ANALYTICAL
96, 64-69 (1979)
BIOCHEMISTRY
A Simple
Infrared Spectroscopic Method for the Measurement of Expired 13C02
SHUSUKE
HIRANO,*
TOMOYUKI KANAMATSU,* AND TOHRU ABEI?
*Department of Physiology Toho University School
YOSHIO
and TDepartment of Internal of Medicine, Ohta-Kus Tokyo,
TAKAGI,*
Medicine, Japan
Received July 27, 1978 For the analysis of *YIOZ and r*COZ in exhaled breath the mass spectrometer has been employed in general, but it is not convenient for clinical use and maintenance. We have been successfully performing the continuous measurement of expired r3COZ and r*COZ with a new analyzer using infrared spectroscopy which is easy to manipulate and maintain. This analyzer measures rzCOZ in a short cell with an absorbancy of around 2360.2 cm-r and r3COZ in a long cell with an absorbancy of around 2272.0 cm-r, recording the volume percentage of l*COZ and the atom percentage excess of r3COZ. In our experiment, using a normal mate rat weighing 210 g, a maximum expired r3COZ of 1.7 atom% excess was recorded 10 min after an intraperitoneal injection of 10 mg of [13C]sodium bicarbonate containing 49.0 atom% of the isotope.
The CO2 in exhaled breath is produced by for the continuous measurement of expired oxidation of various carbon compounds 13C02 or lzC02 is described. This newly dethrough metabolic pathways, or by dehydraveloped instrument is simple to use and tion of sodium bicarbonate present in the maintain. In the experiment, the 13C02 conbody fluids as an acid-base balance-regulattent of exhaled breath of rats given [13C]ing factor. Determination of expired CO2 sodium bicarbonate intraperitoneally was from a certain known source would, there- analyzed, and the fundamental findings for fore, reveal the condition of metabolism of clinical 13C02 measurement were obtained. carbon compounds in the body, or even EXPERIMENTAL metabolic disorders at tissue levels. This purpose is thought to be achieved by givAppurafus. An infrared spectrometer ing subjects some labeled carbon compound (Model EX 130, Japan Spectroscopic Co. Ltd., Tokyo) which can measure 13C02 and and measuring the expired CO2 containing the tracer. In general, the radioactive 12C02 in exhaled breath and record the carbon isotope 14C has been used as such a atom percentage of excess 13C continuously tracer, but its radioactivity makes its use in was used. Figure 1 is the block diagram of man quite difficult because of the possible this instrument. Breath is introduced into radiation damage. In this respect, the stable the two cells (C and D), infrared rays from isotope 13C provides a safe tracer. Howthe light source (A) passing through them. ever, the mass spectrometer generally used The short cell (C) is used for the measurefor the qualitative and quantitative deter- ment of l*CO 2. 13C02 is measured with the mination of stable isotope tracers is not long cell (D) which is about a hundred times convenient for clinical use and maintenance. as long as the short one, because the In the present paper, an experiment with naturally occurring 13C02 is so scarce (only a 13C02 analyzer using infrared spectroscopy 1.108 atom%) that the precise determination 0003-2697/79/090064-06$02.00/O Copyright 0 1979 by Acadenuc F’ress, Inc. All rights of reproduction in any form reserved.
64
NEW METHOD
65
FOR 13COzDETERMINATION
A: Light
SCJWC~
C,D: CC!11 B: Air f,ii: f:
pump
slit
L: mector
M~rrur fntrance
J: Exit K: Lens
M: Amplifier slit
N: hecorder
G: ChODDer I:
Gratjng
FIG. I. Block diagram of optical system.
of the 13C0.J1zCOz ratio requires strengthening of the light absorption by 13COz. Infrared rays passing through the two cells are alternately let through the entrance slit (F) by the chopper (G) and led to the detector (L) with the absorption intensity corresponding
to the length of each cell. Output from the detector is amplified, and ultimately the signal is recorded as the atom percentage excess of 13COz. Figure 2 illustrates the infrared absorption spectrum of CO* obtained with an experi-
Ceil
IOOmm
2360.2
FIG. 2. Infrared absorption IOO-mm cell.
