- Email: [email protected]
Gender and age specific differences in exhaled isoprene levels
Respiratory Physiology & Neurobiology 154 (2006) 478–483
Gender and age specific differences in exhaled isoprene levels Matthias Lechner a,b , Berthold Moser b , David Niederseer b , Alban Karlseder a , Bernhard Holzknecht b , Matthias Fuchs b , Stephan Colvin b , Herbert Tilg c , Josef Rieder b,∗ a b
Department of Hygiene, Microbiology, and Social Medicine, Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria Department of Anaesthesiology and Critical Care Medicine, University Hospital of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria c Department of Medicine, University Hospital of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria Accepted 16 January 2006
Abstract The analysis of volatile organic compounds (VOC) in the human breath has attracted a considerable amount of clinical and scientific interest during the last decade. In our study, we turned our attention to gender and age specific differences of exhaled volatile compounds, particularly on isoprene which is one of the most abundant organic molecules found in human exhaled air. A total of 126 test persons were enrolled in the study: 66 females and 60 males. Moreover, the participants were classified into six groups with regard to their age. In a standardized setting all of them had to exhale the endexpiratory breath into a sample bag. The volatile compounds at m/z values from 21 to 229 were analyzed by using proton-transfer-reaction-mass-spectrometry. Isoprene (at m/z 69) was found to be highly significantly (p < 0.001) elevated in the exhaled air of male subjects. Furthermore, it could be shown that 19–29 years old subjects exhale significantly lower levels of isoprene than older adults (p = 0.002). No significant differences between groups were detected for any other measured mass. In conclusion, the present study demonstrates gender and age specific differences of isoprene levels in the exhaled air. These findings may be of potential clinical relevance regarding the multifaceted roles of isoprene, representing both indicator and effector molecule. © 2006 Elsevier B.V. All rights reserved. Keywords: Isoprene; Breath; Gender; Age; Mass-spectrometry
1. Introduction Abbreviations: m/z, mass-to-charge ratio; ppmv, parts per million by volume; ppbv, parts per billion by volume; PTR-MS, protontransfer-reaction-mass-spectrometry; VOC, volatile organic compounds ∗ Corresponding author. Tel.: +43 51250422400; fax: +43 51250422450. E-mail address: [email protected] (J. Rieder). 1569-9048/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2006.01.007
Breath gas analysis has attracted a considerable amount of scientific and clinical interest during the last decade, as this technique was described as one possible method for non-invasive and rapid diagnosis of a variety of diseases, e.g. lung cancer (Phillips et al.,
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
2003a, 1999), breast cancer (Phillips et al., 2003b), schizophrenia (Phillips et al., 1993), and hyperlipidemia (Rieder et al., 2001). A correlation of these diseases with specific patterns of volatile organic compounds in the exhaled air could be demonstrated. Human breath contains a variety of endogenous volatile compounds, the most abundant ones being acetone ∼1 part per million by volume (ppmv), methanol, ethanol, propanols, and the molecule isoprene (2methylbutadiene-1,3), a reactive aliphatic hydrocarbon with a half-life of a few hours (Fenske and Paulson, 1999). Humans produce isoprene endogenously at a rate of 0.15 mmol/kg/h (Taalman, 1996). In addition to man this compound is also produced by bacteria, plants, and various mammals (Kuzma et al., 1995; Sharkey and Yeh, 2001). Therefore, the amount of isoprene present in the atmosphere is of biological origin. Due to its high volatility (i.e., small Henry’s law constant), its low water solubility and its low boiling point of 34 ◦ C, it is mainly eliminated via the lungs (Dahl et al., 1987). Isoprene concentrations in exhaled air are described to be significantly lower in children than in adults (Taucher et al., 1997). Additional studies showed that neonates had undetectable or very low levels of isoprene in their expired breath during the first postnatal week. Breath isoprene levels increased with age, and healthy school children had higher levels than healthy preschool children (Nelson et al., 1998). However, due to large inter-individual variability, little evidence for an age dependence of exhaled isoprene concentrations has been described among adults. Despite the existence of studies describing the influence of age on exhaled isoprene concentrations, no study has clearly stated whether gender specific differences do exist. In a standardized setting, we analyzed volatile compounds with atomic mass units from 21 to 229, including the molecule isoprene, in the exhaled air of 126 fasting subjects. We particularly turned our attention to gender and age specific differences.
