Nitrate production and phagocyte activation: differences among Sprague-Dawley, Wistar-Furth and Lewis rats.

We have found that activated resident peritoneal macrophages from Wistar-Furth and Sprague-Dawley rats produce significantly more superoxide (2.2 +/- 0.4 and 3.6 +/- 0.8 nmol of cytochrome c reduced/10(6) cells/10 min respectively) than those from Lewis rats (1.0 +/- 0.3 nmol of cytochrome c reduced/10(6) cells/10 min). Similar results are found in macrophages elicited with intraperitoneal thioglycolate. Furthermore, nitrate excreted in the urine increased greatly when either Wistar-Furth or Sprague-Dawley rats are injected i.p. with iota carrageenan (0.25 g), a response that did not occur with Lewis rats. This strain difference in the manifestations of the inflammatory response correlates with previous observations that Wistar-Furth and Sprague-Dawley rats, but not Lewis rats, develop suture-line colonic tumors when the ureters and bladder base are implanted into the colon.

5-Aminosalicylate: oxidation by activated leukocytes and protection of cultured cells from oxidative damage.

It has been postulated that oxygen radicals may play a role in the pathogenesis of inflammatory bowel disease. If so, then a drug like 5-aminosalicylate (5-ASA), which is used to treat such diseases, might work by interacting with oxygen-derived species. We found that activated mononuclear cells and activated granulocytes, as well as the products of the Fenton reaction, transformed [14C]5-ASA to a number of metabolites, among which we have characterized salicylate and gentisate. We also found that the lethal effect on cultured Chinese hamster ovary cells of adding either superoxide radical or hydrogen peroxide, components of the respiratory burst of activated white blood cells, was diminished by the addition of 100 micrograms/ml (0.65 mM) of 5-ASA. Thus, we have demonstrated that 5-ASA was oxidized by the oxidative burst of white blood cells and that 5-ASA protected cells from damage by oxygen-derived species, two findings which may offer an explanation for the role of 5-ASA in the treatment of inflammatory bowel disease.

Basal N-nitrosoproline formation and excretion in the ferret.

Quantitative balance studies indicated that ferrets (Mustela putorius furo) on purified diets excreted approximately four times more N-nitrosoproline than they ingested. This basal N-nitrosoproline excretion was not dependent on dietary nitrate intake, as high levels of dietary [15N]nitrate did not result in the incorporation of 15N into the excreted N-nitrosoproline. This indicates that there are at least two sources of endogenous N-nitrosoproline formation, one linked to dietary nitrate and one independent of dietary nitrate. The latter may result from an intermediate in the endogenous synthesis of nitrate.

Nitrate balance and biosynthesis in the ferret.

The net biosynthesis of nitrate in the ferret was demonstrated to be 8.89 to 10.3 mumol/kg bw/day. Nitrate balance studies indicated that, because of metabolism, excretion of nitrate was lower than ingestion when oral doses were higher than 6.3 mumol/day. At nitrate ingestion levels of less than 6.3 mumol/day, excretion exceeded intake. Studies with 15N-labeled nitrate indicated that only 36% of the oral dose was recoverable as [15N]nitrate from urine. Oral doses of 15N-labeled ammonia resulted in the incorporation of the 15N label into nitrate in urine and feces. This demonstrates that ammonia can serve as a precursor to biosynthesized nitrate. An increase in nitrate excretion was seen when animals were treated with neomycin sulfate and mycostatin to reduce gut microflora populations. This increase was as large as 200%. Short-term studies indicated that dietary protein quality had little effect on nitrate excretion.

Activated oxygen and mammalian nitrate biosynthesis.

The oxidation of NH3 to NO3- by rat liver in vitro is described. A xanthine-xanthine oxidase reaction also oxidized NH3 to NO3- when H2O2 was added. An in vivo inhibitor of superoxide dismutase enhanced the in vitro liver conversion of NH3 to NO3-. Thus, intracellular oxidation by activated oxygen likely represents the source of endogenously formed NO3- in mammals.

A rapid method for estimating nitrate in biological samples using gas chromatography with a flame-ionization detector or a thermal energy analyzer.

A simple, rapid method for estimating the nitrate content of biological samples was developed and tested. The method was based on the nitration of benzene under acidic conditions. The resulting nitrobenzene was analysed by gas chromatography with either a flame ionization detector (FID) or a Thermal Energy Analyzer (TEA) for detection. Recovery of nitrate added to urine, blood, saliva or faeces was 96, 97, 96 and 98%, respectively. Coefficients of variation for repeated analyses ranged from 2.2% for saliva to 12% for blood. Sensitivity was of the order of 0.5-1.5 mg/kg for the FID and 0.02-0.05 mg/kg for the TEA.

The modulating effect of an inhibitor of cholesterolgenesis present in bovine milk upon the synthesis of cholesterol, dolichol and ubiquinone.

Bovine milk contains two inhibitors of hepatic cholesterol genesis. One of these, identified as orotic acid, influences the early segment of the cholesterol biosynthetic pathway and suppresses the conversion of acetate to mevalonate. In this study the other inhibitor was shown to curtail the formation of compounds past farnesyl pyrophosphate on the squalene-cholesterol branch of the pathway. Thus cholesterol synthesis may be suppressed while the production of two other products of the branched pathway, dolichol and ubiquinone, is allowed to continue. The possible role of these ingested regulators in the metabolism of the young until they achieve sufficient development is discussed.