spectrum of COz. (a) S ~01% COz in S-mm cell. (b) 20 ~01% 120~ in
66
HIRANO
mental analyzer using the same spectroscopy as described above. In Fig. 2a, which was obtained from 5 ~01% CO2 in a short @-mm) cell, apattern specific to lzCOZ with a wave number of around 2360.2-2334.2 cm-l is clearly shown. Figure 2b is a spectrum of 20 ~01% CO2 in a long (lOO-mm) cell, showing a pattern specific to 13COZ with a wave number ofaround 2280.3-2261.3 cm-’ with an apparent magnification. Based on this preliminary finding, we tested the new analyzer using the short cell for the measurement of lzCOZ by the band of around 2360.2 cm-l, and the long cell for the measurement of 13COZ by the band of around 2272.0 cm-l. Preparation of standard sample. The standard sample of 13COZ was produced by acidifying a standard solution of [13C]sodium bicarbonate, which was prepared by dissolving crystalline [13C]sodium bicarbonate (90.6 atom%, Hikari Kogyo Co., Ltd.) in redistilled water kept at pH 10.0 with NaOH. Experimental animals. Adult male rats of Wistar albino strain weighing 200-300 g were placed in individual cages, feed and water being allowed ad lib. Standardization
of atom percentage
ET AL. I --
-.
ex-
cess of 13COZ. It is well known that the fresh air in nature contains 1.108 atom% of 13COZ. Therefore, in order to know the amount of 13COZ in breath produced from a certain 13C-labeled compound, the atom percentage excess of 13COZ must be determined. Figure 3 illustrates an example. In this case, 1.5 ~01% of CO* in natural abundance was introduced into the cells to obtain a baseline. Then, the same volume percentage of standard 13COZ was introduced into the cell, and a peak was observed on the graph as shown in the figure. The peak height from the baseline is the atom percentage excess of the standard 13COz. Experimental procedure. A rat was anesthetized with intraperitoneal injection of pentobarbital sodium, and the head was placed in a vinyl bag prepared in our labora-
FIG. 3. Standardization of atom percentage excess of W02. First, 1.5 ~01% of CO2 in natural abundance was introduced into the cells to obtain a baseline, then 1.5 ~01% of 1.6 atom% excess W02 into the same cells after removal of the above sample. Peak height due to the second sample from the baseline was 1.6 atom% excess of W02.
tory. The exhaled breath in the bag was introduced to the analyzer, while fresh air made CO*-free with NaOH was supplied through another opening of the bag. The breath was constantly inhaled into the analyzer with a motor(B) attached to the air outlet of the instrument. When the handle of the flow-route changer of the analyzer was first pulled, the breath was allowed to flow only in the short cell, and the infra-
NEW METHOD
FOR 13C02 DETERMINATION
red absorbancy of lzCOZ was shown at the recorder, from which the volume percentage of i*COZ could be known using the calibration chart shown in Fig. 4. Next, the handle of the flow-route changer was pushed to obtain the absorbancy of 13COZ in the breath, from which the atom percentage excess of 13COZ could be estimated as previously explained. RESULTS
Figure 5a is an infrared absorption spectrum of the exhaled breath of an untreated normal rat, obtained with this analyzer. Amplification of the signals produced a more detailed chart (Fig. 5b), which shows the absorption by 13COZ at an upper right part of the peak due to lzCOZ. When [13C]sodium bicarbonate was given intraperitoneally to the rat, the absorption by expired 13COZ became obviously greater (Fig. 5~). Similar results were obtained in other normal rats. Next. the time course of 13COZ level in
67
exhaled breath was observed in rats given an intraperitoneal injection of [13C]sodium bicarbonate. Figure 6 illustrates an experiment in which a male rat weighing 210 g was anesthetized with pentobarbital sodium and given an injection of 10 mg [13C]sodium bicarbonate containing 49.0 atom% of 13C. The tracing indicates that the expired 13COZ increased remarkably after injection, reached a maximum (1.7 atom percentage excess) in about 10 min, and then decreased gradually until it became near to the initial natural abundance in about 2 h. DISCUSSION
The tracer method is an important means for studying the dynamic changes in metabolism corresponding to functional activities. Although the radioactive isotope 14C is generally used as such a tracer, the stable isotope 13C, without hazards of radiation damage, is decisively more promising in clinical use (1) if the measurement can be
Peak Height
150 -
IO0 -
50 -
FIG. 4. CO2 calibration curve; plot of peak height of light absorption vs volume percentage of 1*C02 in 1.0-5.0.
.
. .: .---: .:. . .; . .--.....-. :..-.. . . .I . ,. , .. * . . -’ -+--+ . . . . . . . . ,.. . . .-.L .-... . . .