2. Materials and methods 2.1. Subjects The study was approved by the local ethical committee and written informed consent was obtained from each participant. Breath samples were collected from
479
126 subjects, 66 females (mean age = 55.35 years; S.D. = 17.29) and 60 males (mean age = 58.93 years; S.D. = 12.0). Furthermore, all participants were classified into six groups according to their age (Group A: 19–29 years old, n = 11; Group B: 30–39 years old, n = 5; Group C: 40–49 years old, n = 16; Group D: 50–59 years old, n = 29; Group E: 60–69 years old, n = 40; Group F: 70–79 years old, n = 25). All subjects who were enrolled in the study had to fast over night and none of them was on statin therapy. Samples were obtained on the following day between 8:00 and 10:00 a.m. in a standardized setting. The subjects were requested to rest for at least 10 min, thus allowing the pulse rate to stabilize. Then they were asked to exhale into a sample bag (Adtech, Gloucestershire, UK) as previously described (Rieder et al., 2001). In brief, patients had to discard the first part of expired air and exhaled only the deep portion into the sample bag. In addition, room air samples were taken, serving as background controls. 2.2. Analysis Samples were heated to 37 ◦ C. Subsequently, the gas in the sample bags and flasks (room air) was siphoned off by a heated Teflon tube (37 ◦ C). Sample analysis was performed using proton-transferreaction-mass-spectrometry (PTR-MS). This technique uses H3 O+ as a chemical ionization reagent to measure volatile organic compounds (VOC) in the parts per billion by volume (ppbv) to parts per trillion by volume (pptv) range. Protonated water, H3 O+ , reacts with neutral molecules (M) according to H3 O+ + M → MH+ + H2 O. This reaction only occurs if these neutral molecules have larger proton affinities than H2 O. Almost all VOCs have larger affinities and therefore proton transfer occurs on every collision with rate constants k, having typical values of 1.5 × 10−9 cm3 s−1 < k < 4 × 10−9 cm3 s−1 . The count rate of ions (protonated molecules, MH+ ) is determined in the ion detection system. There is a linear relationship between the recorded normalized count rate of ions and the concentration of M in the original trace gas, so that the latter can be calculated. The formula includes the normalized count rate of ions and of the primary ion H3 O+ , the temperature in the drift tube, the pressure in the drift tube, the mass dependent transmission efficiency, the rate constant, and the reaction
480
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
time (Lindinger and Jordan, 1998). The concentration of masses at 209 different m/z values (21–229 atomic mass units) was evaluated. For the most part the corresponding substances are hydrocarbons, in particular alkanes, alkenes, alcohols, ketones, and organic acids. Isoprene is detected at m/z 69 using PTR-MS which is a consequence of the proton transfer reaction and reported elsewhere (Karl et al., 2001; Taucher et al., 1997). After baseline conditions had been established each sample was measured at least five times and the calculated average levels were used for further statistical analysis. 2.3. Statistics Statistical analysis was performed using the software package SPSS (v.11.0, Chicago, USA). The concentrations of the investigated volatile compounds are expressed as mean ± S.D. in ppbv for the subgroups. Statistical significance of difference in the concentrations between the groups was determined with the ANOVA and the t-test for unpaired observations after verifying normal distribution in each group. Statistical significance was assumed at pvalues < 0.05 and p < 0.01 were considered highly significant.
3. Results The mean value of isoprene (m/z 69) in the exhaled air of female subjects (n = 66/126) was 57.46 ± 27.75 and that of male subjects (n = 60/126) 80.57 ± 34.13 ppbv. Comparing the two groups by using the t-test for unpaired observations the measured concentrations of isoprene were shown to be highly significantly (p < 0.001) elevated in the exhaled air of men (Fig. 1 and Table 1). The mean value of isoprene in the exhaled air of Group A (19–29 years old; n = 11/126) was 39.95 ± 23.97 ppbv. These levels of isoprene were found to be increased in Group B (30–39 years old; n = 5/126) with 67.86 ± 33.82. In the subgroup of 40–49 years old subjects (Group C; n = 16/126) a mean value of 75.61 ± 28.70 ppbv was detected. The mean value of isoprene was 75.02 ± 30.76 in Group D (50–59 years old; n = 29/126) and 60–69 years old subjects (Group E; n = 40/126) achieved a mean value
Fig. 1. Concentration of isoprene (m/z 69) in the exhaled air of male subjects (m) compared to female subjects (f). Error plot (squares: mean, whiskers: 95% confidence limits)—X-axis: females (f) vs. males (m); Y-axis: concentration in parts per billion by volume (ppbv).
of 67.18 ± 29.16 ppbv. In the subgroup of 70–79 years old subjects (Group F; n = 25/126) a mean value of 71.01 ± 41.82 was found. Data are illustrated in Fig. 2 and Table 2. Taking Groups B, C, D, E, and F together and comparing them to Group A (19–29 years old; n = 11/126) the measured concentrations of isoprene is found to be highly significantly decreased (p = 0.002; tTable 1 Female subjects (f) vs. male subjects (m) Isoprene(m/z 69) f n Mean S.D. S.E.M.
66 57.46 27.75 3.42
n Mean S.D. S.E.M.
60 80.57 34.13 4.41
m
p-value
0.000**
** Highly significantly elevated (p < 0.01); t-test for unpaired observations.
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
Fig. 2. Concentration of isoprene (m/z 69) comparing the different age groups. Error plot (squares: mean, whiskers: 95% confidence limits)—X-axis: age Groups (A–F); Y-axis: concentration in parts per billion by volume (ppbv).
481
Fig. 3. Mass spectrum detected in the exhaled air of the average subject. X-axis: masses at m/z values 21–229; Y-axis: concentration in parts per billion by volume (ppbv).
test for unpaired observations) in the exhaled air of the younger subjects. The measured concentration of isoprene in the room air was generally very low and often only in the pptv range so that it could be neglected. No significant gender or age specific differences between groups were detected for any other measured mass concentrations at m/z values from 21 to 229. The mass spectrum detected in the exhaled air of the average subject is illustrated in Fig. 3 and that found in the room air, serving as the background control, in Fig. 4. Table 2 Mean concentration of isoprene in the exhaled air, standard deviation (S.D.), standard error of the mean (S.E.M.) comparing the age groups A–F n
19–29 years old 30–39 years old 40–49 years old 50–59 years old 60–69 years old 70–79 years old
11 5 16 29 40 25
Isoprene (m/z 69) concentration in the exhaled air Mean
S.D.
S.E.M.
