title: NO detection in biological samples: differentiation of 14 NO and 15 NO using infrared laser spectroscopy.
abstract: Accurate characterization of the biochemical pathways of nitric oxide (NO) is essential for investigations in the field of NO research. To analyze the different reaction pathways of enzymatic and non-enzymatic NO formation, determination of the source of NO is crucial. Measuring NO-related products in biological samples distinguishing between (14)NO and (15)NO offers the opportunity to specifically analyze NO signaling in blood and tissue. The aim of this study was to establish a highly sensitive technique for the specific measurement of NO in an isotopologue-selective manner in biological samples. With the cavity leak-out spectroscopy setup (CALOS) a differentiation between (14)NO and (15)NO is feasible. We describe here the employment of this method for measurements in biological samples. Certified gas mixtures of (14)NO/N(2) and (15)NO/N(2) were used to calibrate the system. (14)NO2- and (15)NO2- of aqueous and biological samples were reduced in a triiodide solution, and the NO released was detected via CALOS. Gas-phase chemiluminescence detection (CLD) was used for evaluation. The correlation received for both methods for the detection of NO in the gas phase was r=0.999, p<0.0001. Results obtained using aqueous and biological samples verified that CALOS enables NO measurements with high accuracy (detection limit for (14)NO2- 0.3 pmol and (15)NO2- 0.5 pmol; correlation (14)NO: p<0.0001, r=0.975, (15)NO: p<0.0001, r=0.969). The CALOS assay represents an extension of NO measurements in biological samples, allowing specific investigations of enzymatic and non-enzymatic NO formation and metabolism in a variety of samples.
authors.affiliation: Universitätsklinikum Düsseldorf, Institut für Lasermedizin, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Fritsch
authors.forename: Paris
authors.initials: P
authors.lastname: Brouzos
authors.forename: Kathrin
authors.initials: K
authors.lastname: Heinrich
authors.forename: Malte
authors.initials: M
authors.lastname: Kelm
authors.forename: Tienush
authors.initials: T
authors.lastname: Rassaf
authors.forename: Peter
authors.initials: P
authors.lastname: Hering
authors.forename: Petra
authors.initials: P
authors.lastname: Kleinbongard
authors.forename: Manfred
authors.initials: M
authors.lastname: Mürtz
mesh_terms: D000818:Animals; D001826:Body Fluids / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D006801:Humans; D007553:Isotope Labeling; D007834:Lasers; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013055:Spectrophotometry, Infrared / Q000295:instrumentation / Q000379:methods*
chemical_list: D009587:Nitrogen Isotopes; D009569:Nitric Oxide