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Measuring nitrous oxide in exhaled air by gas chromatography and infrared photoacoustic spectrometry.

Nitrous oxide ([N.sub.2]O) is a relatively stable compound, present at ~310 nL/L in the atmosphere. It is produced predominantly by microbial reduction of nitrate (N[O.sub.3.sup.-]). This process, called denitrification, is the conversion of nitrate to gaseous nitrogen compounds, resulting in a product of nitrogen ([N.sub.2]) or nitrous oxide under most conditions. Many kinds of denitrifying bacteria have been isolated from the human oral cavity, upper respiratory tract, and alimentary tract (e.g.) [1-4], including pathogens of Pseudomonas, Neisseria, and Campylobacter. Taking these studies into consideration, it is proper to assume that the concentrations of [N.sub.2]O in exhaled air exceed those in the atmosphere, although no studies have been published related to [N.sub.2]O in exhaled air. The purpose of this study is to establish an analytical method for detection of [N.sub.2]O in exhaled air by using gas chromatography (GC) and infrared-photoacoustic spectrometry (IR-PAS) [5].

Exhaled air samples were collected from 15 healthy subjects, ages 20-60 years. Each subject was fully informed of the experimental procedures before giving consent. Samples were collected with a commercially available breath collection system (a 750-mL gas sampler from Quintron, Milwaukee, WI). The exhalation procedure was as follows: Subjects were to inhale deeply but not to maximum capacity, hold the inhalation for ~5 s, and then exhale into the sampling bag. This procedure was repeated twice for all subjects. The protocol was approved by the Human Research Committee of the Research Center of Health, Physical Fitness, and Sports of Nagoya University.

The exhaled air sample was analyzed within 90 min of collection, because preliminary studies showed that leakage from the sampling bags was negligible over a 2-h period. Emission of [N.sub.2]O was taken as the difference in the concentration between the sample and the room air.

A gas chromatograph (Type GC-14BPE; Shimadzu) equipped with an autosampler, PoraPak (Q 80/100 mesh 1.0 m) columns, and a [sup.63]Ni electron capture detector was used for the GC determinations of [N.sub.2]O concentrations. Methane, at 48. 5 mL/L in argon, was used as a carrier gas at a flow rate of 40 mL/min. The oven, columns, and detector temperatures were regulated at 60[degrees]C, 100[degrees]C, and 300[degrees]C, respectively. Calibration was with a gas of 3.1 [micro]L/L [N.sub.2]O in nitrogen (Nihon Sanso Co., Japan).

A MultiGas Monitor (Type 1302; Bruel & Kjxr, Denmark) equipped with an optical filter (UA0985, 2215 [cm.sup.-1]) was also used to determine [N.sub.2]O concentrations. The high humidity and carbon dioxide content (~40 mL/L) in exhaled air posed a problem, because these interfered with infrared absorption of [N.sub.2]O. To remove this interference, sample gas was passed though a 5 cm X 30 cm pipe containing 2-mm-diameter soda lime granules and a 5 cm X 5 cm pipe containing 2-4-mm-diameter alumina granules in series before the analyzer. The calibration curve was constructed from analyses of pure nitrogen gas (grade S), the calibration gas (at 3.1 and 10.5 [micro]L/L), and various dilutions of these (final concentrations 1.1,1.7, 2.1, and 5.3 [micro]L/L). The calibration curve shown in Fig. 1 (top) reveals good linearity for this range.

We found [N.sub.2]O in exhaled air from all subjects, at concentrations ranging from 60 to 890 nL/L by IR-PAS and from 30 to 730 nL/L by GC. Fig. 1 (bottom) shows the highly linear relationship between the values found by GC and IR-PAS (r = 0.985, P <0.001) and a systematic error of -10%. A possible cause of this difference is the slight absorption of [N.sub.2]O in the C[O.sub.2]/[H.sub.2]O trap. [N.sub.2]O is usually analyzed by GC with electron capture detection for analyses in the range of ppb (nL/L). Because [N.sub.2]O has >200 potent infrared absorption bands compared with C[O.sub.2], one can detect such a low concentration of [N.sub.2]O by IR-PAS with almost the same accuracy as GC. The analytical time required for one sample by GC is ~12 min, whereas IR-PAS takes <2 min. In addition, a gas chromatograph equipped with an electron capture detector contains radioisotope ([sup.63]Ni), so the use of the apparatus is restricted for space. For these reasons, the IR-PAS device is practical enough for measuring [N.sub.2]O at concentrations in the range of <1 [micro]L/L in exhaled air if used with a suitable trap for C[O.sub.2]/[H.sub.2]O.

[FIGURE 1 OMITTED]

References

[1.] Berger UZ. Untersuchungen zur Reduktion von Nitrat and Nitrit durch Neisseria gonorrhoeae and Neisseria meningitidis. Z Med Mikrobiol Immunol 1970;156:86-9.

[2.] Berger UZ. Zur Unterscheidung von Neisseria meningitidis and Neisseria meningococcoides. Med Mikrobiol Immunol 1970;156:90-7.

[3.] Loesche WJ, Gibbons RJ, Socransky SS. Biochemical characteristics of Vibrio sputorum and relationship to Vibrio bubulus and Vibrio fetus. J Bacteriol 1965;89:1109-16.

[4.] Stanier RT, Palleroni NJ, Doudoroff M. The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 1966;43:159-271.

[5.] Rosencwaig A. Photoacoustics and photoacoustic spectroscopy. New York: John Wiley, 1980.

Takahiro Mitsui, (1) * Miharu Miyamura, (1) Aritaka Matsunami, (2) Kuniyuki Kitagawa, (2) and Norio Arai (2) ((1) Res. Center of Health, Physical Fitness, and Sports, (2) Res. Center for Advanced Energy Conversion, Nagoya Univ., Furocho, Chikusaku, Nagoya, 464-01, Japan; * author for correspondence: fax 81-52-789-3957, e-mail g960305d@sunspot. eds.ecip.nagoya-.ac.jp)
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Title Annotation:Technical Briefs
Author:Mitsui, Takahiro; Miyamura, Miharu; Matsunami, Aritaka; Kitagawa, Kuniyuki; Arai, Norio
Publication:Clinical Chemistry
Date:Oct 1, 1997
Words:948
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