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Propofol in bronchoalveolar lavage during anaesthesia
Clinica Chimica Acta 412 (2011) 190–193
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Propofol in bronchoalveolar lavage during anaesthesia☆,☆☆ Martin Grossherr a,⁎, Andreas Hengstenberg c, Hilke Papenberg a, Leif Dibbelt b, Barbara Wollenberg d, Peter Schmucker a, Hartmut Gehring a, Torsten Meier a a
Department of Anaesthesiology, University of Luebeck, Germany Institute of Clinical Chemistry, University of Luebeck, Germany Research Unit, Drägerwerk AG & Co. KGaA, Luebeck, Germany d ENR Department, University of Luebeck, Germany b c
a r t i c l e
i n f o
Article history: Received 22 July 2010 Received in revised form 4 October 2010 Accepted 4 October 2010 Available online 16 October 2010 Keywords: Propofol Bronchoalveolar lavage Analysis Lung protection
a b s t r a c t Background: The lung protecting effect of propofol requires methods to measure the propofol concentration of the epithelial line fluid covering the alveolar surface. We hypothesized that (1) propofol can be determined in bronchoalveolar lavage (BAL) by reversed phase high performance liquid chromatography with fluorescence detection. (2) Positive end-expiratory pressure (PEEP) ventilation may have an effect on propofol concentration in BAL (cpB). Methods: 76 surgical patients were investigated after institutional review board approval. After criteria-based exclusion 45 samples were included. For group I (n = 15) BAL was performed directly after induction, for group Z (n = 15, PEEP = 0 cm H2O) and P (n = 15, PEEP = 10 cm H2O) at the end of anaesthesia. BAL and plasma samples were analysed for propofol by reversed phase high performance liquid chromatography with fluorescence detection. Data from all groups were compared by non-parametric Mann–Whitney U-test. Results: Propofol can be detected in BAL. CpB varied between 23 and 167 μg l− 1 in all groups. Patients ventilated with PEEP (group P) showed significantly higher cpB (median 74.5 μg l− 1) compared to those immediately after induction of anaesthesia (median 42.0 μg l− 1) (group I), but not to those ventilated without PEEP in group Z (median 52.5 μg l− 1). Conclusion: Epithelial line fluid, sampled by BAL, can be used to determine cpB by reversed phase high performance liquid chromatography with fluorescence detection. Continuous propofol infusion and PEEP ventilation may have an effect on cpB. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The antioxidative property of propofol offers the opportunity for lung protection in risk patients compared to volatile anaesthetics [1–5]. Propofol is exhaled by the lungs and, compared to the time-course of its plasma concentrations, appears after a time-interval in breath gas within 41.8 s [6,7]. Recent studies demonstrated that online procedures
☆ Financial support for the work: Martin Grossherr, Hartmut Gehring, Leif Dibbelt, Barbara Wollenberg, Peter Schmucker, and Torsten Meier are employees of the University of Luebeck, Germany. Andreas Hengstenberg is an employee of Draegerwerk AG & Co. KGaA, Luebeck, Schleswig-Holstein, Germany. ☆☆ Parts of the work were presented at the 2007 annual meeting of the American Society of Anesthesiologists (ASA), October, 13–17, 2007, San Francisco, California. ⁎ Corresponding author. Department of Anaesthesiology, University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany. Tel.: +49 451 5003467; fax: +49 451 5003510. E-mail address: [email protected] (M. Grossherr). 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.10.005
using mass spectrometry and electrochemical sensors offer the chance to measure breath gas concentration directly and in real-time [8–12]. However, it is not yet known whether propofol, a volatile phenol, can be measured in the epithelial line fluid of the alveolar space during propofol anaesthesia by taking a bronchoalveolar lavage sample (BAL). It is also unclear if the determination of propofol concentration in BAL (cpB) can be used as a surrogate for the measurement of propofol in epithelial line fluid. Lastly, it is not well known how the blood gas barrier itself is influenced by the applied airway pressure during mechanical ventilation and how alveolar pressure elevation influences the passage of propofol into the alveolar compartment during anaesthesia. The samples in this study had been obtained during an earlier study where the pulmonary cytokine response in healthy lungs was investigated during mechanical ventilation [13]. We therefore investigated as a proof of principle whether cpB could be measured during anaesthesia and compared these values with propofol concentrations measured in plasma (cpPl) at the end of propofol application. Also, we analysed the influence of ventilation without a PEEP (positive end-expiratory pressure) and with a PEEP of 10 cm H2O on cpB at the end of anaesthesia.
