Free Radical Biology and Medicine 85 (2015) 157–164
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Original Contribution
Quantification of propionaldehyde in breath of patients after lung transplantation M.E. Dolch a,n, A. Choukèr a, C. Hornuss a, L. Frey a, M. Irlbeck a, S. Praun b, C. Leidlmair b, J. Villinger b, G. Schelling a a b
Department of Anaesthesiology, University Hospital Munich–Campus Großhadern, Ludwig-Maximilians University, Munich, Germany VF Services GmbH, 6067 Absam, Austria
art ic l e i nf o
a b s t r a c t
Article history: Received 25 March 2015 Accepted 1 April 2015 Available online 8 April 2015
Oxygen-derived free radicals (ROS) have been identified to contribute significantly to ischemia– reperfusion (I/R) injury by initiating chain reactions with polyunsaturated membrane lipids (lipid peroxidation, LPO) resulting in the generation of several aldehydes and ketones. Due to their volatile nature these LPO products can be measured noninvasively in breath. We hypothesized that one of these markers, namely propionaldehyde, will be increased in lung and heart–lung transplant patients where severe oxidative stress due to I/R injury with early graft dysfunction represents one of the major postoperative complications resulting in prolonged ventilation and increased in-hospital morbidity and mortality. Expiratory air measurements for acetone, isoprene, and propionaldehyde were performed in seven patients after lung (n ¼5) or heart–lung (n ¼2) transplantation, ventilated patients (n ¼ 12), and healthy volunteers (n ¼17) using online ion-molecule reaction mass spectrometry. Increased concentrations of acetone (transplanted: 3812 [2347–12498]; ventilated: 1255 [276–1959]; healthy: 631 [520–784] ppbv; P o.001) and propionaldehyde (transplanted: 270 [70–424]; ventilated: 82 [41.8–142]; healthy: 1.7 [0.1–11.8] ppbv; P o.001) were found in expiratory air of transplanted and ventilated patients. Propionaldehyde resulting from spontaneous fragmentation of peroxides due to free radical-induced LPO after I/R injury in patients after lung or heart–lung transplantation can be quantified in expired breath. & 2015 Elsevier Inc. All rights reserved.
Keywords: Ion-molecule reaction mass spectrometry Oxidative stress Lipid peroxidation Propionaldehyde Lung transplantation
Introduction Oxygen-derived free radicals have been identified to contribute significantly to ischemia–reperfusion (I/R) injury [1]. During ischemia, adenosine triphosphate (ATP) is catabolised to hypoxanthine and hypoxia triggers the conversion of xanthine dehydrogenase to oxygen radical-producing xanthine oxidase. Oxygen, entering the ischemic tissue during reperfusion, reacts with hypoxanthine and xanthine oxidase generating superoxide and hydrogen peroxide. Furthermore, superoxide and hydrogen peroxide may form highly reactive hydroxyl radicals via the ironcatalysed Haber-Weiss reaction [2]. These reactive oxygen species (ROS) are extremely reactive and may damage cellular molecules such as lipids, proteins, and deoxyribonucleic acid. Reaction of ROS with lipids, termed lipid peroxidation (LPO), initiates chain reactions resulting in the generation of several products of LPO [3]. Due to the short lifetime of ROS monitoring of
n Correspondence to: Department of Anaesthesiology, University Hospital Munich–Campus Großhadern, Marchioninistr. 15, 81377 Munich, Germany. E-mail address: [email protected] (M.E. Dolch).
http://dx.doi.org/10.1016/j.freeradbiomed.2015.04.003 0891-5849/& 2015 Elsevier Inc. All rights reserved.
oxidative stress usually depends on the measurement of these LPO products [4]. Interestingly, most of these products are volatile and therefore appear in breath where they can be quantified [3,5–9]. Conjugated dienes, for example, generated early during LPO allow a quantitative measurement of LPO. However, measurement of conjugated dienes is exposed to ongoing reactions at sampling and processing [4]. Further products of LPO released at the end of the ROS-induced chain reaction are hydrocarbons like ethane and pentane as well as aldehydes like acetaldehyde, malondialdehyde, and propionaldehyde [3,5]. Among these, ethane, pentane, and malondialdehyde have been investigated intensively during asthma [10], aortic cross clamping [11], cardiopulmonary bypass [12,13], critical illness [14,15], cystic fibrosis [16], liver transplantation [9], oxygen challenge [7], and traumatic brain injury [17]. However, data on the impact of LPO on propionaldehyde generation are limited to reports on experimental myocardial I/R injury and ultraviolet radiation-induced skin LPO [18,19]. Applied diagnostic methods for the analysis of volatile organic compounds (VOCs) in exhaled breath including markers of LPO were gas chromatography and different direct mass spectrometric techniques. Among these, ion-molecule reaction mass spectrometry (IMR-MS) [20], proton transfer reaction mass spectrometry
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(PTR-MS) [21–23], and selected ion-flow tube mass spectrometry (SIFT-MS) have been applied for this purpose [24,25]. Although direct mass spectrometric methods allow on-line monitoring of VOCs in exhaled breath in the range of parts per trillion of volume (pptv) to parts per million of volume (ppmv) differentiation of chemical compounds with the same mass is usually difficult. For example, malondialdehyde and pentane do both appear at the mass over charge ratio (m/z) 72. After IMR-MS ionization both molecules appear at the original m/z 72 and, as the result of ionization-induced dissociative charge transfer at m/z 71. Thus, a simple differentiation between malondialdehyde and pentane by IMR-MS is so far not possible. Therefore, this pilot study was performed to elucidate the usefulness of propionaldehyde as a marker of LPO in exhaled breath. Healthy volunteers, where LPO is considered to be insignificant, were compared to ventilated intensive care unit patients and patients after lung and heart–lung transplantation to test the hypothesis that breath propionaldehyde is increased in patients exposed to LPO. In lung transplant patients significant I/R injury with early graft dysfunction represents one of the major postoperative complications. I/R injury results in prolonged ventilation and increased in-hospital morbidity and mortality in this highly vulnerable patient group [26,27].
Methods The IMR-MS instrument A detailed description of the instrument used has been published previously [20]. In brief, the IMR-MS consists of an ion source where a primary ion beam is generated, an octopole system where ion-molecule reactions take place, and a quadrupole mass spectrometer. The IMR-MS can use krypton, mercury, or xenon gas to form the primary ion beam via electron-impact ionization. Selection of primary ions depends on recombination energy required for sample molecule ionization. For our measurements mercury ions were used with a recombination potential of 10.44 eV. Switching between different primary ion beams and hence energy levels takes 400 ms. Sample gas is transferred in a 2.5 m heated capillary system (Silcosteel, Restek, Bellefonte, PA) at a flow rate of 50 ml/min to the instrument. A constant pressure controller feeds, via a second capillary, a stable amount of 1.5 ml/min into the high vacuum ionization section (10 5 mbar). Gas response times to concentration changes are 50 ms, and gas dead time is 2 s, mainly determined by length and diameter of the capillary used. Mass separation is of 1 u over the mass range, cycle time is 2 ms in average, and the response time less than 100 ms. The concentration drift in signal (measured against a steady test gas concentration) is below 5% over 12 h for acetone [20]. Molecules of interest, calculation, and calibration Molecules of interest, namely acetone, isoprene, and propionaldehyde were measured in a sequential order with a dwell time of 500 ms per compound. Prior to measurements a standardised calibration procedure was performed by analysing diluted test gases in a known concentration (acetaldehyde 1000 ppbv, acetone 1010 ppbv, isoprene 1000 ppbv, and propionaldehyde 12,000 ppbv). Assessment of background signalling was done with nitrogen 5.0. Acetone and propionaldehyde As acetone and propionaldehyde are isobaric molecules (identical molecular weight) their differentiation with direct mass spectrometric methods requires differences in ionization properties, thereby enabling the identification of the underlying molecule.
For the ionization of acetone (C3H6O, ionization potential 9.71 eV) as well as propionaldehyde (C3H6O, ionization potential 9.95 eV) mercury ions (Hg þ ) with an recombination potential of 10.44 eV where chosen [28,29]. Ion-molecule reaction of acetone with Hg þ opens two main reaction channels. About 33% of acetone undergoes a simple charge transfer reaction: C3H6O þHg þ -C3H6O þ þHg.
