enzyme-linked immunosorbent assay for the determination of arantichymotrypsin (ACT) and measured it in serum and cerebrospinal fluid (CSF) collected from some patients without any probed neurological diseases. Using 5 pl of CSF or 100 times diluted serum, as less as 0.4 μg/ml of ACT could be detected by this method. The coeffi-cients of variation for between-run measurement by this method were 3.8% (n=10, X =6.3μg/ml) for ACT in CSF and 3.7% (n=10, X=451μg/ml) for ACT in serum. The inter-ference by bilirubin, hemoglobin turbidity, albu-min, immunoglobulin G, haptoglobin, α1-acid gly-coprotein and prealbumin was negligible. The normal reference values of ACT (mean±SD) were determined as being 447±85 μg/ml (n=86) for serum and 2.23±0.69μg/ ml (n=11) for CSF.

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