39.95 67.86 75.61 75.02 67.18 71.01
23.97 33.82 28.70 30.76 29.16 41.82
7.23 15.12 7.18 5.71 4.61 8.36
Fig. 4. Mass spectrum detected in the room air serving as the background control. X-axis: masses at m/z values 21–229 (m/z 45: 75.14 ppbv; m/z 47: 15.94 ppbv; m/z 55: 10.04 ppbv; m/z 59: 46.22 ppbv; m/z 69: 0.87 ppbv); Y-axis: concentration in parts per billion by volume (ppbv).
4. Discussion The present study reveals gender specific differences of isoprene levels in the exhaled air. Moreover, it
482
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
could be shown that 19–29 years old subjects exhaled highly significant lower levels of isoprene than the older adults. Within the rest of the adult groups high standard deviations were observed and no evidence for an age dependence of isoprene concentration was found, corresponding well with previous results, i.e. by Mendis et al. (1994). The reason for the highly significant increase of isoprene levels in the exhaled air of men most probably underlies an increased concentration of isoprene in the blood. Because of the large alveolar surface with respect to alveolar volume and in each of the partial lung volumina, there exists near equilibrium between the blood isoprene concentration and the breath isoprene concentration. This is due to Henry’s law partitioning of blood gases with alveolar air. Thus, the average isoprene concentration is correlated to an average breath isoprene concentration over the whole lung volume. Although the exact origin of isoprene in the body is not completely understood, there is evidence that it is of cellular origin (Deneris et al., 1984). It is conjectured to be generated along the mevalonic pathway of cholesterol synthesis. Deneris et al. (1984, 1985) were able to demonstrate the in vitro synthesis of isoprene from dl-mevalonate using cytosolic fraction of rat liver, and suggested that isoprene is formed non-enzymatically after conversion of mevalonate to dimethyallyl pyrophosphate (DMAPP) and/or from isopentenyl pyrophosphate . This provided the first evidence that breath isoprene is linked to cholesterol biosynthesis. Further evidence was provided by Stone et al., 1993 showing a parallel decrease in isoprene exhalation and sterol synthesis caused by acute and chronic lovastatin administration . This strongly suggests that breath isoprene is derived from the cholesterol synthesis pathway in humans in vivo. A small fraction of exhaled isoprene may also be of bacterial origin (Kuzma et al., 1995). The gender specific differences observed in the present study might be explained by differences in the blood cholesterol levels (which is not yet reflected in the clinical guidelines but commonly known (Legato, 2000) and thus in the extent of cholesterol synthesis. Cell membranes contain 50% cholesterol being responsible for their stability. Due to the increased muscle mass of men (rich in cell membranes and thus in cholesterol) the increased cholesterol synthesis and the
increased synthesis of the by-product isoprene might be explained. But also the degradation of isoprene should be considered. The metabolism of isoprene by liver microsomes in vitro from a range of species including rat, mouse, and human shows significant differences between species, strains and gender with respect to the diastereoselectivity and enantioselectivity of the metabolic oxidation and hydrolysis reactions (Watson et al., 2001) Moreover, in vivo, decreased concentrations of isoprene in hepatic venous blood suggest that isoprene is metabolized in the liver (Miekisch et al., 2001). Furthermore, differences in lung capacity and/or pulmonary circulation between men and women must also be taken into account. Oxidative stress has been proposed to play a role in the genesis and progression of acute and chronic diseases (Scholpp et al., 2002). Several studies were carried out to assess whether human breath isoprene levels reflect oxidative stress (Mendis et al., 1995a,b; McGrath et al., 2001, 2000). However, as the results are controversial, no conclusive statement can be made. The highly significantly lower levels of isoprene in the youngest age group correspond well with previous studies describing significantly lower isoprene concentrations in children and adolescents (Taucher et al., 1997; Nelson et al., 1998). This finding might be explained by the larger amount of cell membranes (containing 50% cholesterol as described above) found in adults, e.g. due to an increased muscle mass and organ tissue as well as a higher lung capacity. In addition to a potentially decreased production of isoprene in the body of children, it is conceivable that their higher heart rates and respiratory rates lead to lower levels of exhaled isoprene in single breath samples. Moreover, age related changes in cholesterol metabolism might also have an influence on the isoprene production rate. Within the groups of adults, the standard deviation was very high and no clear trend towards elevation or decrease of breath isoprene could be observed, consistent with previous studies (Taucher et al., 1997; Mendis et al., 1994). No significant gender or age specific differences between groups were detected for any other measured mass concentrations at m/z values from 21 to 229, including acetone at m/z 59. Finally, one potential limitation of the method should be briefly addressed. In PTR-MS measurements, each measured mass can potentially represent
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
various substances. As investigated and reported elsewhere, there is compelling evidence that the great majority of the abundant m/z 69 signal is due only to breath isoprene in humans (Karl et al., 2001; Taucher et al., 1997). In conclusion, the present study demonstrates gender specific differences of isoprene levels in the exhaled air. Moreover, significantly lower levels of isoprene were detected in 19–29 years old subjects. These findings may be of potential clinical relevance regarding the multifaceted roles of isoprene, representing both indicator and effector molecule.
Acknowledgements This work was supported by the Austrian Federal Science Fund (FWF) with grant P-14149 MED to Josef Rieder and grant P-17447 to Herbert Tilg. Moreover, we would like to thank our technician Dr. Dietmar Bader and Dr. Alfons Jordan (Institute of Ion Physics, University of Innsbruck, Austria) for their invaluable help.
