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A novel peroxidase activity assay was developed for horseradish peroxidase (HRP) and myeloperoxidase (MPO), in which substrate Eu2+was catalytically oxidized to Eu3+, and the Eu3+luminescence was enhanced by the addition of sensitizer 4,4’-bis(1“,1”,1“,2”,2“,3”,3“-hepatafluoro-4”,6“-hexanedione-6”-yl)chlorosulfo-o-terphenyl (BHHCT) for time-resolved measurement of the BHHCT-Eu3+complex. Since BHHCT-Eu3+has a long lifetime (more than 500 (is), typical of Eu3+oxidation state, and the emission wavelength (615 nm) is totally different from those of Eu2+complexes, time-resolved luminescence measurement of the Eu3+complex enabled suppressed background and high signal/background ratio. The present method was successfully applied to monitor the oxidative stress level, which is closely associated with peroxidase activity level, in rat heart muscle homogenates. Notable parallel temporal change was observed for peroxidase activity and 4-hydroxynonenal (HNE) concentration after lipopolysaccharide (LPS) injection for induction of oxidative stress in rats. Such a relation does not contradict the oxidative stress mechanism that HNE is produced vialipid peroxidation, which is caused by the •OH radical generated by peroxidase activity.

A highly sensitive time-resolved fluoroimmunoassay of human plasma cytokines is described. The cytokines such as interleukin-1 α(IL-1 α), tumor necrosis factor α(TNF α) are known to be acute inflammatory cytokines and it has been reported that these cytokines are secreted into blood by physical exercise. In this study, a sandwich-type immunoassay of cytokines was established using a europium chelate BHHCT-Eu3+as a powerful labeling material. The minimum detection limits of cytokines, i.e.IL-1 α, TNF α, and interferon γ(IFN γ) were about 1/10 smaller than those of enzyme-linked immunosorbent assay currently used. By this immunoassay we investigated cytokine increase/decrease in plasma which was thought to derive from the myocytes damaged by bicycle exercise. Healthy young men performed two kinds of bicycle ergometer exercises, under conditions of an incremental and a constant loading. Blood samples were taken before, during, and after exercises, and the concentration levels of plasma IL-1 α, TNF α, and IFN γwere determined. In the case of incremental exercise, IL-1 αincreased significantly at the first stage but decreased to the basal level from the second stage, in spite of heavier exercise. In the case of 30 min constant exercise, the level of plasma IFN γincreased in recovery period, 2 h after the light-exercise. TNF αlevel was significantly higher in a heavy-exercise. The concentration of IL-1 αpeaked at the early stage of the incremental exercise; this fact has not been reported in previous studies. This cytokine is unique in showing a sudden increase during the early stage, while others increase after the exercise. Our highly sensitive assay made it possible to detect a slight change in plasma cytokines.

In selenium determination by carbon-furnace atomic absorption spectrometry, organic materials severely decrease selenium sensitivity. In order to remove the severe negative interference due to organic materials, matrix modifications by addition of Pd, Pt, Ni and Rh were compared. Palladium was found to be most effective. Addition of 100 μg ml-1Pd completely removes the negative interference due to up to 1.5% albumin and restores the sensitivity to that of a simple inorganic Se(IV) solution without organic materials.

Effect of absorption-tube shape in flame-in-tube atomic absorption spectrometry of selenium was investigated. About ten quartz tubes with various hydrogen and air introduction systems were designed and compared for selenium sensitivity. From the examination of the tube size ranging 6 to 50 cm in length and 1 to 7 mm in i.d., the following conclusion was found. At relatively large i.d. (-7 mm), no sensitivity increase is observed by tube elongation. However, at i.d. of 1 mm, elongation of the tube length from 30 to 50 cm increases the sensitivity in proportion to the tube length. At i.d.’s between 1 and 7 mm, sensitivity increase is certainly observed for tube elongation, but the increase is not so much as expected if it would be in proportion to the tube length. This would be due to the difference of the atomic population in a tube in this medium i.d. range. The best sensitivity and detection limit obtained with a tube of 50 cm long and 1 mm i.d. were 0.021 ng (1% absorption) and 0.047 ng, respectively. The method was successfully applied to the analyses of NBS SRM’s.