Reactive nitrogen species derived from nitric oxide are potent oxidants formed during inflammation that can oxidize membrane and lipoprotein lipids in vivo. Herein, it is demonstrated that several of these species react with unsaturated fatty acid to yield nitrated oxidation products. Using HPLC coupled with both UV detection and electrospray ionization mass spectrometry, products of reaction of ONOO- with linoleic acid displayed mass/charge (m/z) characteristics of LNO2 (at least three products at m/z 324, negative ion mode). Further analysis by MS/MS gave a major fragment at m/z 46. Addition of a NO2 group was confirmed using [15N]ONOO- which gave a product at m/z 325, fragmenting to form a daughter ion at m/z 47. Formation of nitrated lipids was inhibited by bicarbonate, superoxide dismutase (SOD), and Fe3+-EDTA, while the yield of oxidation products was decreased by bicarbonate and SOD, but not by Fe3+-EDTA. Reaction of linoleic acid with both nitrogen dioxide (*NO2) or nitronium tetrafluoroborate (NO2BF4) also yielded nitrated lipid products (m/z 324), with HPLC retention times and MS/MS fragmentation patterns identical to the m/z 324 species formed by reaction of ONOO- with linoleic acid. Finally, reaction of HPODE, but not linoleate, with nitrous acid (HONO) or isobutyl nitrite (BuiONO) yielded a product at m/z 340, or 341 upon reacting with [15N]HONO. MS/MS analysis gave an NO2- fragment, and 15N NMR indicated that the product contained a nitro (RNO2) functional group, suggesting that the product was nitroepoxylinoleic acid [L(O)NO2]. This species could form via homolytic dissociation of LOONO to LO* and *NO2 and rearrangement of LO* to an epoxyallylic radical L(O)* followed by recombination of L(O)* with *NO2. Since unsaturated lipids of membranes and lipoproteins are critical targets of reactive oxygen and nitrogen species, these pathways lend insight into mechanisms for the formation of novel nitrogen-containing lipid products in vivo and provide synthetic strategies for further structural and functional studies.

SummaryNeonatal, adult, and fetal rat lungs of 18,20, and 22 d gestation from four to six litters were examined for cytochrome oxidase, glucose-6-phosphate dehydrogenase, catalase, glutathione peroxidase, copper-zinc and manganese superoxide dismutase activities. All results were corrected for the contribution of enzymes in blood that contaminate homogenates. Because lung protein/ DNA ratios and body water change significantly with gestational age, enzyme activities were expressed as U/mg DNA. All activities were low in d 18 lung and increased with advancing gestational age. Only catalase and copper-zinc superoxide dismutase increased activity in response to air breathing, suggesting that maturation of the antioxidant enzyme system is virtually complete before delivery. Activities of glucose-6-phosphate dehydrogenase, catalase, glutathione peroxidase, and manganese superoxide dismutase were higher in neonatal than in adult lung. AbbreviationsBPD, bronchopulmonary dysplasia CAT, catalase CYT OX, cytochrome oxidase G-6-PD, glucose-6-phosphate dehydrogenase GPX, glutathione peroxidase HBSS, Hank's balanced salt solution SOD, superoxide dismutase Premature delivery and exposure to elevated concentrations of inspired oxygen may be significant risk factors (8,14) for the development of BPD. Successful adaptation to a hyperoxic environment by young or adult animals of a number of species is associated with increased intrapulmonary concentrations of enzymes with antioxidant protective functions (6, 22).There is evidence that the initial cell damage observed in hyperoxic tissue occurs as a consequence of increased production of intracellular oxygen radicals by cell constituents, including mitochondria and endoplasmic reticulum (1 I , 20, 29). Cell survival requires the cell either to have adequate antioxidant defense mechanisms or to rapidly respond to oxidant stress by an increases of such mechanisms that can detoxify reduced species of oxygen and by-products. Because production of superoxide and hydrogen peroxide increases in lung cells as a function of oxygen concentration (1 1, 20, 29), the 5-fold increase in arterial oxygen tension which occurs after delivery (23) would be expected to pose significant oxidant stress to the normal neonatal lung, especially since an acute 5-fold increase in inspiratory oxygen concentration is lethal to adults of most species (20). The normal term newborn human infant, however, shows no clinical evidence of pulmonary oxidant damage, suggesting that adequate antioxidant defences have been induced in the lung before delivery. Gestation-dependent increases in antioxidant enzyme activities have been reported for placenta (30) and for catalase, but not GPX or SOD (18, 34) in liver. In rat brain GPX activity is similar, at birth, to that observed in adults whereas SOD activities are low compared with the adult (19).Postnatal changes in pulmonary antioxidant enzyme activities have been comprehensively documented (5, 15, 33) whereas, to our knowledge, antenatal changes have only been studied i...

SummaryIt was obsegved that the lung catalase activity of premature (day 21 of gestation; term = 22 days) rat pups is affected by maternal iron intake. Pups from control dams receiving Purina Lab Chow and water ad libitum have only 50% of the lung catalase activity of pups from dams who received 1 mg/kg parenteral iron dextran daily from day 7 to day 20 of gestation. Other oxygen-protective enzymes, copper-zinc and manganese superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase, were unaffected by maternal iron supplements. AbbreviationsCuZn SOD, copper zinc superoxide dismutase CAT, catalase G-6-PD, glucose-6-phosphate dehydrogenase GPx, glutathione peroxidase Mn SOD, manganese superoxide dismutase HBSS, Hanks' balanced salt solution Undernutrition is reported to enhance pulmonary oxygen toxicity (10). One potential contributing factor is a loss of metalloprotein antioxidant enzymes activities upon metal depletion. This has been reported with copper (4) for CuZn SOD, with manganese (6) for Mn SOD, with iron and selenium (18) for GPx, and with iron (20) for CAT.We have previously reported that the specific activity of lung catalase of gravid female rats fed Purina Lab Chow ad libitum declined with gestation (13). The fetuses of these animals demonstrated gestation-dependent increases in specific activity of CuZn SOD, Mn SOD, GPx, and G-6-PD in lung between day 18 and day 22 (term) of gestation (24). The specific activity of fetal lung CAT, however, increased between days 18 and 20 of gestation, but showed no significant increase between day 20 and term (24). Since the fall in maternal lung CAT activity towards the end of gestation was reversed by parenteral iron supplements, it is possible that the blunted increase in fetal lung catalase reflected limited iron transport across the placenta.In this report, we examine the effect of maternal iron supplementation on fetal lung growth, and on the specific activity of oxygen-protective enzymes of fetal rats sacrificed prematurely on day 2 1 of gestation. Reported values have been corrected for Received June 13, 1983;accepted February 7, 1984 enzyme activity contributed by contaminating blood by a modification of the method described by Cross et al. (5), using homologous blood supplements. MATERIALS AND METHODSFemale Sprague-Dawley white rats of 275-300 g (Charles River Inc., St. Constant, Quebec) were mated with male hooded rats and delivered to our animal quarters the following day. From day 7 to day 20 of gestation, the gravid rats were injected intramuscularly with either 1 mg/kg/day of iron dextran in isotonic saline or isotonic saline alone, or received no injections. On day 21 of gestation (term = 22 days), the animals were sacrificed by excess chloroform anesthesia; the fetuses were decapitated in utero to prevent respiration and immediately removed from the uterus. Each fetus was weighed and the carcasses were placed in HBSS at 4°C. The chest cage was opened from diaphragm to neck; then the heart and lungs were removed en bloc and...

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