References Dahl, A.R., Birnbaum, L.S., Bond, J.A., Gervasi, P.G., Henderson, R.F., 1987. The fate of isoprene inhaled by rats: comparison to butadiene. Toxicol. Appl. Pharmacol. 89, 237– 248. Deneris, E.S., Stein, R.A., Mead, J.F., 1984. In vitro biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction. Biochem. Biophys. Res. Commun. 123, 691–696. Deneris, E.S., Stein, R.A., Mead, J.F., 1985. Acid-catalyzed formation of isoprene from a mevalonate-derived product using a rat liver cytosolic fraction. J. Biol. Chem. 260, 1382–1385. Fenske, J.D., Paulson, S.E., 1999. Human breath emissions of VOCs. J. Air Waste Manag. Assoc. 49, 594–598. Karl, T., Prazeller, P., Mayr, D., Jordan, A., Rieder, J., Fall, R., Lindinger, W., 2001. Human breath isoprene and its relation to blood cholesterol levels: new measurements and modeling. J. Appl. Physiol. 91, 762–770. Kuzma, J., Nemecek-Marshall, M., Pollock, W.H., Fall, R., 1995. Bacteria produce the volatile hydrocarbon isoprene. Curr. Microbiol. 30, 97–103. Legato, M.J., 2000. Dyslipidemia, gender, and the role of highdensity lipoprotein cholesterol: implications for therapy. Am. J. Cardiol. 86, 15L–18L. Lindinger, W., Jordan, A.H., 1998. On-line monitoring of volatile organic compounds at pptv levels by means of proton-transferreaction mass spectrometry (PTR-MS). Medical applications,
483
food control and environmental research. Int. J. Mass Spectrom. Ion Process. McGrath, L.T., Patrick, R., Mallon, P., Dowey, L., Silke, B., Norwood, W., Elborn, S., 2000. Breath isoprene during acute respiratory exacerbation in cystic fibrosis. Eur. Respir. J. 16, 1065–1069. McGrath, L.T., Patrick, R., Silke, B., 2001. Breath isoprene in patients with heart failure. Eur. J. Heart Fail. 3, 423–427. Mendis, S., Sobotka, P.A., Euler, D.E., 1994. Pentane and isoprene in expired air from humans: gas-chromatographic analysis of single breath. Clin. Chem. 40, 1485–1488. Mendis, S., Sobotka, P.A., Euler, D.E., 1995a. Expired hydrocarbons in patients with acute myocardial infarction. Free Radic. Res. 23, 117–122. Mendis, S.S.P., Leja, F.L., Euler, D.E., 1995b. Breath pentane and plasma lipid peroxides in ischemic heart disease. Free Radic. Biol. Med. 19, 679–684. Miekisch, W., Schubert, J.K., Vagts, D.A., Geiger, K., 2001. Analysis of volatile disease markers in blood. Clin. Chem. 47, 1053–1060. Nelson, N., Lagesson, V., Nosratabadi, A.R., Ludvigsson, J., Tagesson, C., 1998. Exhaled isoprene and acetone in newborn infants and in children with diabetes mellitus. Pediatr. Res. 44, 363–367. Phillips, M., Cataneo, R.N., Cummin, A.R., Gagliardi, A.J., Gleeson, K., Greenberg, J., Maxfield, R.A., Rom, W.N., 2003a. Detection of lung cancer with volatile markers in the breath. Chest 123, 2115–2123. Phillips, M., Cataneo, R.N., Ditkoff, B.A., Fisher, P., Greenberg, J., Gunawardena, R., Kwon, C.S., Rahbari-Oskoui, F., Wong, C., 2003b. Volatile markers of breast cancer in the breath. Breast J. 9, 184–191. Phillips, M., Gleeson, K., Hughes, J.M., Greenberg, J., Cataneo, R.N., Baker, L., McVay, W.P., 1999. Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study. Lancet 353, 1930–1933. Phillips, M., Sabas, M., Greenberg, J., 1993. Increased pentane and carbon disulfide in the breath of patients with schizophrenia. J. Clin. Pathol. 46, 861–864. Rieder, J., Lirk, P., Ebenbichler, C., Gruber, G., Prazeller, P., Lindinger, W., Amann, A., 2001. Analysis of volatile organic compounds: possible applications in metabolic disorders and cancer screening. Wien. Klin. Wochenschr. 113, 181–185. Scholpp, J., Schubert, J.K., Miekisch, W., Geiger, K., 2002. Breath markers and soluble lipid peroxidation markers in critically ill patients. Clin. Chem. Lab. Med. 40, 587–594. Sharkey, T.D., Yeh, S., 2001. Isoprene Emission from Plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 407–436. Stone, B.G., Besse, T.J., Duane, W.C., Evans, C.D., DeMaster, E.G., 1993. Effect of regulating cholesterol biosynthesis on breath isoprene excretion in men. Lipids 28, 705–708. Taalman, R.D., 1996. Isoprene: background and issues. Toxicology 113, 242–246. Taucher, J., Hansel, A., Jordan, A., Fall, R., Futrell, J.H., Lindinger, W., 1997. Detection of isoprene in expired air from human subjects using proton-transfer-reaction mass spectrometry. Rapid Commun. Mass Spectrom. 11, 1230–1234. Watson, W.P., Cottrell, L., Zhang, D., Golding, B.T., 2001. Metabolism and molecular toxicology of isoprene. Chem. Biol. Interact. 135–136, 223–238.
