Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols.

The aim of this study was to compare and improve standard methods to determine nitrite (NO2-), nitrate (NO3-) and S-nitrosothiol (RSNO) levels in cell culture supernatants, sera, and urine. We modified the conventional Griess reaction by replacing sulfanilamide with dapsone (4,4'-diamino-diphenylsulfone) and compared the NO2- levels in our study samples with a commercially available NO2- assay kit. Our modification, along with ultrafiltration of the samples, resulted in an enhanced sensitivity to measure NO2- down to 0.2 microM. The detection limit was further improved to 0.02 microM when NO2- was identified by the fluorochrome 2,3-diaminonaphthalene (DAN). To measure the stable end product NO3- by the Griess reaction or the DAN method, this anion must be reduced to NO2-. We compared the capacity of bacterial nitrate reductase with the reducing metal cadmium to convert NO3- to NO2-. After reduction, NO2- levels were determined either by the DAN method or by our modified Griess reaction. We found that there was a high correlation (r2 = 0.998) in total NO2- concentrations in the study samples using both methods for reducing NO3- to NO2-. The simultaneous determination of NO2- and NO3- was achieved by using anion-exchange chromatography (HPLC; Polyspher IC AN-1 column). The detection limit of this assay for each anion is 0.5 microM, and it can be applied equally well to sera, urine, and culture media. We also adapted the DAN method to determine RSNO levels in our study samples. Using this approach, we were able to measure RSNO levels down to 0.15 microM. As result we discovered that RSNO levels were markedly increased in urine from septic patients and in supernatants from cytokine-stimulated human tumor cell lines. L-Citrulline, a coproduct of NO biosynthesis, was measured using a colorimetric assay with a sensitivity limit of 3.0 microM. Increased L-citrulline levels in media from cultured cells, but not in sera or urine, correlated with increased NO production. Although all methods studied were suitable for quantifying end products of NO in biological fluids and media, the use of bacterial reductase and the modified Griess reaction proved successful to provide the greatest sensitivity and linear range for routine measurements of NO2- and NO3-.

Effect of acadesine treatment on postischemic damage to small intestine.

Hemorrhagic mucosal lesions are produced during intestinal ischemia and after reperfusion due at least in part to the accumulation and activation of polymorphonuclear leukocytes in the tissue. It has been shown in vitro that adenosine is instrumental in attenuating this pathophysiological process. Acadesine [5-amino-4-imidazolecarboxamide (AICA) riboside], a purine nucleoside analogue, increases the availability of adenosine in the tissue. The aim of the study was therefore to assess the influence of acadesine treatment on neutrophil accumulation, purine metabolism, and mucosal damage after intestinal ischemia and reperfusion. Intestinal ischemia was induced in cats by partial occlusion of the superior mesenteric artery for 2 h. Samples of the small intestine were exercised before and at the end of the hypotensive period as well as 10 and 60 min after reperfusion. Conjugated dienes, myeloperoxidase, and reduced and oxidized glutathione, as well as the purine metabolites, were determined in the tissue samples. The tissue was also examined histologically. Six cats received saline, and six cats were treated initially before ischemia with acadesine (2.5 mg/kg body wt i.v.) over 5 min as a bolus. Thereafter, acadesine (0.5 mg.kg-1.min- i.v.) was given continuously during ischemia and 30 min after reperfusion. Acadesine treatment significantly attenuated the mucosal lesions seen during reperfusion. This improvement was due at least in part to the inhibition of neutrophil accumulation, as judged by low myeloperoxidase levels. The prevention of neutrophil activation resulted most likely from increased adenosine concentrations in the intestinal tissue in the early reperfusion period.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative endotoxin determination in blood--chromogenic modification of the limulus amebocyte lysate test.

A newly developed modification of the limulus amebocyte lysate test for quantification of endotoxin levels in blood is described. The chromogenic peptide carbobenzoxy-Gly-Gly-Arg-4-methyl-cumarinyl-7-amid proved to be most suitable. The liberated fluorescent dye is diazotized with N(1-naphtyl-)-ethylen-diamin-dihydrochloride. Using this statistically proved reliable and sensitive test, endotoxin serum levels of healthy persons and patients undergoing major surgical treatment were compared. In the postoperative phase endotoxin serum levels up to 0.5 ng/ml can be detected without clinical signs of septicemia. Healthy persons show endotoxin serum levels up to 0.08 ng/ml. In rats no difference of endotoxin serum levels was detected in the portal vein, and in arterial and venous blood. So a physiological endotoxin resorption from the intestine followed by a clearance during the liver passage seems to be doubtful in this species.

Studies on the primary structure of 14 proteins from the large subunit of Escherichia coli ribosomes with an improved protein sequenator and with mass spectrometry.

Fourteen proteins from the large subunit of Escherichia coli ribosomes were analyzed in an improved sequenator. In addition to our previously described modifications of a Beckman sequenator, new valves which work free of a dead volume were constructed. By this and the previous improvements (e.g., a new vacuum system with a recorder, cool traps, automatic conversion) much better results were obtained than before. It was even possible to use (in addition to the standard methods, e.g., thin-layer chromatography and amino acid analysis) mass spectrometry without preceding gas chromatography for identification of the released PTH amino acids. Our experience with the various methods, especially mass spectrometry, is described and the techniques are compared. The results obtained by the described methods on the amino acid sequences of the 14 ribosomal proteins are summarized.