(1)
In about 67%, the ionization of acetone with Hg þ results in dissociative charge transfer with a methyl group split off leaving behind an acetyl group detectable at m/z 43: C3H6O þHg þ -C2H3O þ þCH3 þHg.
(2)
Furthermore at m/z 59 13C isotopes of acetone are detectable at a rate of approximately 3.5% of the acetone signal at m/z 58 (Fig. 1). In contrast to the ionization of acetone the ion-molecule reaction of propionaldehyde with Hg þ opens two main reaction channels. The principal reaction channel results in charge transfer at a rate of approximately 88.38%: C3H6O þHg þ -C3H6O þ þHg.
(3)
A second reaction channel results in dissociative charge transfer occurring at a rate of approximately 8.45%: C3H6O þHg þ -C3H5O þ þHþHg.
(4)
Furthermore, at m/z 59 the 13C-propionaldehyde isotope appears at a rate of 3.28% of the propionaldehyde signal intensity at m/z 58 which is close to the expected rate of 3.3% from 13C isotope presence. Exemplary mass spectra of test gases are shown in Fig. 1. The fraction of propionaldehyde undergoing dissociative charge transfer resulting in the generation m/z 57 fragments is constant over a propionaldehyde concentration range of 0 to 12,000 ppbv as demonstrated in Fig. 2. For propionaldehyde calibration, only the amount of propionaldehyde appearing at m/z 57 (8.45% of total propionaldehyde) was used. The fixed percentage of propionaldehyde (Cm/z57) appearing at m/z 57 at a rate of 8.45% of total propionaldehyde allowed calculation of whole propionaldehyde present at m/z 57 and 58 (Cm/z57 þ 58): Cm/z57 þ 58 ¼Cm/z57 þCm/z57 100 8.45 1.
(5)
The expected amount of 13C-propionaldehyde isotope of 3.3% detectable at m/z 59 was not taken into account for whole propionaldehyde calculation. Therefore propionaldehyde signalling at m/z 57 and 58 was considered to represent 100% of propionaldehyde signal. The simultaneous presence of acetone and propionaldehyde signaling at m/z 58 in mixed gas (as in exhaled breath for example) necessitated a correction procedure to identify the portion of acetone present at m/z 58. Therefore, the m/z 58 portion of propionaldehyde was calculated and subtracted from whole signal intensity at m/z 58. The remaining signal was then considered to represent the amount of acetone, which does not undergo substantial dissociative charge transfer during Hg þ ionisation in the setting used here. Precision and recovery For the assessment of precision and recovery propionaldehyde test gas was diluted with synthetic air to achieve propionaldehyde concentrations of 24, 48, and 122 ppbv. On each concentration step 5 measurements with a dwell time of 500 ms were performed. Precision was assessed by calculation of the relative standard deviation (%) of repetitive propionaldehyde analyses on each concentration step. Computing the ratio of measured and adjusted propionaldehyde concentrations assessed recovery.
M.E. Dolch et al. / Free Radical Biology and Medicine 85 (2015) 157–164
A standardised procedure was used for airway gas sampling in healthy volunteers and ventilated patients. The study protocol was in accordance with the Helsinki Declaration and approved by the local ethics committee (protocol No. 089/04). In all cases, written informed consent was given by the participants or, in case of critically ill patients, by a legal representative. Additionally, all patients who recovered and regained consciousness with adequate cognitive functioning gave their consent to the study retrospectively. A healthy study population of 17 volunteers was recruited in the age range of 26–55 years (BMI 19.4–31.7; 10 male and 7 female). These were mainly physicians of the department of anesthesiology or colleagues from other departments. To avoid effects of exposure to volatile anesthetics on breath gas analysis none of them was employed within the operation area within 2 days before data collection. Measurements in volunteers were performed in a conference room located on the intensive care unit. Each participant was seated for 5 min before measurements and performed a training period of 2 min with metronome guided breathing. For expiratory air measurements 10 metronome-controlled breath cycles through a Teflon tube (diameter 2 cm, length 4 cm) with a side port enabling the connection to the IMR-MS transfer capillary were analysed online. Accompanying ambient air analysis was performed during the
metronome-training period. Twelve intensive care unit patients in the age range of 18 to 77 years (3 female and 9 male) with the need for artificial ventilation for various conditions served as ventilated 103
Signal intensity at m z 57 [cps]
Sampling of airway gas
159
102
101 y = 0.072·x + 63.73 r = 0.99 p < 2.2·10 100 100
101
102
103
104
Signal intensity at m z 58 [cps] Fig. 2. Correlation of signaling at m/z 58 and m/z 57 during gradual increase in propionaldehyde concentration from 0 to 12,000 ppbv diluted in nitrogen 5.0.
5.105
5.105 Acetone
Signal intensity [cps]
58
4.105
4.105
3.105
3.105
2.105
2.105
1.105
1.105
0
0 20
30
40
50
60
70
80
90
100
1.105
1.105
Signal intensity [cps]
Propionaldehyde
58
8.104
8.104
6.104
6.104
4.104
4.104
2.104
2.104
0
0 20
30
40
50
60
70
80
90
100
m/z Fig. 1. IMR-MS mass spectrums scan of test gas containing acetone (a) and propionaldehyde (b) in nitrogen 5.0 with Hg þ as primary ion (integration time per m/z 500 ms). The parent ion signal of acetone is also detected at m/z 58. Significant fragment ions are detected at m/z 43 and 42. At m/z 57 only negligible 1.3% of total acetone appear as fragment ions. The parent ion signal of propionaldehyde is detected at m/z 58. Fragment ions are present at m/z 57 accounting for about 8.5% of the whole propionaldehyde signal. The chemical selectivity of the IMR-MS is illustrated by the absence of any signal at m/z values of 28 (nitrogen, N2). This is explained by the ionization potential of nitrogen (15.58 eV), which is above the recombination energy potential of mercury (10.44 eV) used as primary ion for these measurements.
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control group (ICU). Only patients who had total intravenous anesthesia for surgery prior to breath gas analysis were included. A group of 7 patients in the age range of 33–57 years (5 female and 2 female) after lung (n¼ 5) and heart–lung transplantation (n ¼2) were studied (TX). In general all lung and heart transplant procedures at our institution are performed under total intravenous anesthesia. Measurements were performed on postoperative day 1 followed by measurements on days 2 and 3 if mechanically ventilation was still necessary. In mechanically ventilated critically ill patients ventilation parameters were kept constant 60 min prior and during airway gas sampling. In addition disinfectants were not used for the same time period. All patients were sedated with sufentanil and either midazolam or propofol. Volatile anesthetics were not used prior to measurements. For gas sampling, a sterilized stainless steel T-piece was placed at the end of the endotracheal tube within the respirator circuit and connected to the capillary system of the IMR-MS for on-line analysis. For inspiratory gas analysis, the T-piece was inserted within the inspiratory limb of the respiratory circuit after the active humidifier. Measurements were performed over 9 min for expiratory and 3 min for inspiratory gas. Identification of the end-expiratory phase was performed by simultaneous analysis of CO2 concentration in airway gas with a conventional electron-impact MS integrated into the IMR-MS [20]. Statistical analysis All data were tested for normal distribution using the ShapiroWilk test. Parametric data were reported as mean 7standard deviation; nonparametric data as median with 25–75% percentiles. Statistical significance of differences was tested with the KruskalWallis rank sum test followed by post hoc Nemenyi test if indicated. Intragroup changes were tested by paired t test or Wilcoxon signed-rank test where appropriate. Relationships between parameters were computed using simple linear regression. A P value o0.05 was considered to be significant. Statistical analyses were carried out with R version 2.11.1 [30].
