Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].

Macrophage oxidation of l-arginine to nitrite and nitrate

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate.

NITRIC oxide is an important bioregulatory molecule, being responsible, for example, for activity of endothelium-derived relaxing factor (EDRF)1–4. Acute hypertension5, diabetes6, ischaemia7and atherosclerosis8 are associated with abnormalities of EDRF. Nitric oxide is thought to be a retrograde messenger in the central nervous system9. The technology is not yet available for rapid detection of NO released by a single cell in the presence of oxygen and/or nitrite, so the release, distribution and reactivity of endogenous NO in biological systems cannot be analysed. Here we describe a porphyrinic microsensor that we have developed and applied to monitoring NO release in a microsystem. We selectively measured in situ the NO released from a single cell with a response time of less than 10 ms. The microsensor consists of p-type semiconducting polymeric porphyrin and a cationic exchanger (Nation) deposited on a thermally sharpened carbon fibre with a tip diameter of ∼0.5 (μm. The microsensor, which can be operated in either the amperometric or voltammetric mode, is characterized by a linear response up to 300 μM and a detection limit of 10 nM. Nitric oxide at the level of 10−20 mols can be detected in a single cell.

Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor

Nitric oxide (NO) is a small, gaseous, paramagnetic radical with a high affinity for interaction with ferrous hemoproteins such as soluble guanylate cyclase and hemoglobin. Interest in NO measurement increased exponentially with the discovery that NO or a related compound is the endothelium-derived relaxing factor (EDRF). In addition to being a potent endogenous vasodilator, NO has a role in inflammation, thrombosis, immunity, and neurotransmission. Measurement of NO is important as many of its effects (e.g., vasodilatation, inhibition of platelet aggregation) are similar to those of other substances produced by the endothelium, such as prostacyclin. NO is formed in small amounts in vivo and is rapidly destroyed by interaction with oxygen, making measurement difficult. A computerized search of the past five year's literature found NO measurements reported in fewer than 50 of 955 articles dealing with EDRF. Inhibitors of NO synthesis such as the arginine analogs or agents that inactivate NO, such as reduce...

Measurement of nitric oxide in biological models.

Background Persistent pulmonary hypertension of the newborn causes systemic arterial hypoxemia because of increased pulmonary vascular resistance and right-to-left shunting of deoxygenated blood. Inhaled nitric oxide decreases pulmonary vascular resistance in newborns. We studied whether inhaled nitric oxide decreases severe hypoxemia in infants with persistent pulmonary hypertension. Methods In a prospective, multicenter study, 58 full-term infants with severe hypoxemia and persistent pulmonary hypertension were randomly assigned to breathe either a control gas (nitrogen) or nitric oxide (80 parts per million), mixed with oxygen from a ventilator. If oxygenation increased after 20 minutes and systemic blood pressure did not decrease, the treatment was considered successful and was continued at lower concentrations. Otherwise, it was discontinued and alternative therapies, including extracorporeal membrane oxygenation, were used. Results Inhaled nitric oxide successfully doubled systemic oxygenation in 16 of 30 infants (53 percent), whereas conventional therapy without inhaled nitric oxide increased oxygenation in only 2 of 28 infants (7 percent). Long-term therapy with inhaled nitric oxide sustained systemic oxygenation in 75 percent of the infants who had initial improvement. Extracorporeal membrane oxygenation was required in 71 percent of the control group and 40 percent of the nitric oxide group (P=0.02). The number of deaths was similar in the two groups. Inhaled nitric oxide did not cause systemic hypotension or increase methemoglobin levels. Conclusions Inhaled nitric oxide improves systemic oxygenation in infants with persistent pulmonary hypertension and may reduce the need for more invasive treatments.

Inhaled Nitric Oxide and Persistent Pulmonary Hypertension of the Newborn

A method devised for the determination of N-nitrososarcosine, in which the N-nitrosamine in solution is denitrosated with hydrogen bromide to form volatile products that are rapidly removed and determined in a chemiluminescence analyser, has been applied successfully to the same compound on powdered corn flakes. Differentiation of N-nitrososarcosine and a number of other N-nitrosamines and N-nitrosamides from inorganic nitrite was achieved by decomposing the nitrite with acetic acid prior to the denitrosation of the N-nitroso compounds. In the presence of a secondary-amine receptor limited nitrosation can occur during the process of differentiation but this can be prevented through the use of ascorbyl palmitate. In differentiating between large amounts of nitrite and much lower levels of N-nitrososarcosine on corn flakes, using a chemiluminescence analyser, the duration of the response from the nitrite can be shortened by freeze-drying the food matrix in the presence of ascorbic acid. The spectrophotometric determination of N-nitrososarcosine as nitrosyl bromide released into solution by the action of hydrogen bromide was hindered by the presence of powdered corn flakes.

Determination of a non-volatile N-nitrosamine on a food matrix

A method has been devised for the determination of N-nitrososarcosine, representative of the group of non-volatile nitrosamines of physiological origin, whereby the nitrosamine is denitrosated with hydrogen bromide to form the volatile nitrosyl bromide. After dehalogenation in a heated converter, the nitrosamine can be determined as nitric oxide by using a chemiluminescence analyser, the response being linear up to at least 200 µg. The limit of detection is below 6 ng, and is therefore much lower than that for the determination of N-nitrososarcosine as inorganic nitrite in solution after denitrosation. A response is also obtained from inorganic nitrite but this can be differentiated from that of N-nitrososarcosine by the use of acetic acid alone, prior to denitrosation with hydrogen bromide in the same solvent.

Determination of a non-volatile nitrosamine by using denitrosation and a chemiluminescence analyser

In model system studies simulating the frying of bacon a considerable variation in the production of N-nitrosopyrrolidine (NOPyr) has been observed, as occurs in sliced bacon fried conventionally. The inclusion of ascorbate or erythorbate (isoascorbate) has led consistently to a fall in or an elimination of NOPyr production up to an addition of 300 ppm (1,5 mM). Above this concentration of ascorbate, the elimination of NOPyr formation persisted in some instances but in others an increased production of the nitrosamine occurred. Usingα-tocopherol, a fall of NOPyr production during the simulated frying of bacon was obtained with increase of concentration of the antioxidant, there being a consistent elimination of the volatile nitrosamine following an addition of 500 ppm (1.16 mM). Whilst ascorbyl palmitate had a similar action in reducing or eliminating NOPyr production, no synergistic effect between it and a-tocopherol was noted in an experiment combining the two antioxidants. When bacon was fried in fat containing 400 or 800 ppmα-tocopherol, the total production of NOPyr and DMN in the fried bacon, cooked out fat and condensate was markedly reduced but not completely eliminated as in the model system studies. Similarly, the curing of bacon with a brine containing a mixture ofα-tocopherol, ascorbyl palmitate and citric acid has led to considerable reductions in NOPyr and DMN output on frying when the combined concentrations of the two anti-oxidants were calculated to be 800 ppm.

M. W. Edwards

Papers

Determination of a non-volatile N-nitrosamine on a food matrix

Determination of a non-volatile nitrosamine by using denitrosation and a chemiluminescence analyser

The effect of antioxidants on the production of volatile nitrosamines during the frying of bacon.