,
1
NEW METHOD
FOR ‘%O* DETERMINATION
simplified. The study of CO2 levels in exhaled breath is not only important in analyzing the respiratory function, but also indicative of the changes in tissue metabolism involving carbon compounds, since the expired CO2 is derived from metabolism in the body. Thus far some relevant isotopic investigations by continuous measurement of 14COZ or by discontinuous measurement after administration of 14C- or 13Clabeled compounds (2-5) have been carried out; however, there have been no reports describing continuous measurement of expired 13COZby a method similar to ours. In our experiment to obtain basic findings for clinical application, the time course of expired 13COZ levels in rats given an intraperitoneal injection of [13C]sodium bicarbonate was studied with a new 13COZ analyzer. When 5 mg of labeled sodium bicarbonate containing 49.0 atom% of 13C/ 100 g of body weight was administered, levels of 13COZ peaking at 1.7 atom% excess were observed in exhaled breath. Since this experiment the analyzer has been much improved, and the current model is 15 times as sensitive as the original one used in the present study. The sensitivity is expected to be further improved before the instrument is commercially available, and it is thought that an extremely lower dose (perhaps decreased to one fiftieth or less of that described above) of the tracer would produce well measurable levels of expired 13COZ. This high sensitivity, together with the simplicity to use and maintain, would make
69
this analyzer valuable for clinical application. In our recent experiment using the above analyzer, in which the time course of the expired 13COZ level was continuously studied in rats given [13C]ethanol, a highly significant difference in the expired 13COZ levels between the animals with an experimentally induced hepatic disorder and normal controls was observed 10-20 min after the carrier administration (6). We expect that this method will be extensively applied to clinical examinations in the future. ACKNOWLEDGMENTS The authors thank Miss Kaoru Wakabayashi for her technical assistance and ate indebted to Mt. Tadashi Miyazaki, Japan Spectroscopic Co. Ltd., for developing the instrument and making it available for this study.
REFERENCES
4.
5.
6.
Matwiyoff, N. A., and Ott, D. G. (1973) Science 181, 1125-1133. DaCosta, H., Shteeve, W. W., and Merchant, S. (1976) J. Nucl. Med. 17, 218-219. Shteeve, W. W., Shoop, J. D., Ott, D. G., and McInteer, B. B. (1976) Gmtroenterdogy 71, 98-101. Sasaki, Y. (1974) in Recent Advances in Nuclear Medicine, Proceedings of the 1st World Congress on Nuclear Medicine, pp. 66-70. Sasaki, Y. (197s) in Nuclear Medicine in Japan (Iio, M., ed.), pp. 249-278, International Medical Foundation of Japan, Tokyo. Abei, T., Takane, T., and Hitano, S., unpublished data.
96, 64-69 (1979)
BIOCHEMISTRY
A Simple
Infrared Spectroscopic Method for the Measurement of Expired 13C02
SHUSUKE
HIRANO,*
TOMOYUKI KANAMATSU,* AND TOHRU ABEI?
*Department of Physiology Toho University School
YOSHIO
and TDepartment of Internal of Medicine, Ohta-Kus Tokyo,
TAKAGI,*
Medicine, Japan
Received July 27, 1978 For the analysis of *YIOZ and r*COZ in exhaled breath the mass spectrometer has been employed in general, but it is not convenient for clinical use and maintenance. We have been successfully performing the continuous measurement of expired r3COZ and r*COZ with a new analyzer using infrared spectroscopy which is easy to manipulate and maintain. This analyzer measures rzCOZ in a short cell with an absorbancy of around 2360.2 cm-r and r3COZ in a long cell with an absorbancy of around 2272.0 cm-r, recording the volume percentage of l*COZ and the atom percentage excess of r3COZ. In our experiment, using a normal mate rat weighing 210 g, a maximum expired r3COZ of 1.7 atom% excess was recorded 10 min after an intraperitoneal injection of 10 mg of [13C]sodium bicarbonate containing 49.0 atom% of the isotope.
The CO2 in exhaled breath is produced by for the continuous measurement of expired oxidation of various carbon compounds 13C02 or lzC02 is described. This newly dethrough metabolic pathways, or by dehydraveloped instrument is simple to use and tion of sodium bicarbonate present in the maintain. In the experiment, the 13C02 conbody fluids as an acid-base balance-regulattent of exhaled breath of rats given [13C]ing factor. Determination of expired CO2 sodium bicarbonate intraperitoneally was from a certain known source would, there- analyzed, and the fundamental findings for fore, reveal the condition of metabolism of clinical 13C02 measurement were obtained. carbon compounds in the body, or even EXPERIMENTAL metabolic disorders at tissue levels. This purpose is thought to be achieved by givAppurafus. An infrared spectrometer ing subjects some labeled carbon compound (Model EX 130, Japan Spectroscopic Co. Ltd., Tokyo) which can measure 13C02 and and measuring the expired CO2 containing the tracer. In general, the radioactive 12C02 in exhaled breath and record the carbon isotope 14C has been used as such a atom percentage of excess 13C continuously tracer, but its radioactivity makes its use in was used. Figure 1 is the block diagram of man quite difficult because of the possible this instrument. Breath is introduced into radiation damage. In this respect, the stable the two cells (C and D), infrared rays from isotope 13C provides a safe tracer. Howthe light source (A) passing through them. ever, the mass spectrometer generally used The short cell (C) is used for the measurefor the qualitative and quantitative deter- ment of l*CO 2. 13C02 is measured with the mination of stable isotope tracers is not long cell (D) which is about a hundred times convenient for clinical use and maintenance. as long as the short one, because the In the present paper, an experiment with naturally occurring 13C02 is so scarce (only a 13C02 analyzer using infrared spectroscopy 1.108 atom%) that the precise determination 0003-2697/79/090064-06$02.00/O Copyright 0 1979 by Acadenuc F’ress, Inc. All rights of reproduction in any form reserved.