Gender and age specific differences in exhaled isoprene levels Matthias Lechner a,b , Berthold Moser b , David Niederseer b , Alban Karlseder a , Bernhard Holzknecht b , Matthias Fuchs b , Stephan Colvin b , Herbert Tilg c , Josef Rieder b,∗ a b
Department of Hygiene, Microbiology, and Social Medicine, Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria Department of Anaesthesiology and Critical Care Medicine, University Hospital of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria c Department of Medicine, University Hospital of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria Accepted 16 January 2006
Abstract The analysis of volatile organic compounds (VOC) in the human breath has attracted a considerable amount of clinical and scientific interest during the last decade. In our study, we turned our attention to gender and age specific differences of exhaled volatile compounds, particularly on isoprene which is one of the most abundant organic molecules found in human exhaled air. A total of 126 test persons were enrolled in the study: 66 females and 60 males. Moreover, the participants were classified into six groups with regard to their age. In a standardized setting all of them had to exhale the endexpiratory breath into a sample bag. The volatile compounds at m/z values from 21 to 229 were analyzed by using proton-transfer-reaction-mass-spectrometry. Isoprene (at m/z 69) was found to be highly significantly (p < 0.001) elevated in the exhaled air of male subjects. Furthermore, it could be shown that 19–29 years old subjects exhale significantly lower levels of isoprene than older adults (p = 0.002). No significant differences between groups were detected for any other measured mass. In conclusion, the present study demonstrates gender and age specific differences of isoprene levels in the exhaled air. These findings may be of potential clinical relevance regarding the multifaceted roles of isoprene, representing both indicator and effector molecule. © 2006 Elsevier B.V. All rights reserved. Keywords: Isoprene; Breath; Gender; Age; Mass-spectrometry
1. Introduction Abbreviations: m/z, mass-to-charge ratio; ppmv, parts per million by volume; ppbv, parts per billion by volume; PTR-MS, protontransfer-reaction-mass-spectrometry; VOC, volatile organic compounds ∗ Corresponding author. Tel.: +43 51250422400; fax: +43 51250422450. E-mail address: [email protected] (J. Rieder). 1569-9048/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2006.01.007
Breath gas analysis has attracted a considerable amount of scientific and clinical interest during the last decade, as this technique was described as one possible method for non-invasive and rapid diagnosis of a variety of diseases, e.g. lung cancer (Phillips et al.,
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
2003a, 1999), breast cancer (Phillips et al., 2003b), schizophrenia (Phillips et al., 1993), and hyperlipidemia (Rieder et al., 2001). A correlation of these diseases with specific patterns of volatile organic compounds in the exhaled air could be demonstrated. Human breath contains a variety of endogenous volatile compounds, the most abundant ones being acetone ∼1 part per million by volume (ppmv), methanol, ethanol, propanols, and the molecule isoprene (2methylbutadiene-1,3), a reactive aliphatic hydrocarbon with a half-life of a few hours (Fenske and Paulson, 1999). Humans produce isoprene endogenously at a rate of 0.15 mmol/kg/h (Taalman, 1996). In addition to man this compound is also produced by bacteria, plants, and various mammals (Kuzma et al., 1995; Sharkey and Yeh, 2001). Therefore, the amount of isoprene present in the atmosphere is of biological origin. Due to its high volatility (i.e., small Henry’s law constant), its low water solubility and its low boiling point of 34 ◦ C, it is mainly eliminated via the lungs (Dahl et al., 1987). Isoprene concentrations in exhaled air are described to be significantly lower in children than in adults (Taucher et al., 1997). Additional studies showed that neonates had undetectable or very low levels of isoprene in their expired breath during the first postnatal week. Breath isoprene levels increased with age, and healthy school children had higher levels than healthy preschool children (Nelson et al., 1998). However, due to large inter-individual variability, little evidence for an age dependence of exhaled isoprene concentrations has been described among adults. Despite the existence of studies describing the influence of age on exhaled isoprene concentrations, no study has clearly stated whether gender specific differences do exist. In a standardized setting, we analyzed volatile compounds with atomic mass units from 21 to 229, including the molecule isoprene, in the exhaled air of 126 fasting subjects. We particularly turned our attention to gender and age specific differences.