Results The lower limits of detection (LOD) and quantification (LLOQ) of acetone (3.9 and 11.9 ppbv) and isoprene (2.6 and 7.9 ppbv) were published previously [20]. Values found for propionaldehyde are LOD 1.4 ppbv and LLOQ 4.2 ppbv. LOD and LLOQ measurements were performed at the laboratory of the system manufacturer. LOD and LLOQ were defined as k times of the estimated standard deviation of blank samples (SDbl). From the detector signal LOD and LLOQ were calculated using the slope of the calibration curve (S) as given in Fig. 3a with the following formula: LOD or LLOQ¼k∙SDbl/S (for LOD calculation k¼3.3 and for LLOQ calculation k¼10 was used) [31,32]. Fig. 3b shows the linear response of the IMR-MS against changes in propionaldehyde concentration up to 12,000 ppbv. In Fig. 3c the linear response of the IMR-MS at m/z 57 against changes in propionaldehyde concentration and the influence of humidification are demonstrated. Detected signal intensity at m/z 57 showed a linear response to changes in propionaldehyde concentration over the full range of applied propionaldehyde concentration. The effects of humidification on propionaldehyde signal intensity at m/z 57 were only minor with deviations of 1.5% at the 122 ppbv and 4.7% at the 12,000 ppbv level. Table 1 shows data of precision and recovery of propionaldehyde analysis on three different concentration steps. On each concentration step a sufficient precision and recovery of propionaldehyde were found. Precision ranged from 3.4 to 7.3% whereas recovery varied from 93.5 to 99.9%. The characteristics of the ICU
and TX population are given in Tables 2 and 3. The median time from the last surgery to exhaled breath gas measurement was 19.0 [15.0–49.0] h in ICU patients and 11.0 [7.5–13.0] h in TX patients. With the exception of propionaldehyde measurements in healthy volunteers, significant differences were present for all VOCs analysed with regard to inspiratory and expiratory concentrations (Fig. 4). Furthermore, the results of expiratory propionaldehyde in healthy volunteers were close to the corresponding inspiratory level. Acetone was found in significantly higher concentrations in TX patients when compared to ICU patients and healthy volunteers. Propionaldehyde was found in significantly higher amounts of ICU und TX patients when compared to healthy volunteers. Although a marked difference was notable for breath propionaldehyde comparing ICU und TX patients, this did not reach statistical significance (Fig. 4). Isoprene was found decreased in ICU patients when compared to TX patients. In TX patients number one, two, four, and five, repetitive measurements on postoperative days 2 and 3 were possible due to the need for prolonged mechanical ventilation. During consecutive measurements on postoperative days a steady but not significant decrease of expiratory air acetone (day 1, 3812 [2347–12498]; day 2, 936 [496–3543]; day 3, 841 [271–1553]; ppbv) and propionaldehyde (day 1, 270 [70–424]; day 2, 65 [41–172]; day 3, 53 [41–68] ppbv) was notable whereas isoprene values slightly increased over the time (day 1, 181 [141–337]; day 2, 188 [165–305]; day 3, 203 [166–328]; ppbv) without reaching statistical significance.
Discussion In a patient population with a significant risk for I/R injury increased amounts of acetone and propionaldehyde were found in expiratory breath when compared to a group of ICU patients and healthy volunteers. The concentrations of exhaled breath acetone and isoprene collected in this study are in good agreement with previously reported results for healthy volunteers [23,33,34] and for critically ill patients in the presence of severe respiratory dysfunction which was also present in our population of patients after lung and heart–lung transplantation [14,35,36]. To the best of our knowledge there are currently no published data available regarding exhaled breath propionaldehyde in healthy individuals or a critically ill patient population. However, our data are in good agreement with results obtained during ultraviolet-induced LPO of the skin [19,37]. The event of primary graft failure after lung transplantation and the development of bronchiolitis obliterative syndrome are well known to be associated with I/R injury and oxidative stress [27,38]. Studer et al. observed increased amounts of breath carbonyl sulfide in lung transplant patients with acute rejection and suggested a different pathway of carbonyl sulfide generation compared to markers of LPO [39]. Differences in breath ethane were not found, possibly attributable to the presence of chronic oxidative stress in even stable lung transplant recipients making the absolute change in pro-oxidant biomarkers undetectable [39,40]. Phillips et al. noted changes in the breath methylated alkane contour (BMAC) patients after heart transplantation with either no or mild rejection and severe rejection. No or mild rejection was associated with increases in BMACs attributable to the presence of chronic oxidative stress. Whereas in the case of severe rejection the BMAC decreased which is possibly accountable to accelerated alkane catabolism [41]. Both studies give evidence that chronic oxidative stress is present in transplanted patients and acute rejection induces changes in exhaled breath VOC composition. In contrast to Studer et al. and Phillips et al. the focus of the present work is on oxidative stress in response to the initial I/R injury where markers of oxidative stress are released
M.E. Dolch et al. / Free Radical Biology and Medicine 85 (2015) 157–164
161
25000 400 20000 300 15000 200
y = 2.09·x + 159.44
100
y = 2.08·x + 201.20
10000
r = 0.99
r = 0.99
5000
p < 2.2·10
p < 2.2·10 0
0
Signal intensities at m z 57 [cps]
0
25
50
75
100
125
0
3000
6000
9000
12000
open symbols, dashed line,
25000
dry conditions 400 y = 2.02·x + 161.70 300
20000
r = 1.0 200 p < 2.2·10 100 15000
0 0
50
100 closed symbols, solid line, humidifaction with 5 vol%
10000
y = 1.99·x + 175.10 r = 1.0 5000
p < 2.2·10
0 0
2000
4000
6000
8000
10000
12000
Propionaldehyde [ppbv] Fig. 3. For LOD and LLOQ determination of the IMR-MS the propionaldehyde signal at m/z 57 (a, open circles) was measured on 6 levels with 10 replicates per level by diluting the propionaldehyde standard gas with varying amounts of nitrogen 5.0 (dwell time 500 ms per compound). For signals at m/z 57 a highly linear response was found. The linearity of the system covers a range from 0 to 12,000 ppbv (b). Panel c demonstrates the influence of humidification at a level of 5 vol% H2O on propionaldehyde signal at m/z 57 in nitrogen 5.0 (c) at 0, 48, 122 (small scale figure), and 2416, 4818, 9592, 12,000 ppbv (large scale figure). Data are given as means with standard deviations.
Table 1 Precision and recovery of propionaldehyde in spiked synthetic air at three different concentrations with 5 replicate measurements per concentration step. Concentrations
Precision [%]
Spiked [ppbv]
Measured [ppbv]
24 48 122
23.98 7 1.65 46.277 1.51 114.487 6.71
7.30 3.44 5.70
Recovery [%]
99.92 96.40 93.53
Table 2 Characteristics of mixed intensive care patient population (ICU). Pat. Age Gender Diagnosis [No.] [year] 1
64
M
2
59
W
3 4
51 67
M M
5 6 7
77 63 27
M M W
8
23
M
9
42
W
10
42
M
11 12
49 18
M M
Data of measured concentrations are means with standard deviation.
acutely which could be shown by the appearance of propionaldehyde in breath. Furthermore, it seems to be unlikely that the observed increase in propionaldehyde is caused by the immunosuppressive medication itself as in contrast to azathioprine the use of tacrolimus alleviates oxidative stress in response to I/R injury [42]. Consistent with this, increased amounts of propionaldehyde were present in ICU patients. Two patients suffered from sepsis and 10 patients had a severely compromised oxygenation, which is in agreement with previous findings, reporting an increased level of oxidative stress in critically ill patients [14,35,36]. In those patients from our study who required prolonged ventilation after TX a decrease in acetone and propionaldehyde over time was observable. This finding for acetone has previously
Heart surgery Heart surgery COPD ORL abscess Peritonitis Pneumonia Lung fibrosis Tracheal surgery ORL abscess ORL surgery ORL tumor Fibroma
PaO2/FIO2 [mm Hg]
LIS
178.0
1.75 38
1
91
136.0
2.5
28
0
49
116.0 206.0
2.25 23 1.25 27
1 0
– 50
242.0 95.3 103.8
1.5 46 3.25 50 3.75 21
1 0 0
9 – –
236.9
1.0
11
0
21
204.3
1.5
21
0
19
184.6
0.75 21
0
4
483.3 583.3
0.25 16 0.5 5
0 0
15 19
SAPS Sepsis [y/n]
Surgery [h]
Pat, patient; f, female; m, male; ORL, otorhinolaryngologic; PaO2, partial pressure of arterial oxygen; FIO2, fraction of inspired oxygen; LIS, lung injury severity score; SAPS II, simplified acute physiology score; surgery, time between last surgical procedure to measurement.
