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We report a simple, enzymatic, end point method for determining nitrate in serum and urine with use of nitrate reductase from Aspergillus sp. (EC 1.6.6.2). The decrease in absorbance at 340 nm as a result of the oxidation of beta-NADPH is measured. We used FAD as a supplementary electron carrier and added an internal standard to avoid interference from possible inhibitors of the enzymatic reaction. The method was linear from 5 to 200 mumol/L nitrate in serum. Within-run (n = 12) and total (n = 14 days) imprecisions (CV) were 5.1-7.7% and 6.2-9.8%, respectively, at 20-90 mumol/L nitrate in serum. Recoveries of added nitrate were 80-106%. The median (range) concentration in serum of 20 healthy subjects was 16 (0-42) mumol/L.

Although the physiological role of alpha 1-acid glycoprotein (AGP), an acute-phase protein, is poorly understood, several lines of evidence support a modulatory action on the immune response. In this study, we investigated the effect of AGP on the production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by human monocytes, macrophages and the monocytic THP-1 cell line. AGP significantly enhanced (2- to 7-fold) the production of these cytokines in monocytes induced by suboptimal concentrations of lipopolysaccharide [E. coli lipopolysaccharide (LPS): 100 ng/ml] in serum-free conditions, whereas it had little or no effect in the absence of LPS. The potentiating effect of AGP was inhibited by specific antibodies. It was concentration dependent and the greatest enhancement was observed with 250-500 micrograms/ml. Moreover, AGP only potentiated the effect of suboptimal concentrations of LPS. AGP did not alter the time course of LPS-induced IL-1 beta, IL-6 or TNF-alpha secretion. AGP acts as a co-inducer and could also potentiate cytokine secretion triggered by Neisseria meningitidis LPS and muramyl dipeptide. The glycan moiety of AGP did not seem to be involved in its potentiating effect, since both its major glycoforms and asialo-AGP potentiated the effect of LPS to the same extent as native AGP. Possible differences in the effect of AGP according to cell maturation were investigated using isolated human macrophages: AGP potentiated LPS-induced cytokine production by both peritoneal and alveolar macrophages. These data suggest that AGP can modulate monocyte/macrophage functions, thereby contributing to the amplification and regulation of immune and inflammatory responses.

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when cirrhosis is decompensated. [2][3][4][5][6] In vivo studies using veNitric oxide production was studied in cirrhotic panous occlusion plethysmography have shown an overproductients with spontaneous bacterial peritonitis (SBP) or tion of NO related to the hyporesponsiveness to vasoconstricwith other infections. We followed up on the time course tors in cirrhotic patients. 7 However, which specific isoform of of serum nitrate levels in 51 hospitalized patients aged NOS accounts for this increment has not been clearly deterbetween 34 and 81 years. Four groups were defined: pamined. To explain vasodilation in cirrhotic patients, Vallance tients with SBP (group 1, n Å 14), patients with bacterand Moncada have hypothesized that endotoxemia might inemia (group 2, n Å 11), patients with urinary tract infecduce the synthesis and release of NO by iNOS, either directly tion (group 3, n Å 11) and patients in a stable clinical or indirectly via cytokines, and this might therefore account condition (group 4, n Å 20). The four groups did not differ for the hyperdynamic circulatory syndrome associated with in terms of Pugh score (11 { 1, 10 { 1, 11 { 1, and 10 { cirrhosis. 8 This is supported by the findings of Guarner et al. 1, respectively). Serum nitrate levels averaged 31 { 2 that serum levels of nitrate correlated with those found in mmol/L in group 4 (84 samples). On the day results of endotoxemia. 4 On the other hand, endothelial constitutive cytobacteriological examination were positive, mean se-NOS was reported to be up-regulated in rats with CCl 4 -inrum nitrate levels were 75 { 17, 63 { 9, and 36 { 9 mmol/ duced cirrhosis, which suggests a major role of this isoform L, respectively, in groups 1 (17 cases), 2 (11 cases), and in the increased production of NO in cirrhotic rats. 9 The last 3 (11 cases) (P õ .001). The maximum nitrate values repossibility is that both isoforms are involved: a mechanical corded during follow-up were higher in groups 1 (149 { stimulation of endothelial constitutive NOS might be second-15 mmol/L) and 2 (112 { 11 mmol/L) than in group 3 (66 ary to increased cardiac output and shear stress, whereas { 7 mmol/L; P õ .001 and õ.01, respectively). These maxianother source of NO might be from iNOS stimulated biomum values were recorded in all groups approximately chemically through cytokines or endotoxins. weeks after the infection was diagnosed. The meanIn a previous study, we found that 1) patients with ascites duration of NO overproduction, as defined by nitrate had higher nitrate levels than those without ascites and 2) level 3 90 mmol/L, was 15 { 3 days in group 1 and 5 { 1 nitrate levels correlated with orosomucoid, a protein of the day in group 2. When the nitrate concentration was studacute-phase response, which suggests that increased nitrate ied in serum and ascitic fluid sampled on the same day, levels were related to activation of iNOS. 6 The high prevait was found to be higher in ascitic fluid than in serum lence of infectious complications in cirrhotic patients with in eight ca...

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