64
NEW METHOD
65
FOR 13COzDETERMINATION
A: Light
SCJWC~
C,D: CC!11 B: Air f,ii: f:
pump
slit
L: mector
M~rrur fntrance
J: Exit K: Lens
M: Amplifier slit
N: hecorder
G: ChODDer I:
Gratjng
FIG. I. Block diagram of optical system.
of the 13C0.J1zCOz ratio requires strengthening of the light absorption by 13COz. Infrared rays passing through the two cells are alternately let through the entrance slit (F) by the chopper (G) and led to the detector (L) with the absorption intensity corresponding
to the length of each cell. Output from the detector is amplified, and ultimately the signal is recorded as the atom percentage excess of 13COz. Figure 2 illustrates the infrared absorption spectrum of CO* obtained with an experi-
Ceil
IOOmm
2360.2
FIG. 2. Infrared absorption IOO-mm cell.
spectrum of COz. (a) S ~01% COz in S-mm cell. (b) 20 ~01% 120~ in
66
HIRANO
mental analyzer using the same spectroscopy as described above. In Fig. 2a, which was obtained from 5 ~01% CO2 in a short @-mm) cell, apattern specific to lzCOZ with a wave number of around 2360.2-2334.2 cm-l is clearly shown. Figure 2b is a spectrum of 20 ~01% CO2 in a long (lOO-mm) cell, showing a pattern specific to 13COZ with a wave number ofaround 2280.3-2261.3 cm-’ with an apparent magnification. Based on this preliminary finding, we tested the new analyzer using the short cell for the measurement of lzCOZ by the band of around 2360.2 cm-l, and the long cell for the measurement of 13COZ by the band of around 2272.0 cm-l. Preparation of standard sample. The standard sample of 13COZ was produced by acidifying a standard solution of [13C]sodium bicarbonate, which was prepared by dissolving crystalline [13C]sodium bicarbonate (90.6 atom%, Hikari Kogyo Co., Ltd.) in redistilled water kept at pH 10.0 with NaOH. Experimental animals. Adult male rats of Wistar albino strain weighing 200-300 g were placed in individual cages, feed and water being allowed ad lib. Standardization
of atom percentage
ET AL. I --
-.
ex-
cess of 13COZ. It is well known that the fresh air in nature contains 1.108 atom% of 13COZ. Therefore, in order to know the amount of 13COZ in breath produced from a certain 13C-labeled compound, the atom percentage excess of 13COZ must be determined. Figure 3 illustrates an example. In this case, 1.5 ~01% of CO* in natural abundance was introduced into the cells to obtain a baseline. Then, the same volume percentage of standard 13COZ was introduced into the cell, and a peak was observed on the graph as shown in the figure. The peak height from the baseline is the atom percentage excess of the standard 13COz. Experimental procedure. A rat was anesthetized with intraperitoneal injection of pentobarbital sodium, and the head was placed in a vinyl bag prepared in our labora-
FIG. 3. Standardization of atom percentage excess of W02. First, 1.5 ~01% of CO2 in natural abundance was introduced into the cells to obtain a baseline, then 1.5 ~01% of 1.6 atom% excess W02 into the same cells after removal of the above sample. Peak height due to the second sample from the baseline was 1.6 atom% excess of W02.