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been observed in critically ill patients by Sturney et al. and was ascribed to the effects of treatment and feeding as well to a decline in systemic inflammation and exposure to oxidative stress [27,36]. In contrast to the findings for acetone and propionaldehyde, isoprene concentrations slightly increased over time in the TX population. Due to the small number of TX patients requiring prolonged ventilation in our study, the interpretation of this observation is difficult. A possible explanation for this phenomenon could be improvements in respiratory function or increases in cardiac output [14,43] but none of these changes were seen in our patients. Distinguishing isobaric molecules using direct mass spectrometric techniques is usually challenging and requires different reagent ions together with differences in ionization characteristics of the targeted molecules. During IMR-MS chemical ionization with Hg þ at a recombination potential of 10.44 eV each, acetone and propionaldehyde present unique ionization characteristics that finally allow distinguishing them from each other. The reaction of Table 3 Characteristics of patients with lung and heart–lung transplantation (TX). Pat. Sample [No.] [day]
Age Gender TX PaO2/ FIO2 [year] [mm Hg]
LIS
SAPS II
CBP Ischemia [min] [min]
1
43 – – 41 – – 57 33 – – 47 – – 42 49
3.0 2.5 2.7 1.5 1.5 1.5 1.75 2.0 2.0 1.75 2.5 2,0 2,25 3.00 3.00
31 36 39 28 31 38 31 32 39 36 25 22 30 33 25
98 – – 314 – – – 300 – – 239 – – – –
2
3 4
5
6 7
1 2 3 1 2 3 1 1 2 3 1 2 3 1 1
f – – f – – m f – – f – – m f
SL – – HL – – DL DL – – HL – – DL DL
267 166 202 393 387 325 263 284 289 317 200 213 215 200 156
289 – – 305 – – 480 513 – – 249 – – 600 442
Pat, patient; f, female; m, male; TX, type of transplantation; SL, single lung; DL, bilateral lung; HL, heart–lung; PaO2, partial pressure of arterial oxygen; FIO2, fraction of inspired oxygen; LIS, lung injury severity score; SAPS II, simplified acute physiology score; CBP, cardiopulmonary bypass; duration of ischemia refers to the lung reperfused at last.
VOC concentration [ppbv]
105
acetone with Hg þ proceeds via charge transfer to either C3H6O þ at m/z of 58 (33%) or dissociative charge transfer to C2H3O þ at m/z 43 Hg (67%). In contrast, the reaction of propionaldehyde with Hg þ proceeds via charge transfer to C3H6O þ þ Hg at m/z 58 (88.4%) or via dissociative charge transfer to C3H5O þ at m/z 57 (8.45%). Thus, acetone product ions appear at m/z's 58 and 43 whereas at m/z 57 only the production ion of propionaldehyde appears which makes acetone and propionaldehyde distinguishable using IMR-MS chemical ionization. The constancy of these chemical ionization characteristics was observed over a concentration range of 12,000 ppbv and the effects of humidification were found to be negligible, which allows the use of propionaldehyde as a marker of LPO in exhaled breath. A major problem when carrying out breath gas analysis studies within a hospital environment and especially in intensive care units is the ever-present use of disinfectants resulting in a substantial risk for sample contamination. Sample contamination might occur either via diffusion of disinfectant ingredients into the patients ventilator tubing or, alternatively, by skin absorption. At our institution, a disinfectant solution containing 45 g 2-propanol, 10 g 1-propanol, and 0.2 g 2-biphenylole per 100 g of the solution is used for surgical skin scrubbing and topical skin disinfection. Primary alcohols as 1-propanol undergo alcohol dehydrogenase catalysed oxidation to propionaldehyde whereas secondary alcohols as 2-propanol are oxidized to acetone [44,45]. Reported elimination half-times are 45 min for 1-propanol and 150 min for 2-propanol [44,45]. Given the possible confounding effects of disinfectants on breath propionaldehyde strict control with regard to their use must be implicated in breath gas analysis. During our study the use of disinfectants was restricted within 60 min prior to measurements. A pervious study investigating the effects of surgical hand rubs which is far more intense compared to the usual topical disinfection of catheter insertion sites in intensive care units found systemic increases in the 2-propanol metabolite acetone but not for the 1-propanol metabolite propionaldehyde [46]. Thus, we assume that the observed increase in exhaled breath propionaldehyde in TX patients is related to I/R injuryinduced LPO. This appears to restrict the validity of propionaldehyde as a biomarker of LPO to periods of 60 min following disinfectant use. This delay seems to be acceptable in clinical practice, however. The limitation of our study lies in the small number of patients investigated for the presence for markers of oxidative stress in
*#
*
#
105
#
104
104
103
103
102
102
101
101
100
100
10−1
10−1
Acetone
Isoprene
Propionaldehyde
Fig. 4. Exhaled breath acetone, isoprene, and propionaldehyde in healthy volunteers (dark grey boxes, n¼ 17), mixed intensive care unit population (light grey boxes, n ¼12), and patients on day 1 after lung and heart–lung transplantation (white boxes, n¼ 7). Measurements in inspiratory air (ambient air for healthy volunteers) are hatched. With the exception of propionaldehyde in healthy volunteers, for all compounds a significant difference comparing expiratory and inspiratory measurements was present (Wilcoxon signed-rank test; Po 0.05). Intergroup differences were present for acetone, isoprene, and propionaldehyde measurements (Kruskal-Wallis rank sum test with post hoc Nemenyi test; nPo .05, #P o.001).
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exhaled breath. However, the present study was intended as a pilot study to confirm our hypothesis that propionaldehyde is detectable within the exhaled breath of TX patients who are exposed to I/R injury and oxidative stress. Further studies will have to confirm these initial results in a larger population. Furthermore, we were not able to show any relationship among I/R, primary graft failure, and secondary organ dysfunction with exhaled breath propionaldehyde levels. To answer these questions a further study within a larger population will be necessary. In summary and despite the above-noted limitations of our study, we have shown that propionaldehyde resulting from spontaneous fragmentation of peroxides originating from free radical-induced LPO after I/R injury in TX patients can be quantified in expired breath. Due to the noninvasive nature of measurements, exhaled breath propionaldehyde could possibly serve as a biomarker for the reliable estimate of LPO extent and, possibly, as an additional risk assessment parameter in patients after chest organ transplantation.
Conflict of interest and source of funding VF services GmbH provided the IMR-MS system used in this study. Johannes Villinger is senior scientist of VF Services GmbH and Siegfried Praun and Christian Leidlmair are scientists employed by this company with financial interests regarding the IMR-MS system. Michael E. Dolch and Cyrill Hornuss received traveling funds from VF Services GmbH for research presentations at the annual meeting of the American Society of Anesthesiologists. None of the other authors has received funding or other financial support from VF services GmbH; this includes contracts, equity interest, stock option(s), direct or indirect salary support, consultant fee(s), lecture fees, or honoraria received within a period of 3 years of the date of submission of this manuscript. The funding sources had no role in study design, in the collection, analysis, and interpretation of data and in the decision to submit the paper for publication.