2. Materials and methods 2.1. Subjects The study was approved by the local ethical committee and written informed consent was obtained from each participant. Breath samples were collected from
479
126 subjects, 66 females (mean age = 55.35 years; S.D. = 17.29) and 60 males (mean age = 58.93 years; S.D. = 12.0). Furthermore, all participants were classified into six groups according to their age (Group A: 19–29 years old, n = 11; Group B: 30–39 years old, n = 5; Group C: 40–49 years old, n = 16; Group D: 50–59 years old, n = 29; Group E: 60–69 years old, n = 40; Group F: 70–79 years old, n = 25). All subjects who were enrolled in the study had to fast over night and none of them was on statin therapy. Samples were obtained on the following day between 8:00 and 10:00 a.m. in a standardized setting. The subjects were requested to rest for at least 10 min, thus allowing the pulse rate to stabilize. Then they were asked to exhale into a sample bag (Adtech, Gloucestershire, UK) as previously described (Rieder et al., 2001). In brief, patients had to discard the first part of expired air and exhaled only the deep portion into the sample bag. In addition, room air samples were taken, serving as background controls. 2.2. Analysis Samples were heated to 37 ◦ C. Subsequently, the gas in the sample bags and flasks (room air) was siphoned off by a heated Teflon tube (37 ◦ C). Sample analysis was performed using proton-transferreaction-mass-spectrometry (PTR-MS). This technique uses H3 O+ as a chemical ionization reagent to measure volatile organic compounds (VOC) in the parts per billion by volume (ppbv) to parts per trillion by volume (pptv) range. Protonated water, H3 O+ , reacts with neutral molecules (M) according to H3 O+ + M → MH+ + H2 O. This reaction only occurs if these neutral molecules have larger proton affinities than H2 O. Almost all VOCs have larger affinities and therefore proton transfer occurs on every collision with rate constants k, having typical values of 1.5 × 10−9 cm3 s−1 < k < 4 × 10−9 cm3 s−1 . The count rate of ions (protonated molecules, MH+ ) is determined in the ion detection system. There is a linear relationship between the recorded normalized count rate of ions and the concentration of M in the original trace gas, so that the latter can be calculated. The formula includes the normalized count rate of ions and of the primary ion H3 O+ , the temperature in the drift tube, the pressure in the drift tube, the mass dependent transmission efficiency, the rate constant, and the reaction
480
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
time (Lindinger and Jordan, 1998). The concentration of masses at 209 different m/z values (21–229 atomic mass units) was evaluated. For the most part the corresponding substances are hydrocarbons, in particular alkanes, alkenes, alcohols, ketones, and organic acids. Isoprene is detected at m/z 69 using PTR-MS which is a consequence of the proton transfer reaction and reported elsewhere (Karl et al., 2001; Taucher et al., 1997). After baseline conditions had been established each sample was measured at least five times and the calculated average levels were used for further statistical analysis. 2.3. Statistics Statistical analysis was performed using the software package SPSS (v.11.0, Chicago, USA). The concentrations of the investigated volatile compounds are expressed as mean ± S.D. in ppbv for the subgroups. Statistical significance of difference in the concentrations between the groups was determined with the ANOVA and the t-test for unpaired observations after verifying normal distribution in each group. Statistical significance was assumed at pvalues < 0.05 and p < 0.01 were considered highly significant.
3. Results The mean value of isoprene (m/z 69) in the exhaled air of female subjects (n = 66/126) was 57.46 ± 27.75 and that of male subjects (n = 60/126) 80.57 ± 34.13 ppbv. Comparing the two groups by using the t-test for unpaired observations the measured concentrations of isoprene were shown to be highly significantly (p < 0.001) elevated in the exhaled air of men (Fig. 1 and Table 1). The mean value of isoprene in the exhaled air of Group A (19–29 years old; n = 11/126) was 39.95 ± 23.97 ppbv. These levels of isoprene were found to be increased in Group B (30–39 years old; n = 5/126) with 67.86 ± 33.82. In the subgroup of 40–49 years old subjects (Group C; n = 16/126) a mean value of 75.61 ± 28.70 ppbv was detected. The mean value of isoprene was 75.02 ± 30.76 in Group D (50–59 years old; n = 29/126) and 60–69 years old subjects (Group E; n = 40/126) achieved a mean value
Fig. 1. Concentration of isoprene (m/z 69) in the exhaled air of male subjects (m) compared to female subjects (f). Error plot (squares: mean, whiskers: 95% confidence limits)—X-axis: females (f) vs. males (m); Y-axis: concentration in parts per billion by volume (ppbv).
of 67.18 ± 29.16 ppbv. In the subgroup of 70–79 years old subjects (Group F; n = 25/126) a mean value of 71.01 ± 41.82 was found. Data are illustrated in Fig. 2 and Table 2. Taking Groups B, C, D, E, and F together and comparing them to Group A (19–29 years old; n = 11/126) the measured concentrations of isoprene is found to be highly significantly decreased (p = 0.002; tTable 1 Female subjects (f) vs. male subjects (m) Isoprene(m/z 69) f n Mean S.D. S.E.M.
66 57.46 27.75 3.42
n Mean S.D. S.E.M.
60 80.57 34.13 4.41
m
p-value
0.000**
** Highly significantly elevated (p < 0.01); t-test for unpaired observations.
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
Fig. 2. Concentration of isoprene (m/z 69) comparing the different age groups. Error plot (squares: mean, whiskers: 95% confidence limits)—X-axis: age Groups (A–F); Y-axis: concentration in parts per billion by volume (ppbv).
481
Fig. 3. Mass spectrum detected in the exhaled air of the average subject. X-axis: masses at m/z values 21–229; Y-axis: concentration in parts per billion by volume (ppbv).
test for unpaired observations) in the exhaled air of the younger subjects. The measured concentration of isoprene in the room air was generally very low and often only in the pptv range so that it could be neglected. No significant gender or age specific differences between groups were detected for any other measured mass concentrations at m/z values from 21 to 229. The mass spectrum detected in the exhaled air of the average subject is illustrated in Fig. 3 and that found in the room air, serving as the background control, in Fig. 4. Table 2 Mean concentration of isoprene in the exhaled air, standard deviation (S.D.), standard error of the mean (S.E.M.) comparing the age groups A–F n
19–29 years old 30–39 years old 40–49 years old 50–59 years old 60–69 years old 70–79 years old
11 5 16 29 40 25
Isoprene (m/z 69) concentration in the exhaled air Mean
S.D.
S.E.M.
