myeloperoxidase-dependent pathway. J. Lipid Res. 2011. 52: 87-97. Supplementary key words secosterol • neutrophils • myeloperoxidase • infl ammationA reactive species with a chemical signature similar to that of ozone has been proposed to be generated by the antibody-catalyzed oxidation of water with singlet oxygen during the oxidative burst of activated human neutrophils and in infl amed tissues ( 1, 2 ). The formation of an ozonelike oxidant from singlet oxygen by neutrophils can be catalyzed not only by antibodies but also by amino acids such as tryptophan, methionine, and cysteine ( 3 ). To prove the formation of an ozone-like oxidant by neutrophils, previous studies used chemical reactions, such as the conversion of indigo carmine to isatin sulfonic acid or the oxidation of vinylbenzoic acid to 4-carboxybenzaldehyde. However, these reactions are not suffi ciently specifi c to ozone to conclude an ozone production by neutrophils ( 4, 5 ).Further evidence for ozone formation in vivo was based on the detection and formation of the cholesterol ozonolysis products 3 ␤ -hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A, also called atheronal-A) and its aldolization product 3 ␤ -hydroxy-5 ␤ -hydroxy-B-norcholestane-6 ␤ -carboxaldehyde (secosterol-B, also called atheronal-B) in human tissues. These secosterols were previously reported to be formed only by ozone among the various reactive oxygen species (ROS) such as singlet oxygen, superoxide anion, hydroxyl Abstract 3 ␤ -Hydroxy-5-oxo-5,6-secocholestan-6-al (sec osterol-A) and its aldolization product 3 ␤ -hydroxy-5 ␤ -hydroxy-B-norcholestane-6 ␤ -carboxaldehyde (secosterol-B) were recently detected in human atherosclerotic tissues and brain specimens, and they may play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. However, as their origin remains unidentifi ed, we examined the formation mechanism, the stability, and the fate of secosterols in vitro and in vivo. About 40% of secosterol-A remained unchanged after 3 h incubation in the FBS-free medium, whereas 20% and 40% were converted to its aldehyde-oxidation product, 3 ␤ -hydroxy-5-oxo-secocholestan-6-oic acid, and secosterol-B, respectively. In the presence of FBS, almost all secosterol-A was converted immediately to these compounds. Secosterol-B in the medium, with and without FBS, was relatively stable, but ‫ف‬ 30% was converted to its aldehyde-oxidation product, 3 ␤ -hydroxy-5 ␤ -hydroxy-B-norcholestane-6-oic acid (secoB-COOH). When neutrophillike differentiated human leukemia HL-60 (nHL-60) cells activated with PMA were cultured in the FBS-free medium containing cholesterol, signifi cantly increased levels of secosterol-A and its aldehyde-oxidation product, but not secosterol-B, were formed. This secosterol-A formation was decreased in the culture of PMA-activated nHL-60 cells containing several reactive oxygen species (ROS) inhibitors and scavengers or in the culture of PMA-activated neutrophils isolated from myeloperoxidase (MPO)-defi cient mice. Our results demonst...

An impaired generation of nitric oxide has been associated with diabetic renal disease. In order to elucidate the underlying molecular mechanisms into how nitric oxide synthesis is impaired in diabetic renal disease, we examined changes in activities and expressions of some renal enzymes involved in nitric oxide production during the development of diabetic nephropathy in type II diabetic Otsuka Long-Evans Tokushima Fatty rats. Ten-week old Otsuka Long-Evans Tokushima Fatty (n = 40) and control Long-Evans Tokushima Otsuka rats (n = 20) were given drinking water containing 20% sucrose to accelerate the development of diabetic nephropathy. Otsuka Long-Evans Tokushima Fatty rats developed diabetic nephropathy in an age-dependent manner. Renal nitric oxide synthase activities in Otsuka Long-Evans Tokushima Fatty rats gradually declined with the progression of diabetic mellitus and were significantly lower than those of age-matched Long-Evans Tokushima Otsuka rats after 22 weeks of age. The lower activities of renal nitric oxide synthase in Otsuka Long-Evans Tokushima Fatty rats were correlated with relatively higher levels of renal free asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, and were also correlated with decreased activities of dimethylargininedimethylaminohydrolase which metabolizes asymmetric dimethylarginine to citrulline. These results imply that dimethylargininedimethylaminohydrolase dysregulation may play an important role in the development of diabetic nephropathy by increasing asymmetric dimethylarginine levels, which leads to inhibition of renal nitric oxide synthesis.