M. Grossherr et al. / Clinica Chimica Acta 412 (2011) 190–193
level for all BALs, study patients with a positive microbiology test, a recovery rate of ≤30% or a macrophage ratio of ≤78% in the differential cell count were excluded.
2. Materials and methods 2.1. Study protocol After having obtained approval from the local ethical committee as well as written informed consent, 76 patients scheduled for elective otorhinolaryngology surgery with expected minor systemic trauma (e.g. rhinoplastic surgery and total auricular reconstruction surgery) were enrolled in a prospective, randomized controlled study in the period between February 2002 and November 2003. Patients were eligible if they were aged between 18 and 70 years, classified with ASA status I/II, had an expected surgery time N90 min and a non-smoking history (minimum N1 year). Exclusion criteria included a body mass index (BMI) N35, inflammatory disease or elevation of leukocyte counts, signs of pulmonary disease (pneumonia, COPD, etc.), malignant disease, immunomodulation or -suppression therapy (e.g. steroid medication) and a history of bronchoscopy, BAL or ventilation therapy within the preceding two months. Before induction of anaesthesia, patients were randomized into three groups: the induction group (group I), the total intravenous anaesthesia (TIVA) group ventilated without PEEP (group Z), or the TIVA group ventilated with a PEEP of 10 cm H2O (group P). After premedication, anaesthesia was induced using propofol (2 mg kg− 1 i.v., Fa. Astra Zeneca, Hamburg, Germany), sufentanil (0.5 μg kg − 1 ; Fa. Janssen, Neuss, Germany) and rocuronium (0.5 mg kg− 1, Fa. Schering-Plough, Oberschleissheim, Germany) for muscle relaxation. For maintenance of anaesthesia, propofol was continuously applied (4–6 mg kg− 1 h−1) and further sufentanil (0.2 μg kg− 1) was given as deemed necessary. Mechanical ventilation was performed in a volume controlled mode with a tidal volume (VT) of 8 ml kg− 1 ideal bodyweight, an inspiratory/ expiratory ratio (I:E) of 1:2, a fraction of inspired oxygen (FiO2) of 0.5, and a respiratory rate adjusted to maintain paCO2 between 35 and 45 mm Hg with a standard anaesthesia ventilator (Primus, Draeger Medical, Luebeck, Germany; fresh gas flow: 1 l min−1). 2.2. Bronchoalveolar lavage protocol For the study, each BAL was performed by the same investigator (T.M.). A fractionated BAL was performed immediately, which was on average 4 min after induction of anaesthesia (group I), or at the end of anaesthesia, ventilated without a PEEP (group Z) or with a PEEP of 10 cm H2O (group P). Bronchoscopy was performed via the endotracheal tube with the end of the scope wedged into the right middle lobe. Six 20-ml-aliquots of sterile saline solution were instilled and gently aspirated. The first recovered BAL-aliquot, which contained the bronchiolar fraction, was not included in the analysis. 2.3. Preparation of BAL samples for the determination of propofol, BAL cell count and differentiation The alveolar cells were separated from the BAL fluids by centrifugation (150 ×g, 10 min). The supernatant was then stored at −80 °C until further analysis. Following this procedure approximately 105 cells ml−1 were separated from the cell pellet for cell counting and Pappenheim's staining. In order to achieve a comparable quality
Table 1 Demographic data (mean ± standard deviation (SD)).