Acknowledgments This work was supported by intramural funding of the Department of Anaesthesiology and the German Space Agency (DLR) on behalf of the Federal Ministry of Economics and Technology (BMWi 50WB0719, 50WB0919) and the European Space Agency (ESA, ELIPS 3 programme) (regarding precursor studies). References [1] McCord, J. M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159–163; 1985. [2] Granger, D. N. Role of xanthine oxidase and granulocytes in ischemiareperfusion injury. Am. J. Physiol. 255:H1269–H1275; 1988. [3] Frankel, E. N. Volatile lipid oxidation products. Prog Lipid Res. 22:1–33; 1983. [4] Brown, R. H.; Risby, T. H. Monitoring distant organ reperfusion injury by measurement of volatile organic compounds. In: Marczin, N. N., Kharitonov, S. A., Yacoub, M. H., Barnes, P. J., editors. Disease markers in exhaled breath. New York: Marcel Dekker; 2003. p. 281–304. [5] Kneepkens, C. M. F.; Lepage, G.; Roy, C. C. The potential of the hydrocarbon breath test as a measure of lipid peroxidation. Free Radic. Biol. Med. 17:127–160; 1994. [6] Phillips, M. Method for the collection and assay of volatile organic compounds in breath. Anal. Biochem. 247:272–278; 1997. [7] Phillips, M.; Cataneo, R. N.; Greenberg, J.; Grodman, R.; Gunawardena, R.; Naidu, A. Effect of oxygen on breath markers of oxidative stress. Eur. Respir. J. 21:48–51; 2003. [8] Risby, T. H.; Sehnert, S. S. Clinical application of breath biomarkers of oxidative stress status. Free Radic. Biol. Med. 27:1182–1192; 1999. [9] Kazui, M.; Andreoni, K. A.; Norris, E. J.; Klein, A. S.; Burdick, J. F.; Beattie, C.; Sehnert, S. S.; Bell, W. R.; Bulkley, G. B.; Risby, T. H. Breath ethane: a specific
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Original Contribution
Quantification of propionaldehyde in breath of patients after lung transplantation M.E. Dolch a,n, A. Choukèr a, C. Hornuss a, L. Frey a, M. Irlbeck a, S. Praun b, C. Leidlmair b, J. Villinger b, G. Schelling a a b
Department of Anaesthesiology, University Hospital Munich–Campus Großhadern, Ludwig-Maximilians University, Munich, Germany VF Services GmbH, 6067 Absam, Austria
art ic l e i nf o
a b s t r a c t
Article history: Received 25 March 2015 Accepted 1 April 2015 Available online 8 April 2015
Oxygen-derived free radicals (ROS) have been identified to contribute significantly to ischemia– reperfusion (I/R) injury by initiating chain reactions with polyunsaturated membrane lipids (lipid peroxidation, LPO) resulting in the generation of several aldehydes and ketones. Due to their volatile nature these LPO products can be measured noninvasively in breath. We hypothesized that one of these markers, namely propionaldehyde, will be increased in lung and heart–lung transplant patients where severe oxidative stress due to I/R injury with early graft dysfunction represents one of the major postoperative complications resulting in prolonged ventilation and increased in-hospital morbidity and mortality. Expiratory air measurements for acetone, isoprene, and propionaldehyde were performed in seven patients after lung (n ¼5) or heart–lung (n ¼2) transplantation, ventilated patients (n ¼ 12), and healthy volunteers (n ¼17) using online ion-molecule reaction mass spectrometry. Increased concentrations of acetone (transplanted: 3812 [2347–12498]; ventilated: 1255 [276–1959]; healthy: 631 [520–784] ppbv; P o.001) and propionaldehyde (transplanted: 270 [70–424]; ventilated: 82 [41.8–142]; healthy: 1.7 [0.1–11.8] ppbv; P o.001) were found in expiratory air of transplanted and ventilated patients. Propionaldehyde resulting from spontaneous fragmentation of peroxides due to free radical-induced LPO after I/R injury in patients after lung or heart–lung transplantation can be quantified in expired breath. & 2015 Elsevier Inc. All rights reserved.
Keywords: Ion-molecule reaction mass spectrometry Oxidative stress Lipid peroxidation Propionaldehyde Lung transplantation
Introduction Oxygen-derived free radicals have been identified to contribute significantly to ischemia–reperfusion (I/R) injury [1]. During ischemia, adenosine triphosphate (ATP) is catabolised to hypoxanthine and hypoxia triggers the conversion of xanthine dehydrogenase to oxygen radical-producing xanthine oxidase. Oxygen, entering the ischemic tissue during reperfusion, reacts with hypoxanthine and xanthine oxidase generating superoxide and hydrogen peroxide. Furthermore, superoxide and hydrogen peroxide may form highly reactive hydroxyl radicals via the ironcatalysed Haber-Weiss reaction [2]. These reactive oxygen species (ROS) are extremely reactive and may damage cellular molecules such as lipids, proteins, and deoxyribonucleic acid. Reaction of ROS with lipids, termed lipid peroxidation (LPO), initiates chain reactions resulting in the generation of several products of LPO [3]. Due to the short lifetime of ROS monitoring of
n Correspondence to: Department of Anaesthesiology, University Hospital Munich–Campus Großhadern, Marchioninistr. 15, 81377 Munich, Germany. E-mail address: [email protected] (M.E. Dolch).
http://dx.doi.org/10.1016/j.freeradbiomed.2015.04.003 0891-5849/& 2015 Elsevier Inc. All rights reserved.
oxidative stress usually depends on the measurement of these LPO products [4]. Interestingly, most of these products are volatile and therefore appear in breath where they can be quantified [3,5–9]. Conjugated dienes, for example, generated early during LPO allow a quantitative measurement of LPO. However, measurement of conjugated dienes is exposed to ongoing reactions at sampling and processing [4]. Further products of LPO released at the end of the ROS-induced chain reaction are hydrocarbons like ethane and pentane as well as aldehydes like acetaldehyde, malondialdehyde, and propionaldehyde [3,5]. Among these, ethane, pentane, and malondialdehyde have been investigated intensively during asthma [10], aortic cross clamping [11], cardiopulmonary bypass [12,13], critical illness [14,15], cystic fibrosis [16], liver transplantation [9], oxygen challenge [7], and traumatic brain injury [17]. However, data on the impact of LPO on propionaldehyde generation are limited to reports on experimental myocardial I/R injury and ultraviolet radiation-induced skin LPO [18,19]. Applied diagnostic methods for the analysis of volatile organic compounds (VOCs) in exhaled breath including markers of LPO were gas chromatography and different direct mass spectrometric techniques. Among these, ion-molecule reaction mass spectrometry (IMR-MS) [20], proton transfer reaction mass spectrometry
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(PTR-MS) [21–23], and selected ion-flow tube mass spectrometry (SIFT-MS) have been applied for this purpose [24,25]. Although direct mass spectrometric methods allow on-line monitoring of VOCs in exhaled breath in the range of parts per trillion of volume (pptv) to parts per million of volume (ppmv) differentiation of chemical compounds with the same mass is usually difficult. For example, malondialdehyde and pentane do both appear at the mass over charge ratio (m/z) 72. After IMR-MS ionization both molecules appear at the original m/z 72 and, as the result of ionization-induced dissociative charge transfer at m/z 71. Thus, a simple differentiation between malondialdehyde and pentane by IMR-MS is so far not possible. Therefore, this pilot study was performed to elucidate the usefulness of propionaldehyde as a marker of LPO in exhaled breath. Healthy volunteers, where LPO is considered to be insignificant, were compared to ventilated intensive care unit patients and patients after lung and heart–lung transplantation to test the hypothesis that breath propionaldehyde is increased in patients exposed to LPO. In lung transplant patients significant I/R injury with early graft dysfunction represents one of the major postoperative complications. I/R injury results in prolonged ventilation and increased in-hospital morbidity and mortality in this highly vulnerable patient group [26,27].
Methods The IMR-MS instrument A detailed description of the instrument used has been published previously [20]. In brief, the IMR-MS consists of an ion source where a primary ion beam is generated, an octopole system where ion-molecule reactions take place, and a quadrupole mass spectrometer. The IMR-MS can use krypton, mercury, or xenon gas to form the primary ion beam via electron-impact ionization. Selection of primary ions depends on recombination energy required for sample molecule ionization. For our measurements mercury ions were used with a recombination potential of 10.44 eV. Switching between different primary ion beams and hence energy levels takes 400 ms. Sample gas is transferred in a 2.5 m heated capillary system (Silcosteel, Restek, Bellefonte, PA) at a flow rate of 50 ml/min to the instrument. A constant pressure controller feeds, via a second capillary, a stable amount of 1.5 ml/min into the high vacuum ionization section (10 5 mbar). Gas response times to concentration changes are 50 ms, and gas dead time is 2 s, mainly determined by length and diameter of the capillary used. Mass separation is of 1 u over the mass range, cycle time is 2 ms in average, and the response time less than 100 ms. The concentration drift in signal (measured against a steady test gas concentration) is below 5% over 12 h for acetone [20]. Molecules of interest, calculation, and calibration Molecules of interest, namely acetone, isoprene, and propionaldehyde were measured in a sequential order with a dwell time of 500 ms per compound. Prior to measurements a standardised calibration procedure was performed by analysing diluted test gases in a known concentration (acetaldehyde 1000 ppbv, acetone 1010 ppbv, isoprene 1000 ppbv, and propionaldehyde 12,000 ppbv). Assessment of background signalling was done with nitrogen 5.0. Acetone and propionaldehyde As acetone and propionaldehyde are isobaric molecules (identical molecular weight) their differentiation with direct mass spectrometric methods requires differences in ionization properties, thereby enabling the identification of the underlying molecule.
