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Technical Briefs |
1
Res. Center of Health, Physical Fitness, and Sports,
2
Res. Center for Advanced Energy Conversion, Nagoya Univ., Furocho, Chikusaku, Nagoya, 464–01, Japan;
a author for correspondence: fax 81-52-789-3957, e-mail g960305d{at}sunspot.eds.ecip.nagoya-.ac.jp
Nitrous oxide (N2O) is a relatively stable compound, present at ~310 nL/L in the atmosphere. It is produced predominantly by microbial reduction of nitrate (NO3-). This process, called denitrification, is the conversion of nitrate to gaseous nitrogen compounds, resulting in a product of nitrogen (N2) 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)(2)(3)(4), including pathogens of Pseudomonas, Neisseria, and Campylobacter. Taking these studies into consideration, it is proper to assume that the concentrations of N2O in exhaled air exceed those in the atmosphere, although no studies have been published related to N2O in exhaled air. The purpose of this study is to establish an analytical method for detection of N2O 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 N2O 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 63Ni electron capture detector was used for the GC determinations of N2O 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 °C, 100 °C, and 300 °C, respectively. Calibration was with a gas of 3.1 µL/L N2O in nitrogen (Nihon Sanso Co., Japan).
A MultiGas Monitor (Type 1302; Brüel & Kjær, Denmark) equipped
with an optical filter (UA0985, 2215 cm-1) was also
used to determine N2O concentrations. The high humidity and
carbon dioxide content (~40 mL/L) in exhaled air posed a problem,
because these interfered with infrared absorption of N2O.
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 µL/L), and various dilutions of these (final concentrations
1.1, 1.7, 2.1, and 5.3 µL/L). The calibration curve shown in Fig. 1
(top) reveals good linearity for this range.
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We found N2O 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 N2O in
the CO2/H2O trap. N2O is usually
analyzed by GC with electron capture detection for analyses in the
range of ppb (nL/L). Because N2O has >200 potent infrared
absorption bands compared with CO2, one can detect such a
low concentration of N2O 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 (63Ni), so the use of the apparatus is
restricted for space. For these reasons, the IR-PAS device is practical
enough for measuring N2O at concentrations in the range of
<1 µL/L in exhaled air if used with a suitable trap for
CO2/H2O.
References
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