Age (year) Gender, f/m BMI (kg/m2) ASA, I/II
191
Group I (n = 15)
Group Z (n = 15)
Group P (n = 15)
35 (± 10.0) 8/7 25.2 (± 3.5) 13/2
35 (± 12.4) 7/8 24.2 (± 3.5) 12/3
39 (± 15.3) 6/9 24.2 (± 3.7) 13/2
Body mass index (BMI), ASA (American Society of Anesthesiologists).
2.4. Measured plasma propofol concentration At the end of surgery, venous blood samples were collected alongside the BAL in groups Z and P so that cpPl could be measured. 2.5. Measurement of cpB and cpPl After thawing, 200 μl plasma were mixed with a twofold volume of methanol containing the internal standard (2,4-di-t-butylphenol). Following centrifugation, propofol was determined in the supernatant (10 μl) by reversed phase high performance liquid chromatography with fluorimetric detection as described in more detail elsewhere [14]. The assay range extended from 10 to 6000 μg l− 1. Using this method, the obtained intra-assay coefficients of variation (CV) were 2.6, 0.5 and 0.7% at mean propofol concentrations of 40, 490 and 2850 μg l− 1 while the inter-assay CV values were 8.7 and 4.5% at mean concentrations of 50 and 1000 μg l− 1, respectively; the mean recovery of propofol added to blank plasma samples ranged between 91 and 115%. In order to quantify propofol in the BAL samples, the method described above was slightly modified: 200 μl BAL fluid was mixed with 50 μl methanol containing the internal standard before the mixture was centrifuged, and 50 μl was injected for chromatographic analysis as cited above. The lower limit of detection for this modified method was 2 μg l− 1, while the linear range extended to 1500 μg l− 1. At mean propofol concentrations of 48.9 and 982 μg l− 1 the intra-assay CV values were 2.9 and 3.1% and the inter-assay CV values were 3.6 and 5.6%, respectively; recovery of added propofol ranged between 97 and 105%. 2.6. Statistical analysis The non-parametric Mann–Whitney U-test was used to compare the data of the three groups. Data are presented as median and as 25 and 75% quartiles. Wherever a significant difference between groups is stated, the corresponding test statistic produced a p-value of less than 5%. For each sampling point for cpB and cpPl, measured values were compared by regression analysis. 3. Results Samples of 45 patients were included for the evaluation of propofol concentration (group I, n = 15; group Z, n = 15; group P, n = 15), because of the exclusion criteria and limited available reserves. Table 1 presents the demographic data of the patients. There were no significant differences in the distribution of age, gender, body mass index and ASA status between the randomized patients. The BAL recovery rates were comparable for group I with 70.5 (56/75) ml, group Z with 75 (69/80) ml and group P with 73 (61/77) ml, here presented as median and 25%/75% quartiles. Between groups Z and P no differences in the length of the anaesthesia were observed (group Z: median 147.5 min, range 90–320 min; group P: median 155.2 min, range 95–252 min). Propofol was detected in every BAL and serum sample, while for group P one blood sample could not be analysed for technical reasons (Table 2).
Table 2 Propofol concentration in plasma at the end of anaesthesia.
CpPl [μg l− 1]
Group Z (n = 15)
Group P (n = 14)
3000 (2800; 4200)
3500 (3100; 3600)
CpPl: propofol concentration in plasma. Data are presented as medians (25%; 75% quartiles). The difference of CpPl between groups Z and P was not statistically significant.
192
M. Grossherr et al. / Clinica Chimica Acta 412 (2011) 190–193 Table 3 Correlation between propofol concentration in BAL (cpB) and plasma (cpPl) for the groups Z and P.
Z: cpB/cpPl P: cpB/cpPl
n
r
p
15 14
−0.157 0.379
0.576 0.182
r: correlation coefficient.