39.95 67.86 75.61 75.02 67.18 71.01
23.97 33.82 28.70 30.76 29.16 41.82
7.23 15.12 7.18 5.71 4.61 8.36
Fig. 4. Mass spectrum detected in the room air serving as the background control. X-axis: masses at m/z values 21–229 (m/z 45: 75.14 ppbv; m/z 47: 15.94 ppbv; m/z 55: 10.04 ppbv; m/z 59: 46.22 ppbv; m/z 69: 0.87 ppbv); Y-axis: concentration in parts per billion by volume (ppbv).
4. Discussion The present study reveals gender specific differences of isoprene levels in the exhaled air. Moreover, it
482
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
could be shown that 19–29 years old subjects exhaled highly significant lower levels of isoprene than the older adults. Within the rest of the adult groups high standard deviations were observed and no evidence for an age dependence of isoprene concentration was found, corresponding well with previous results, i.e. by Mendis et al. (1994). The reason for the highly significant increase of isoprene levels in the exhaled air of men most probably underlies an increased concentration of isoprene in the blood. Because of the large alveolar surface with respect to alveolar volume and in each of the partial lung volumina, there exists near equilibrium between the blood isoprene concentration and the breath isoprene concentration. This is due to Henry’s law partitioning of blood gases with alveolar air. Thus, the average isoprene concentration is correlated to an average breath isoprene concentration over the whole lung volume. Although the exact origin of isoprene in the body is not completely understood, there is evidence that it is of cellular origin (Deneris et al., 1984). It is conjectured to be generated along the mevalonic pathway of cholesterol synthesis. Deneris et al. (1984, 1985) were able to demonstrate the in vitro synthesis of isoprene from dl-mevalonate using cytosolic fraction of rat liver, and suggested that isoprene is formed non-enzymatically after conversion of mevalonate to dimethyallyl pyrophosphate (DMAPP) and/or from isopentenyl pyrophosphate . This provided the first evidence that breath isoprene is linked to cholesterol biosynthesis. Further evidence was provided by Stone et al., 1993 showing a parallel decrease in isoprene exhalation and sterol synthesis caused by acute and chronic lovastatin administration . This strongly suggests that breath isoprene is derived from the cholesterol synthesis pathway in humans in vivo. A small fraction of exhaled isoprene may also be of bacterial origin (Kuzma et al., 1995). The gender specific differences observed in the present study might be explained by differences in the blood cholesterol levels (which is not yet reflected in the clinical guidelines but commonly known (Legato, 2000) and thus in the extent of cholesterol synthesis. Cell membranes contain 50% cholesterol being responsible for their stability. Due to the increased muscle mass of men (rich in cell membranes and thus in cholesterol) the increased cholesterol synthesis and the
increased synthesis of the by-product isoprene might be explained. But also the degradation of isoprene should be considered. The metabolism of isoprene by liver microsomes in vitro from a range of species including rat, mouse, and human shows significant differences between species, strains and gender with respect to the diastereoselectivity and enantioselectivity of the metabolic oxidation and hydrolysis reactions (Watson et al., 2001) Moreover, in vivo, decreased concentrations of isoprene in hepatic venous blood suggest that isoprene is metabolized in the liver (Miekisch et al., 2001). Furthermore, differences in lung capacity and/or pulmonary circulation between men and women must also be taken into account. Oxidative stress has been proposed to play a role in the genesis and progression of acute and chronic diseases (Scholpp et al., 2002). Several studies were carried out to assess whether human breath isoprene levels reflect oxidative stress (Mendis et al., 1995a,b; McGrath et al., 2001, 2000). However, as the results are controversial, no conclusive statement can be made. The highly significantly lower levels of isoprene in the youngest age group correspond well with previous studies describing significantly lower isoprene concentrations in children and adolescents (Taucher et al., 1997; Nelson et al., 1998). This finding might be explained by the larger amount of cell membranes (containing 50% cholesterol as described above) found in adults, e.g. due to an increased muscle mass and organ tissue as well as a higher lung capacity. In addition to a potentially decreased production of isoprene in the body of children, it is conceivable that their higher heart rates and respiratory rates lead to lower levels of exhaled isoprene in single breath samples. Moreover, age related changes in cholesterol metabolism might also have an influence on the isoprene production rate. Within the groups of adults, the standard deviation was very high and no clear trend towards elevation or decrease of breath isoprene could be observed, consistent with previous studies (Taucher et al., 1997; Mendis et al., 1994). No significant gender or age specific differences between groups were detected for any other measured mass concentrations at m/z values from 21 to 229, including acetone at m/z 59. Finally, one potential limitation of the method should be briefly addressed. In PTR-MS measurements, each measured mass can potentially represent
M. Lechner et al. / Respiratory Physiology & Neurobiology 154 (2006) 478–483
various substances. As investigated and reported elsewhere, there is compelling evidence that the great majority of the abundant m/z 69 signal is due only to breath isoprene in humans (Karl et al., 2001; Taucher et al., 1997). In conclusion, the present study demonstrates gender specific differences of isoprene levels in the exhaled air. Moreover, significantly lower levels of isoprene were detected in 19–29 years old subjects. These findings may be of potential clinical relevance regarding the multifaceted roles of isoprene, representing both indicator and effector molecule.
Acknowledgements This work was supported by the Austrian Federal Science Fund (FWF) with grant P-14149 MED to Josef Rieder and grant P-17447 to Herbert Tilg. Moreover, we would like to thank our technician Dr. Dietmar Bader and Dr. Alfons Jordan (Institute of Ion Physics, University of Innsbruck, Austria) for their invaluable help.