For the ionization of acetone (C3H6O, ionization potential 9.71 eV) as well as propionaldehyde (C3H6O, ionization potential 9.95 eV) mercury ions (Hg þ ) with an recombination potential of 10.44 eV where chosen [28,29]. Ion-molecule reaction of acetone with Hg þ opens two main reaction channels. About 33% of acetone undergoes a simple charge transfer reaction: C3H6O þHg þ -C3H6O þ þHg.
(1)
In about 67%, the ionization of acetone with Hg þ results in dissociative charge transfer with a methyl group split off leaving behind an acetyl group detectable at m/z 43: C3H6O þHg þ -C2H3O þ þCH3 þHg.
(2)
Furthermore at m/z 59 13C isotopes of acetone are detectable at a rate of approximately 3.5% of the acetone signal at m/z 58 (Fig. 1). In contrast to the ionization of acetone the ion-molecule reaction of propionaldehyde with Hg þ opens two main reaction channels. The principal reaction channel results in charge transfer at a rate of approximately 88.38%: C3H6O þHg þ -C3H6O þ þHg.
(3)
A second reaction channel results in dissociative charge transfer occurring at a rate of approximately 8.45%: C3H6O þHg þ -C3H5O þ þHþHg.
(4)
Furthermore, at m/z 59 the 13C-propionaldehyde isotope appears at a rate of 3.28% of the propionaldehyde signal intensity at m/z 58 which is close to the expected rate of 3.3% from 13C isotope presence. Exemplary mass spectra of test gases are shown in Fig. 1. The fraction of propionaldehyde undergoing dissociative charge transfer resulting in the generation m/z 57 fragments is constant over a propionaldehyde concentration range of 0 to 12,000 ppbv as demonstrated in Fig. 2. For propionaldehyde calibration, only the amount of propionaldehyde appearing at m/z 57 (8.45% of total propionaldehyde) was used. The fixed percentage of propionaldehyde (Cm/z57) appearing at m/z 57 at a rate of 8.45% of total propionaldehyde allowed calculation of whole propionaldehyde present at m/z 57 and 58 (Cm/z57 þ 58): Cm/z57 þ 58 ¼Cm/z57 þCm/z57 100 8.45 1.
(5)
The expected amount of 13C-propionaldehyde isotope of 3.3% detectable at m/z 59 was not taken into account for whole propionaldehyde calculation. Therefore propionaldehyde signalling at m/z 57 and 58 was considered to represent 100% of propionaldehyde signal. The simultaneous presence of acetone and propionaldehyde signaling at m/z 58 in mixed gas (as in exhaled breath for example) necessitated a correction procedure to identify the portion of acetone present at m/z 58. Therefore, the m/z 58 portion of propionaldehyde was calculated and subtracted from whole signal intensity at m/z 58. The remaining signal was then considered to represent the amount of acetone, which does not undergo substantial dissociative charge transfer during Hg þ ionisation in the setting used here. Precision and recovery For the assessment of precision and recovery propionaldehyde test gas was diluted with synthetic air to achieve propionaldehyde concentrations of 24, 48, and 122 ppbv. On each concentration step 5 measurements with a dwell time of 500 ms were performed. Precision was assessed by calculation of the relative standard deviation (%) of repetitive propionaldehyde analyses on each concentration step. Computing the ratio of measured and adjusted propionaldehyde concentrations assessed recovery.
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A standardised procedure was used for airway gas sampling in healthy volunteers and ventilated patients. The study protocol was in accordance with the Helsinki Declaration and approved by the local ethics committee (protocol No. 089/04). In all cases, written informed consent was given by the participants or, in case of critically ill patients, by a legal representative. Additionally, all patients who recovered and regained consciousness with adequate cognitive functioning gave their consent to the study retrospectively. A healthy study population of 17 volunteers was recruited in the age range of 26–55 years (BMI 19.4–31.7; 10 male and 7 female). These were mainly physicians of the department of anesthesiology or colleagues from other departments. To avoid effects of exposure to volatile anesthetics on breath gas analysis none of them was employed within the operation area within 2 days before data collection. Measurements in volunteers were performed in a conference room located on the intensive care unit. Each participant was seated for 5 min before measurements and performed a training period of 2 min with metronome guided breathing. For expiratory air measurements 10 metronome-controlled breath cycles through a Teflon tube (diameter 2 cm, length 4 cm) with a side port enabling the connection to the IMR-MS transfer capillary were analysed online. Accompanying ambient air analysis was performed during the
metronome-training period. Twelve intensive care unit patients in the age range of 18 to 77 years (3 female and 9 male) with the need for artificial ventilation for various conditions served as ventilated 103
Signal intensity at m z 57 [cps]
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101 y = 0.072·x + 63.73 r = 0.99 p < 2.2·10 100 100
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Signal intensity at m z 58 [cps] Fig. 2. Correlation of signaling at m/z 58 and m/z 57 during gradual increase in propionaldehyde concentration from 0 to 12,000 ppbv diluted in nitrogen 5.0.
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m/z Fig. 1. IMR-MS mass spectrums scan of test gas containing acetone (a) and propionaldehyde (b) in nitrogen 5.0 with Hg þ as primary ion (integration time per m/z 500 ms). The parent ion signal of acetone is also detected at m/z 58. Significant fragment ions are detected at m/z 43 and 42. At m/z 57 only negligible 1.3% of total acetone appear as fragment ions. The parent ion signal of propionaldehyde is detected at m/z 58. Fragment ions are present at m/z 57 accounting for about 8.5% of the whole propionaldehyde signal. The chemical selectivity of the IMR-MS is illustrated by the absence of any signal at m/z values of 28 (nitrogen, N2). This is explained by the ionization potential of nitrogen (15.58 eV), which is above the recombination energy potential of mercury (10.44 eV) used as primary ion for these measurements.
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control group (ICU). Only patients who had total intravenous anesthesia for surgery prior to breath gas analysis were included. A group of 7 patients in the age range of 33–57 years (5 female and 2 female) after lung (n¼ 5) and heart–lung transplantation (n ¼2) were studied (TX). In general all lung and heart transplant procedures at our institution are performed under total intravenous anesthesia. Measurements were performed on postoperative day 1 followed by measurements on days 2 and 3 if mechanically ventilation was still necessary. In mechanically ventilated critically ill patients ventilation parameters were kept constant 60 min prior and during airway gas sampling. In addition disinfectants were not used for the same time period. All patients were sedated with sufentanil and either midazolam or propofol. Volatile anesthetics were not used prior to measurements. For gas sampling, a sterilized stainless steel T-piece was placed at the end of the endotracheal tube within the respirator circuit and connected to the capillary system of the IMR-MS for on-line analysis. For inspiratory gas analysis, the T-piece was inserted within the inspiratory limb of the respiratory circuit after the active humidifier. Measurements were performed over 9 min for expiratory and 3 min for inspiratory gas. Identification of the end-expiratory phase was performed by simultaneous analysis of CO2 concentration in airway gas with a conventional electron-impact MS integrated into the IMR-MS [20]. Statistical analysis All data were tested for normal distribution using the ShapiroWilk test. Parametric data were reported as mean 7standard deviation; nonparametric data as median with 25–75% percentiles. Statistical significance of differences was tested with the KruskalWallis rank sum test followed by post hoc Nemenyi test if indicated. Intragroup changes were tested by paired t test or Wilcoxon signed-rank test where appropriate. Relationships between parameters were computed using simple linear regression. A P value o0.05 was considered to be significant. Statistical analyses were carried out with R version 2.11.1 [30].