Fig. 1. Propofol concentration in bronchoalveolar lavage performed immediately after intubation (group I, n = 15) and at the end of anaesthesia without PEEP ventilation (group Z, PEEP = 0 cm H2O, n = 15) or with PEEP ventilation (group P, PEEP = 10 cm H2O, n = 15). Differences between groups I and P are demonstrated (*: p = 0.03).
CpB for all three groups ranged between 23 and 167 μg l− 1. At the end of anaesthesia, patients ventilated with PEEP (group P) showed significantly higher cpB (median 74.5 μg l− 1) compared to those measured immediately after induction of anaesthesia (median 42.0 μg l− 1) (group I) (p: 0.03). At the end of anaesthesia, patients in group Z (median 52.5 μg l− 1) however showed no difference compared to groups I (p: 0.14) and P (p: 0.16) (Fig. 1). Only a poor correlation between the cpB and measured cpPl could be observed (Table 3).
4. Discussion In the presented study, the cpB was successfully determined by reversed phase high performance liquid chromatography. CpB ranged between 23 and 167 μg l− 1. Compared to the corresponding cpPls, cpBs were lower by a factor of 50. Because of different dilution factors in the BAL, the real concentration in the epithelial line fluid of the alveolus is probably higher. Our results demonstrate that propofol can be measured in the BAL with the same method for the analysis of propofol concentration in plasma. A significantly increased cpB was found at the end of anaesthesia with PEEP ventilation compared to the time point immediately after induction. For ventilation without PEEP during anaesthesia (group Z), no difference was found for cpB compared to alveolar propofol concentration after induction of anaesthesia (group I) or at the end of surgery in patients ventilated with PEEP (group P). CpPl could not be determined after induction in this investigation. Nevertheless, the timeeffect of ongoing propofol infusion on propofol concentration in the lung tissue itself should be investigated in further studies. For group P a higher variability in the cpB was found. It can be assumed that PEEP ventilation increases the proportion of open, nonatelectatic lung tissue to a different extent [15–17]. The individual lung mechanics of the patient lead to an individual response to the PEEP ventilation and therefore more lung tissue can be reached by BAL fluids. So, in some patients a higher cpB can be detected. Consequently, in the whole study group ventilated with PEEP a wider range of cpB can be identified. This may also explain that Meier et al. observed an increased mediator level with a wider range under PEEP ventilation [13]. The correlation between cpB and measured cpPl is rather low due to the diluting effect of the BAL (Table 2). Besides the distribution and redistribution of propofol between plasma and lung tissue [18–22] and the exhalation of propofol [6–12,23] the lung protective effect of propofol has been discussed because of the possible antioxidative capacity [1,2].
Antioxidative properties of propofol can be used for lung protection. Chen et al. described diminished or reversed effects of oleate-induced acute lung injury by pre- or post-treatment with high doses of propofol (30 mg kg− 1) under experimental conditions [1]. These pathological changes depressed Na+-K+-ATPase activity and upregulated inducible nitric oxide synthetase. Allaouchiche et al. observed decreased oxidative markers in plasma and BAL under anaesthesia with propofol, thus revealing a further effect of its oxidative capacity [2]. A quantification of propofol in epithelial line fluid as shown in our study might confirm the existence of antioxidative propofol in the whole lung tissue since it completely traverses the blood gas barrier [3,4]. However, only standard doses were used in our study to induce anaesthesia, and these were only one tenth of the doses applied in the latter experimental study by Allaouchiche et al. In a recent investigation Abou-Elenain et al. demonstrated the antioxidative properties of propofol in comparison to the volatile anaesthetic sevoflurane and asked for