References Dahl, A.R., Birnbaum, L.S., Bond, J.A., Gervasi, P.G., Henderson, R.F., 1987. The fate of isoprene inhaled by rats: comparison to butadiene. Toxicol. Appl. Pharmacol. 89, 237– 248. Deneris, E.S., Stein, R.A., Mead, J.F., 1984. In vitro biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction. Biochem. Biophys. Res. Commun. 123, 691–696. Deneris, E.S., Stein, R.A., Mead, J.F., 1985. Acid-catalyzed formation of isoprene from a mevalonate-derived product using a rat liver cytosolic fraction. J. Biol. Chem. 260, 1382–1385. Fenske, J.D., Paulson, S.E., 1999. Human breath emissions of VOCs. J. Air Waste Manag. Assoc. 49, 594–598. Karl, T., Prazeller, P., Mayr, D., Jordan, A., Rieder, J., Fall, R., Lindinger, W., 2001. Human breath isoprene and its relation to blood cholesterol levels: new measurements and modeling. J. Appl. Physiol. 91, 762–770. Kuzma, J., Nemecek-Marshall, M., Pollock, W.H., Fall, R., 1995. Bacteria produce the volatile hydrocarbon isoprene. Curr. Microbiol. 30, 97–103. Legato, M.J., 2000. Dyslipidemia, gender, and the role of highdensity lipoprotein cholesterol: implications for therapy. Am. J. Cardiol. 86, 15L–18L. Lindinger, W., Jordan, A.H., 1998. On-line monitoring of volatile organic compounds at pptv levels by means of proton-transferreaction mass spectrometry (PTR-MS). Medical applications,
483
food control and environmental research. Int. J. Mass Spectrom. Ion Process. McGrath, L.T., Patrick, R., Mallon, P., Dowey, L., Silke, B., Norwood, W., Elborn, S., 2000. Breath isoprene during acute respiratory exacerbation in cystic fibrosis. Eur. Respir. J. 16, 1065–1069. McGrath, L.T., Patrick, R., Silke, B., 2001. Breath isoprene in patients with heart failure. Eur. J. Heart Fail. 3, 423–427. Mendis, S., Sobotka, P.A., Euler, D.E., 1994. Pentane and isoprene in expired air from humans: gas-chromatographic analysis of single breath. Clin. Chem. 40, 1485–1488. Mendis, S., Sobotka, P.A., Euler, D.E., 1995a. Expired hydrocarbons in patients with acute myocardial infarction. Free Radic. Res. 23, 117–122. Mendis, S.S.P., Leja, F.L., Euler, D.E., 1995b. Breath pentane and plasma lipid peroxides in ischemic heart disease. Free Radic. Biol. Med. 19, 679–684. Miekisch, W., Schubert, J.K., Vagts, D.A., Geiger, K., 2001. Analysis of volatile disease markers in blood. Clin. Chem. 47, 1053–1060. Nelson, N., Lagesson, V., Nosratabadi, A.R., Ludvigsson, J., Tagesson, C., 1998. Exhaled isoprene and acetone in newborn infants and in children with diabetes mellitus. Pediatr. Res. 44, 363–367. Phillips, M., Cataneo, R.N., Cummin, A.R., Gagliardi, A.J., Gleeson, K., Greenberg, J., Maxfield, R.A., Rom, W.N., 2003a. Detection of lung cancer with volatile markers in the breath. Chest 123, 2115–2123. Phillips, M., Cataneo, R.N., Ditkoff, B.A., Fisher, P., Greenberg, J., Gunawardena, R., Kwon, C.S., Rahbari-Oskoui, F., Wong, C., 2003b. Volatile markers of breast cancer in the breath. Breast J. 9, 184–191. Phillips, M., Gleeson, K., Hughes, J.M., Greenberg, J., Cataneo, R.N., Baker, L., McVay, W.P., 1999. Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study. Lancet 353, 1930–1933. Phillips, M., Sabas, M., Greenberg, J., 1993. Increased pentane and carbon disulfide in the breath of patients with schizophrenia. J. Clin. Pathol. 46, 861–864. Rieder, J., Lirk, P., Ebenbichler, C., Gruber, G., Prazeller, P., Lindinger, W., Amann, A., 2001. Analysis of volatile organic compounds: possible applications in metabolic disorders and cancer screening. Wien. Klin. Wochenschr. 113, 181–185. Scholpp, J., Schubert, J.K., Miekisch, W., Geiger, K., 2002. Breath markers and soluble lipid peroxidation markers in critically ill patients. Clin. Chem. Lab. Med. 40, 587–594. Sharkey, T.D., Yeh, S., 2001. Isoprene Emission from Plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 407–436. Stone, B.G., Besse, T.J., Duane, W.C., Evans, C.D., DeMaster, E.G., 1993. Effect of regulating cholesterol biosynthesis on breath isoprene excretion in men. Lipids 28, 705–708. Taalman, R.D., 1996. Isoprene: background and issues. Toxicology 113, 242–246. Taucher, J., Hansel, A., Jordan, A., Fall, R., Futrell, J.H., Lindinger, W., 1997. Detection of isoprene in expired air from human subjects using proton-transfer-reaction mass spectrometry. Rapid Commun. Mass Spectrom. 11, 1230–1234. Watson, W.P., Cottrell, L., Zhang, D., Golding, B.T., 2001. Metabolism and molecular toxicology of isoprene. Chem. Biol. Interact. 135–136, 223–238.