tory. The exhaled breath in the bag was introduced to the analyzer, while fresh air made CO*-free with NaOH was supplied through another opening of the bag. The breath was constantly inhaled into the analyzer with a motor(B) attached to the air outlet of the instrument. When the handle of the flow-route changer of the analyzer was first pulled, the breath was allowed to flow only in the short cell, and the infra-
NEW METHOD
FOR 13C02 DETERMINATION
red absorbancy of lzCOZ was shown at the recorder, from which the volume percentage of i*COZ could be known using the calibration chart shown in Fig. 4. Next, the handle of the flow-route changer was pushed to obtain the absorbancy of 13COZ in the breath, from which the atom percentage excess of 13COZ could be estimated as previously explained. RESULTS
Figure 5a is an infrared absorption spectrum of the exhaled breath of an untreated normal rat, obtained with this analyzer. Amplification of the signals produced a more detailed chart (Fig. 5b), which shows the absorption by 13COZ at an upper right part of the peak due to lzCOZ. When [13C]sodium bicarbonate was given intraperitoneally to the rat, the absorption by expired 13COZ became obviously greater (Fig. 5~). Similar results were obtained in other normal rats. Next. the time course of 13COZ level in
67
exhaled breath was observed in rats given an intraperitoneal injection of [13C]sodium bicarbonate. Figure 6 illustrates an experiment in which a male rat weighing 210 g was anesthetized with pentobarbital sodium and given an injection of 10 mg [13C]sodium bicarbonate containing 49.0 atom% of 13C. The tracing indicates that the expired 13COZ increased remarkably after injection, reached a maximum (1.7 atom percentage excess) in about 10 min, and then decreased gradually until it became near to the initial natural abundance in about 2 h. DISCUSSION
The tracer method is an important means for studying the dynamic changes in metabolism corresponding to functional activities. Although the radioactive isotope 14C is generally used as such a tracer, the stable isotope 13C, without hazards of radiation damage, is decisively more promising in clinical use (1) if the measurement can be
Peak Height
150 -
IO0 -
50 -
FIG. 4. CO2 calibration curve; plot of peak height of light absorption vs volume percentage of 1*C02 in 1.0-5.0.
.
. .: .---: .:. . .; . .--.....-. :..-.. . . .I . ,. , .. * . . -’ -+--+ . . . . . . . . ,.. . . .-.L .-... . . .
,
1
NEW METHOD
FOR ‘%O* DETERMINATION
simplified. The study of CO2 levels in exhaled breath is not only important in analyzing the respiratory function, but also indicative of the changes in tissue metabolism involving carbon compounds, since the expired CO2 is derived from metabolism in the body. Thus far some relevant isotopic investigations by continuous measurement of 14COZ or by discontinuous measurement after administration of 14C- or 13Clabeled compounds (2-5) have been carried out; however, there have been no reports describing continuous measurement of expired 13COZby a method similar to ours. In our experiment to obtain basic findings for clinical application, the time course of expired 13COZ levels in rats given an intraperitoneal injection of [13C]sodium bicarbonate was studied with a new 13COZ analyzer. When 5 mg of labeled sodium bicarbonate containing 49.0 atom% of 13C/ 100 g of body weight was administered, levels of 13COZ peaking at 1.7 atom% excess were observed in exhaled breath. Since this experiment the analyzer has been much improved, and the current model is 15 times as sensitive as the original one used in the present study. The sensitivity is expected to be further improved before the instrument is commercially available, and it is thought that an extremely lower dose (perhaps decreased to one fiftieth or less of that described above) of the tracer would produce well measurable levels of expired 13COZ. This high sensitivity, together with the simplicity to use and maintain, would make
69
this analyzer valuable for clinical application. In our recent experiment using the above analyzer, in which the time course of the expired 13COZ level was continuously studied in rats given [13C]ethanol, a highly significant difference in the expired 13COZ levels between the animals with an experimentally induced hepatic disorder and normal controls was observed 10-20 min after the carrier administration (6). We expect that this method will be extensively applied to clinical examinations in the future. ACKNOWLEDGMENTS The authors thank Miss Kaoru Wakabayashi for her technical assistance and ate indebted to Mt. Tadashi Miyazaki, Japan Spectroscopic Co. Ltd., for developing the instrument and making it available for this study.
REFERENCES
4.
5.
6.
Matwiyoff, N. A., and Ott, D. G. (1973) Science 181, 1125-1133. DaCosta, H., Shteeve, W. W., and Merchant, S. (1976) J. Nucl. Med. 17, 218-219. Shteeve, W. W., Shoop, J. D., Ott, D. G., and McInteer, B. B. (1976) Gmtroenterdogy 71, 98-101. Sasaki, Y. (1974) in Recent Advances in Nuclear Medicine, Proceedings of the 1st World Congress on Nuclear Medicine, pp. 66-70. Sasaki, Y. (197s) in Nuclear Medicine in Japan (Iio, M., ed.), pp. 249-278, International Medical Foundation of Japan, Tokyo. Abei, T., Takane, T., and Hitano, S., unpublished data.