more methods to monitor the antioxidative status [5]. Therefore, the measurement of local propofol in the BAL is a further step to verify sufficient propofol concentration in the lung itself. Further studies are required to investigate the effects of various doses on propofol concentrations in BAL. Limitations of the present study were the technical factors and errors in the BAL procedure which may have generated differences in the composition of the epithelial line fluid, which is influenced by various pulmonary physicochemical factors [16]. These factors were not controlled in this study. Also, because of the study protocol employed, no blood samples were available after the induction of anaesthesia. The invasive nature of BAL sampling demanded the evaluation of surplus samples by new methods so that their usefulness could be tested as we did here. In summary, propofol concentrations in BAL can be determined by a modified reversed phase high performance liquid chromatography. Furthermore, differences in applied airway pressure during mechanical ventilation may have an effect on propofol concentration in the epithelial line fluid. Acknowledgement The authors thank Ellen Spies, Technician, Institute of Clinical Chemistry, University of Luebeck, Schleswig-Holstein, Germany for technical support. References [1] Chen HI, Hsieh NK, Kao SJ, Su CF. Protective effects of propofol on acute lung injury induced by oleic acid in conscious rats. Crit Care Med 2008;36:1214–21. [2] Allaouchiche B, Debon R, Goudable J, Chassard D, Duflo F. Oxidative stress status during exposure to propofol, sevoflurane and desflurane. Anesth Analg 2001;93: 981–5. [3] Haitsma JJ, Lachmann B, Papadakos PJ. Additives in intravenous anesthesia modulate pulmonary inflammation in a model of LPS-induced respiratory distress. Acta Anaesthesiol Scand 2009;53:176–82. [4] Chu CH, David Liu D, Hsu YH, Lee KC, Chen HI. Propofol exerts protective effects on the acute lung injury induced by endotoxin in rats. Pulm Pharmacol Ther 2007;20:503–12. [5] Abou-Elenain K. Study of the systemic and pulmonary oxidative stress status during exposure to propofol and sevoflurane anaesthesia during thoracic surgery. Eur J Anaesthesiol 2010;27:566–71. [6] Takita A, Masui K, Kazama T. On-line monitoring of end-tidal propofol concentration in anesthetized patients. Anesthesiology 2007;106:659–64. [7] Grossherr M, Hengstenberg A, Meier T, et al. Propofol concentration in exhaled air and arterial plasma in mechanically ventilated patients undergoing cardiac surgery. Br J Anaesth 2009;102:608–13.
M. Grossherr et al. / Clinica Chimica Acta 412 (2011) 190–193 [8] Harrison GR, Critchley AD, Mayhew CA, Thompson JM. Real-time breath monitoring of propofol and its volatile metabolites during surgery using a novel mass spectrometric technique: a feasibility study. Br J Anaesth 2003;91:797–9. [9] Grossherr M, Hengstenberg A, Meier T, Dibbelt L, Gerlach K, Gehring H. Discontinuous monitoring of propofol concentrations in expired alveolar gas and in arterial and venous plasma during artificial ventilation. Anesthesiology 2006;104:786–9. [10] Miekisch W, Fuchs P, Kamysek S, Neumann C, Schubert JK. Assessment of propofol concentrations in human breath and blood by means of HS-SPME–GC–MS. Clin Chim Acta 2008;395:32–7. [11] Hornuss C, Praun S, Villinger J, et al. Real-time monitoring of propofol in expired air in humans undergoing total intravenous anesthesia. Anesthesiology 2007;106:665–74. [12] Hengstenberg A, Grossherr M, Meier T, Dibbelt L, Gehring H. Continuous real-time monitoring of propofol in breathing gas during anesthesia. Anesthesiology 2006;A 577. [13] Meier T, Lange A, Rose H, et al. Pulmonary cytokine responses during mechanical ventilation of non-injured lungs with and without end-expiratory pressure. Anesth Analg 2008;107:1265–75. [14] Grossherr M, Spies E, Scheel A, Hengstenberg A, Gehring H, Dibbelt L. Differences of propofol concentration in mammalian whole blood and in corresponding plasma samples analyzed by high performance liquid chromatography. Clin Lab 2007;53: 315–9.