Results The lower limits of detection (LOD) and quantification (LLOQ) of acetone (3.9 and 11.9 ppbv) and isoprene (2.6 and 7.9 ppbv) were published previously [20]. Values found for propionaldehyde are LOD 1.4 ppbv and LLOQ 4.2 ppbv. LOD and LLOQ measurements were performed at the laboratory of the system manufacturer. LOD and LLOQ were defined as k times of the estimated standard deviation of blank samples (SDbl). From the detector signal LOD and LLOQ were calculated using the slope of the calibration curve (S) as given in Fig. 3a with the following formula: LOD or LLOQ¼k∙SDbl/S (for LOD calculation k¼3.3 and for LLOQ calculation k¼10 was used) [31,32]. Fig. 3b shows the linear response of the IMR-MS against changes in propionaldehyde concentration up to 12,000 ppbv. In Fig. 3c the linear response of the IMR-MS at m/z 57 against changes in propionaldehyde concentration and the influence of humidification are demonstrated. Detected signal intensity at m/z 57 showed a linear response to changes in propionaldehyde concentration over the full range of applied propionaldehyde concentration. The effects of humidification on propionaldehyde signal intensity at m/z 57 were only minor with deviations of 1.5% at the 122 ppbv and 4.7% at the 12,000 ppbv level. Table 1 shows data of precision and recovery of propionaldehyde analysis on three different concentration steps. On each concentration step a sufficient precision and recovery of propionaldehyde were found. Precision ranged from 3.4 to 7.3% whereas recovery varied from 93.5 to 99.9%. The characteristics of the ICU
and TX population are given in Tables 2 and 3. The median time from the last surgery to exhaled breath gas measurement was 19.0 [15.0–49.0] h in ICU patients and 11.0 [7.5–13.0] h in TX patients. With the exception of propionaldehyde measurements in healthy volunteers, significant differences were present for all VOCs analysed with regard to inspiratory and expiratory concentrations (Fig. 4). Furthermore, the results of expiratory propionaldehyde in healthy volunteers were close to the corresponding inspiratory level. Acetone was found in significantly higher concentrations in TX patients when compared to ICU patients and healthy volunteers. Propionaldehyde was found in significantly higher amounts of ICU und TX patients when compared to healthy volunteers. Although a marked difference was notable for breath propionaldehyde comparing ICU und TX patients, this did not reach statistical significance (Fig. 4). Isoprene was found decreased in ICU patients when compared to TX patients. In TX patients number one, two, four, and five, repetitive measurements on postoperative days 2 and 3 were possible due to the need for prolonged mechanical ventilation. During consecutive measurements on postoperative days a steady but not significant decrease of expiratory air acetone (day 1, 3812 [2347–12498]; day 2, 936 [496–3543]; day 3, 841 [271–1553]; ppbv) and propionaldehyde (day 1, 270 [70–424]; day 2, 65 [41–172]; day 3, 53 [41–68] ppbv) was notable whereas isoprene values slightly increased over the time (day 1, 181 [141–337]; day 2, 188 [165–305]; day 3, 203 [166–328]; ppbv) without reaching statistical significance.
Discussion In a patient population with a significant risk for I/R injury increased amounts of acetone and propionaldehyde were found in expiratory breath when compared to a group of ICU patients and healthy volunteers. The concentrations of exhaled breath acetone and isoprene collected in this study are in good agreement with previously reported results for healthy volunteers [23,33,34] and for critically ill patients in the presence of severe respiratory dysfunction which was also present in our population of patients after lung and heart–lung transplantation [14,35,36]. To the best of our knowledge there are currently no published data available regarding exhaled breath propionaldehyde in healthy individuals or a critically ill patient population. However, our data are in good agreement with results obtained during ultraviolet-induced LPO of the skin [19,37]. The event of primary graft failure after lung transplantation and the development of bronchiolitis obliterative syndrome are well known to be associated with I/R injury and oxidative stress [27,38]. Studer et al. observed increased amounts of breath carbonyl sulfide in lung transplant patients with acute rejection and suggested a different pathway of carbonyl sulfide generation compared to markers of LPO [39]. Differences in breath ethane were not found, possibly attributable to the presence of chronic oxidative stress in even stable lung transplant recipients making the absolute change in pro-oxidant biomarkers undetectable [39,40]. Phillips et al. noted changes in the breath methylated alkane contour (BMAC) patients after heart transplantation with either no or mild rejection and severe rejection. No or mild rejection was associated with increases in BMACs attributable to the presence of chronic oxidative stress. Whereas in the case of severe rejection the BMAC decreased which is possibly accountable to accelerated alkane catabolism [41]. Both studies give evidence that chronic oxidative stress is present in transplanted patients and acute rejection induces changes in exhaled breath VOC composition. In contrast to Studer et al. and Phillips et al. the focus of the present work is on oxidative stress in response to the initial I/R injury where markers of oxidative stress are released
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161
25000 400 20000 300 15000 200
y = 2.09·x + 159.44
100
y = 2.08·x + 201.20
10000
r = 0.99
r = 0.99
5000
p < 2.2·10
p < 2.2·10 0
0
Signal intensities at m z 57 [cps]
0
25
50
75
100
125
0
3000
6000
9000
12000
open symbols, dashed line,
25000
dry conditions 400 y = 2.02·x + 161.70 300
20000
r = 1.0 200 p < 2.2·10 100 15000
0 0
50
100 closed symbols, solid line, humidifaction with 5 vol%
10000
y = 1.99·x + 175.10 r = 1.0 5000
p < 2.2·10
0 0
2000
4000
6000
8000
10000
12000
Propionaldehyde [ppbv] Fig. 3. For LOD and LLOQ determination of the IMR-MS the propionaldehyde signal at m/z 57 (a, open circles) was measured on 6 levels with 10 replicates per level by diluting the propionaldehyde standard gas with varying amounts of nitrogen 5.0 (dwell time 500 ms per compound). For signals at m/z 57 a highly linear response was found. The linearity of the system covers a range from 0 to 12,000 ppbv (b). Panel c demonstrates the influence of humidification at a level of 5 vol% H2O on propionaldehyde signal at m/z 57 in nitrogen 5.0 (c) at 0, 48, 122 (small scale figure), and 2416, 4818, 9592, 12,000 ppbv (large scale figure). Data are given as means with standard deviations.
Table 1 Precision and recovery of propionaldehyde in spiked synthetic air at three different concentrations with 5 replicate measurements per concentration step. Concentrations
Precision [%]
Spiked [ppbv]
Measured [ppbv]
24 48 122
23.98 7 1.65 46.277 1.51 114.487 6.71
7.30 3.44 5.70
Recovery [%]
99.92 96.40 93.53
Table 2 Characteristics of mixed intensive care patient population (ICU). Pat. Age Gender Diagnosis [No.] [year] 1
64
M
2
59
W
3 4
51 67
M M
5 6 7
77 63 27
M M W
8
23
M
9
42
W
10
42
M
11 12
49 18
M M
Data of measured concentrations are means with standard deviation.
acutely which could be shown by the appearance of propionaldehyde in breath. Furthermore, it seems to be unlikely that the observed increase in propionaldehyde is caused by the immunosuppressive medication itself as in contrast to azathioprine the use of tacrolimus alleviates oxidative stress in response to I/R injury [42]. Consistent with this, increased amounts of propionaldehyde were present in ICU patients. Two patients suffered from sepsis and 10 patients had a severely compromised oxygenation, which is in agreement with previous findings, reporting an increased level of oxidative stress in critically ill patients [14,35,36]. In those patients from our study who required prolonged ventilation after TX a decrease in acetone and propionaldehyde over time was observable. This finding for acetone has previously
Heart surgery Heart surgery COPD ORL abscess Peritonitis Pneumonia Lung fibrosis Tracheal surgery ORL abscess ORL surgery ORL tumor Fibroma
PaO2/FIO2 [mm Hg]
LIS
178.0
1.75 38
1
91
136.0
2.5
28
0
49
116.0 206.0
2.25 23 1.25 27
1 0
– 50
242.0 95.3 103.8
1.5 46 3.25 50 3.75 21
1 0 0
9 – –
236.9
1.0
11
0
21
204.3
1.5
21
0
19
184.6
0.75 21
0
4
483.3 583.3
0.25 16 0.5 5
0 0
15 19
SAPS Sepsis [y/n]
Surgery [h]
Pat, patient; f, female; m, male; ORL, otorhinolaryngologic; PaO2, partial pressure of arterial oxygen; FIO2, fraction of inspired oxygen; LIS, lung injury severity score; SAPS II, simplified acute physiology score; surgery, time between last surgical procedure to measurement.
