The spin-trapping method with 5,5-dimethyl-1-pyrroline Noxide (DMPO) has been widely accepted as an assay method to measure hydroxyl radical formation, and to detect the hydroxyl radical scavenging activity of a compound. Although unstability of DMPO is claimed because it turns yellow with time even at -20˚C in a sealed tube under vacuum, 1 the basic chemistry of DMPO and DMPO-OH has not been well understood.In a previous work, 2 we noticed very weak DMPO-OH signals in the baseline of a negative control spectrum which was obtained after standing DMPO solution at 37˚C for 24 h. We suspected that in a DMPO aqueous solution at a higher concentration and at a higher temperature, an appropriate amount of DMPO-OH might generate. And we considered that this reaction should provide a practical preparation method to obtain DMPO-OH radical as a simple aqueous solution which contains no other materials except unreacted DMPO. Also this DMPO-OH aqueous solution may be utilized in the investigation of the chemical or physicochemical properties of DMPO-OH radicals.When DMPO was dissolved in purified water and the solution was heated at 70˚C for 30 min, a sufficient amount of DMPO-OH radical formation was observed by ESR ( Fig. 1). Under an argon atmosphere, DMPO-OH formation was minimized to 1/4, and this slightly increased under a dioxygen atmosphere. This indicates that dioxygen participates in the reaction. Previously, Makino et al. observed DMPO-OH formation in an aqueous mixture of DMPO and 1 mM FeCl3. Based on this observation of the formation of iron chelate with DMPO at 77 K, and DMPO-OCH3 formation in the presence of CH3OH, researchers elucidated that nucleophilic attachment of water to DMPO should occur in the presence of Fe 3+ ion. 3 To study the effect of a small number of metal ions in purified water, we replaced the purified water with ultra-pure water for ICP-MS measurements; the latter should contain iron of no more than 1 ppb. In ultrapure water, and under an argon atmosphere, DMPO-OH signal was still observed after heating, although its intensity decreased to about 1/20. In this communication, we describe DMPO-OH radical formation in hot water, in which dissolved dioxygen participates in the reaction and metal ions such as Fe 3+ might catalyze DMPO-OH formation. University, Japan *3 Tokyo Metropolitan Industrial Technology Research Institute, Itabashi, Japan *4 Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho, Japan When an aqueous solution of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was heated at 70˚C for 30 min, formation of DMPO-OH was observed by ESR. This DMPO-OH radical formation was suppressed under an argon atmosphere. When water was replaced with ultra-pure water for ICP-MS experiments, DMPO-OH radical formation was also diminished. Under an argon atmosphere in ultra-pure water, the intensity of the DMPO-OH signal decreased to about 1/20 of that observed under aerobic conditions with regular purified water. The addition of hydroxyl radical scavengers such as mannitol did not a...

Temporary cessation of milking is widely used during the dry period of dairy cows. Temporary cessation of milking induces an increase in the somatic cell count (SCC) and level of several inflammatory components of milk, which is believed to be a local adaptation and defense mechanism of the mammary gland. In Japan, temporary cessation of milking combined with antibiotic administration is widely used to treat mastitis. The present study aimed to elucidate the role of the innate immune system during temporary cessation of milking in a goat model by investigating the concentration of several innate immune components in milk during and around the temporary cessation. In experiment 1, 6 goats were subjected to cessation of milking for 3 d in both udder halves, whereas in experiment 2, 6 other goats were subjected to cessation of milking for 3 d only in 1 udder half. In experiment 1, the milk yield was lower on d 5 and 6, whereas the mean SCC was higher on d 5 compared with d 0 before temporary milking cessation. The concentrations of goat DEFB1, S100A7, cathelicidin-2 and 7 (CATHL-2 and 7), IgA, and lactoferrin were increased after temporary cessation of milking. In experiment 2, the milk yield was lower between d 5 and 7, whereas the mean SCC was higher between d 4 and 7 compared with d 0. The concentrations of CATHL-2, IgA, and lactoferrin were increased after temporary cessation of milking only in the udder half subjected to milking cessation. These results suggest that temporary cessation of milking increase the SCC and concentration of several innate immune components in milk without infection, which may contribute to mastitis treatment.

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