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The use of stable isotope labeled substrates and subsequent analysis of urinary nitrate, forms a noninvasive test for evaluation of the in vivo NO metabolism. The present paper describes a new method for simultaneous quantification of [sup.15]N-nitrate and total nitrate with gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). Nitrate, isolated from urine with a nitrate selective resin, was reduced to nitrite using copperized cadmium. Subsequently, Sudan I was formed by diazotation. Sudan II was added as internal standard, and both molecules were analyzed with GC-C-IRMS as tert-butyldimethylsilyl derivatives. The accuracy was determined during a recovery study of two different known nitrate concentrations and two [sup.15]N-enrichments. A recovery of 101.6% and 103.9% for total nitrate and 107.6% and 91.2% for [sup.15]N-nitrate was obtained, respectively. The validated method was applied on complete 72 h urine collections after intravenous administration of [sup.15]N-nitrate and [sup.15]N-arginine in humans. On average, 51.8% (47.0-71.0%) of administered [sup.15]N-nitrate was excreted, while 0.68% (0.44-1.17%) of [sup.15]N-arginine was metabolized to nitrate. In conclusion, this method can be used for accurate simultaneous determination of [sup.15]N-nitrate and total nitrate concentrations in urine and can be applied in clinical studies for noninvasive evaluation of NO metabolism in vivo. 10.1021/ac9019208

Pre- and/or probiotics can cause changes in the ecological balance of intestinal microbiota and hence influence microbial metabolic activities. In the present study, the influence of oligofructose-enriched inulin (OF-IN), Lactobacillus casei Shirota, and Bifidobacterium breve Yakult on the colonic fate of N[H.sub.3] and p-cresol was investigated. A randomized, placebo-controlled, crossover study was performed in 20 healthy volunteers to evaluate the influence of short- and long-term administration of OF-IN, L. casei Shirota, B. breve Yakult, and the synbiotic L. casei Shirota + OF-IN. The lactose[[sup.15]N, [sup.15]N]ureide biomarker was used to study the colonic fate of N[H.sub.3]. Urine and fecal samples were analyzed for [sup.15]N content by combustion-isotope ratio mass spectrometery and for p-cresol content by gas chromatography-mass spectrometry. RT-PCR was applied to determine the levels of total bifidobacteria. Both short- and long-term administration of OF-IN resulted in significantly decreased urinary p-cresol and [sup.15]N content. The reduction of urinary [sup.15]N excretion after short-term OF-IN intake was accompanied by a significant increase in the [sup.15]N content of the fecal bacterial fraction. However, this effect was not observed after long-term OF-IN intake. In addition, RT-PCR results indicated a significant increase in total fecal bifidobacteria after long-term OF-IN intake. Long-term L. casei Shirota and B. breve Yakult intake showed a tendency to decrease urinary [sup.15]N excretion, whereas a significant decrease was noted in p-cresol excretion. In conclusion, dietary addition of OF-IN, L. casei Shirota, and B. breve Yakult results in a favorable effect on colonic N[H.sub.3] and p-cresol metabolism, which, in the case of OF-IN, was accompanied by an increase in total fecal bifidobacteria. ammonia; p-cresol; bifidobacteria; biomarker

In vitro experiments have shown that fermentation of carbohydrates prevents accumulation of nitrogen in the colon. Variable results have been obtained on modulation of dietary intakes in vivo. Lactose[[sup.15]N, [sup.15]N]-labeled ureide has been proposed as a tool to study colonic nitrogen metabolism. However, on oral administration of the marker, different urinary excretion patterns of the [sup.15]N label have been found. In this study, 50 mg lactose[[sup.15]N, [sup.15]N]ureide was directly instilled in the colon through an orocecal tube to investigate the colonic handling of this molecule in a direct way. In basal conditions, 42% (range, 37-48%) of labeled nitrogen administered as lactose[[sup.15]N, [sup.15]N]ureide was retrieved in urine after 72 h. A substantial variability in total urinary excretion of the label was found, but the urinary excretion pattern of the label was similar in all volunteers. When inulin, a fermentable carbohydrate, was administered together with the labeled marker, a significant decrease in urinary excretion of [sup.15]N after 72 h was found, to 29% (range, 23-34%). The effect of a smaller dose of inulin (250 mg) on colonic handling of lactose[[sup.15]N, [sup.15]N]ureide (50 mg), was investigated in another group of volunteers, and this time, fecal excretion of the marker was also evaluated. The results seem to indicate that fermentation of inulin causes an increased fecal excretion of the marker, thereby reducing urinary excretion but not retention in the human nitrogen pool. This instillation study shows that lactose[[sup.15]N, [sup.15]N]ureide is a tool with good properties to investigate the effect of different types of carbohydrates on nitrogen metabolism in the proximal colon in vivo. carbohydrate fermentation; stable isotopes

The kinetics of inulin-type fructan degradation is examined by representatives of the genera Anaerostipes and Roseburia and a method based on online gas chromatography (GC) is developed for assessing gas production in a continuously sparged fermentation vessel for complete coverage of metabolite production. The complete recovery of metabolites during fermentations of Clostridial cluster XIVa butyrate-producing colon bacteria has allowed stoichiometric balancing of the metabolite pathway for butyrate production, including the formation of [H.sub.2].