162
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been observed in critically ill patients by Sturney et al. and was ascribed to the effects of treatment and feeding as well to a decline in systemic inflammation and exposure to oxidative stress [27,36]. In contrast to the findings for acetone and propionaldehyde, isoprene concentrations slightly increased over time in the TX population. Due to the small number of TX patients requiring prolonged ventilation in our study, the interpretation of this observation is difficult. A possible explanation for this phenomenon could be improvements in respiratory function or increases in cardiac output [14,43] but none of these changes were seen in our patients. Distinguishing isobaric molecules using direct mass spectrometric techniques is usually challenging and requires different reagent ions together with differences in ionization characteristics of the targeted molecules. During IMR-MS chemical ionization with Hg þ at a recombination potential of 10.44 eV each, acetone and propionaldehyde present unique ionization characteristics that finally allow distinguishing them from each other. The reaction of Table 3 Characteristics of patients with lung and heart–lung transplantation (TX). Pat. Sample [No.] [day]
Age Gender TX PaO2/ FIO2 [year] [mm Hg]
LIS
SAPS II
CBP Ischemia [min] [min]
1
43 – – 41 – – 57 33 – – 47 – – 42 49
3.0 2.5 2.7 1.5 1.5 1.5 1.75 2.0 2.0 1.75 2.5 2,0 2,25 3.00 3.00
31 36 39 28 31 38 31 32 39 36 25 22 30 33 25
98 – – 314 – – – 300 – – 239 – – – –
2
3 4
5
6 7
1 2 3 1 2 3 1 1 2 3 1 2 3 1 1
f – – f – – m f – – f – – m f
SL – – HL – – DL DL – – HL – – DL DL
267 166 202 393 387 325 263 284 289 317 200 213 215 200 156
289 – – 305 – – 480 513 – – 249 – – 600 442
Pat, patient; f, female; m, male; TX, type of transplantation; SL, single lung; DL, bilateral lung; HL, heart–lung; PaO2, partial pressure of arterial oxygen; FIO2, fraction of inspired oxygen; LIS, lung injury severity score; SAPS II, simplified acute physiology score; CBP, cardiopulmonary bypass; duration of ischemia refers to the lung reperfused at last.
VOC concentration [ppbv]
105
acetone with Hg þ proceeds via charge transfer to either C3H6O þ at m/z of 58 (33%) or dissociative charge transfer to C2H3O þ at m/z 43 Hg (67%). In contrast, the reaction of propionaldehyde with Hg þ proceeds via charge transfer to C3H6O þ þ Hg at m/z 58 (88.4%) or via dissociative charge transfer to C3H5O þ at m/z 57 (8.45%). Thus, acetone product ions appear at m/z's 58 and 43 whereas at m/z 57 only the production ion of propionaldehyde appears which makes acetone and propionaldehyde distinguishable using IMR-MS chemical ionization. The constancy of these chemical ionization characteristics was observed over a concentration range of 12,000 ppbv and the effects of humidification were found to be negligible, which allows the use of propionaldehyde as a marker of LPO in exhaled breath. A major problem when carrying out breath gas analysis studies within a hospital environment and especially in intensive care units is the ever-present use of disinfectants resulting in a substantial risk for sample contamination. Sample contamination might occur either via diffusion of disinfectant ingredients into the patients ventilator tubing or, alternatively, by skin absorption. At our institution, a disinfectant solution containing 45 g 2-propanol, 10 g 1-propanol, and 0.2 g 2-biphenylole per 100 g of the solution is used for surgical skin scrubbing and topical skin disinfection. Primary alcohols as 1-propanol undergo alcohol dehydrogenase catalysed oxidation to propionaldehyde whereas secondary alcohols as 2-propanol are oxidized to acetone [44,45]. Reported elimination half-times are 45 min for 1-propanol and 150 min for 2-propanol [44,45]. Given the possible confounding effects of disinfectants on breath propionaldehyde strict control with regard to their use must be implicated in breath gas analysis. During our study the use of disinfectants was restricted within 60 min prior to measurements. A pervious study investigating the effects of surgical hand rubs which is far more intense compared to the usual topical disinfection of catheter insertion sites in intensive care units found systemic increases in the 2-propanol metabolite acetone but not for the 1-propanol metabolite propionaldehyde [46]. Thus, we assume that the observed increase in exhaled breath propionaldehyde in TX patients is related to I/R injuryinduced LPO. This appears to restrict the validity of propionaldehyde as a biomarker of LPO to periods of 60 min following disinfectant use. This delay seems to be acceptable in clinical practice, however. The limitation of our study lies in the small number of patients investigated for the presence for markers of oxidative stress in
*#
*
#
105
#
104
104
103
103
102
102
101
101
100
100
10−1
10−1
Acetone
Isoprene
Propionaldehyde
Fig. 4. Exhaled breath acetone, isoprene, and propionaldehyde in healthy volunteers (dark grey boxes, n¼ 17), mixed intensive care unit population (light grey boxes, n ¼12), and patients on day 1 after lung and heart–lung transplantation (white boxes, n¼ 7). Measurements in inspiratory air (ambient air for healthy volunteers) are hatched. With the exception of propionaldehyde in healthy volunteers, for all compounds a significant difference comparing expiratory and inspiratory measurements was present (Wilcoxon signed-rank test; Po 0.05). Intergroup differences were present for acetone, isoprene, and propionaldehyde measurements (Kruskal-Wallis rank sum test with post hoc Nemenyi test; nPo .05, #P o.001).
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exhaled breath. However, the present study was intended as a pilot study to confirm our hypothesis that propionaldehyde is detectable within the exhaled breath of TX patients who are exposed to I/R injury and oxidative stress. Further studies will have to confirm these initial results in a larger population. Furthermore, we were not able to show any relationship among I/R, primary graft failure, and secondary organ dysfunction with exhaled breath propionaldehyde levels. To answer these questions a further study within a larger population will be necessary. In summary and despite the above-noted limitations of our study, we have shown that propionaldehyde resulting from spontaneous fragmentation of peroxides originating from free radical-induced LPO after I/R injury in TX patients can be quantified in expired breath. Due to the noninvasive nature of measurements, exhaled breath propionaldehyde could possibly serve as a biomarker for the reliable estimate of LPO extent and, possibly, as an additional risk assessment parameter in patients after chest organ transplantation.
Conflict of interest and source of funding VF services GmbH provided the IMR-MS system used in this study. Johannes Villinger is senior scientist of VF Services GmbH and Siegfried Praun and Christian Leidlmair are scientists employed by this company with financial interests regarding the IMR-MS system. Michael E. Dolch and Cyrill Hornuss received traveling funds from VF Services GmbH for research presentations at the annual meeting of the American Society of Anesthesiologists. None of the other authors has received funding or other financial support from VF services GmbH; this includes contracts, equity interest, stock option(s), direct or indirect salary support, consultant fee(s), lecture fees, or honoraria received within a period of 3 years of the date of submission of this manuscript. The funding sources had no role in study design, in the collection, analysis, and interpretation of data and in the decision to submit the paper for publication.
Acknowledgments This work was supported by intramural funding of the Department of Anaesthesiology and the German Space Agency (DLR) on behalf of the Federal Ministry of Economics and Technology (BMWi 50WB0719, 50WB0919) and the European Space Agency (ESA, ELIPS 3 programme) (regarding precursor studies). References [1] McCord, J. M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159–163; 1985. [2] Granger, D. N. Role of xanthine oxidase and granulocytes in ischemiareperfusion injury. Am. J. Physiol. 255:H1269–H1275; 1988. [3] Frankel, E. N. Volatile lipid oxidation products. Prog Lipid Res. 22:1–33; 1983. [4] Brown, R. H.; Risby, T. H. Monitoring distant organ reperfusion injury by measurement of volatile organic compounds. In: Marczin, N. N., Kharitonov, S. A., Yacoub, M. H., Barnes, P. J., editors. Disease markers in exhaled breath. New York: Marcel Dekker; 2003. p. 281–304. [5] Kneepkens, C. M. F.; Lepage, G.; Roy, C. C. The potential of the hydrocarbon breath test as a measure of lipid peroxidation. Free Radic. Biol. Med. 17:127–160; 1994. [6] Phillips, M. Method for the collection and assay of volatile organic compounds in breath. Anal. Biochem. 247:272–278; 1997. [7] Phillips, M.; Cataneo, R. N.; Greenberg, J.; Grodman, R.; Gunawardena, R.; Naidu, A. Effect of oxygen on breath markers of oxidative stress. Eur. Respir. J. 21:48–51; 2003. [8] Risby, T. H.; Sehnert, S. S. Clinical application of breath biomarkers of oxidative stress status. Free Radic. Biol. Med. 27:1182–1192; 1999. [9] Kazui, M.; Andreoni, K. A.; Norris, E. J.; Klein, A. S.; Burdick, J. F.; Beattie, C.; Sehnert, S. S.; Bell, W. R.; Bulkley, G. B.; Risby, T. H. Breath ethane: a specific
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