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[15] Reinius H, Jonsson L, Gustafsson S, et al. Prevention of atelectasis in morbidly obese patients during general anesthesia and paralysis: a computerized tomography study. Anesthesiology 2009;111:979–87. [16] Hedenstierna G, Edmark L. The effects of anaesthesia and muscle paralysis on the respiratory system. Intensiv Care Med 2005;31:1327–35. [17] Terragni PP, Rosboch G, Tealdi A, et al. Tidal hyperinflation durino low tidal volume ventilation in acute respiratory distress syndrome. Am J Resp Crit Care Med 2007;175:160–6. [18] Kuipers JA, Boer F, Olieman W, Burm AG, Bovill JG. First-pass lung uptake and pulmonary clearance of propofol: assessment with a recirculatory indocyanine green pharmacokinetic model. Anesthesiology 1999;91:1780–7. [19] Boer F. Drug handling by the lungs. Br J Anaesth 2003;91:50–60. [20] Kurita T, Morita K, Kazama T, Sato S. Influence of cardiac output on plasma propofol concentrations during constant infusion in swine. Anesthesiology 2002;96: 1498–503. [21] Matot I, Neely CF, Katz RY, Neufeld GR. Pulmonary uptake of propofol in cats. Effect of fentanyl and halothane. Anesthesiology 1993;78:1157–65. [22] Matot I, Neely CF, Katz RY, Marshall BE. Fentanyl and propofol uptake by the lung: effect of time between injections. Acta Anaesthesiol Scand 1994;38:711–5. [23] Grossherr M, Hengstenberg A, Dibbelt L, et al. Blood gas partition coefficient and pulmonary extraction ratio for propofol in goats and pigs. Xenobiotica 2009;39: 782–7.
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Propofol in bronchoalveolar lavage during anaesthesia☆,☆☆ Martin Grossherr a,⁎, Andreas Hengstenberg c, Hilke Papenberg a, Leif Dibbelt b, Barbara Wollenberg d, Peter Schmucker a, Hartmut Gehring a, Torsten Meier a a
Department of Anaesthesiology, University of Luebeck, Germany Institute of Clinical Chemistry, University of Luebeck, Germany Research Unit, Drägerwerk AG & Co. KGaA, Luebeck, Germany d ENR Department, University of Luebeck, Germany b c
a r t i c l e
i n f o
Article history: Received 22 July 2010 Received in revised form 4 October 2010 Accepted 4 October 2010 Available online 16 October 2010 Keywords: Propofol Bronchoalveolar lavage Analysis Lung protection
a b s t r a c t Background: The lung protecting effect of propofol requires methods to measure the propofol concentration of the epithelial line fluid covering the alveolar surface. We hypothesized that (1) propofol can be determined in bronchoalveolar lavage (BAL) by reversed phase high performance liquid chromatography with fluorescence detection. (2) Positive end-expiratory pressure (PEEP) ventilation may have an effect on propofol concentration in BAL (cpB). Methods: 76 surgical patients were investigated after institutional review board approval. After criteria-based exclusion 45 samples were included. For group I (n = 15) BAL was performed directly after induction, for group Z (n = 15, PEEP = 0 cm H2O) and P (n = 15, PEEP = 10 cm H2O) at the end of anaesthesia. BAL and plasma samples were analysed for propofol by reversed phase high performance liquid chromatography with fluorescence detection. Data from all groups were compared by non-parametric Mann–Whitney U-test. Results: Propofol can be detected in BAL. CpB varied between 23 and 167 μg l− 1 in all groups. Patients ventilated with PEEP (group P) showed significantly higher cpB (median 74.5 μg l− 1) compared to those immediately after induction of anaesthesia (median 42.0 μg l− 1) (group I), but not to those ventilated without PEEP in group Z (median 52.5 μg l− 1). Conclusion: Epithelial line fluid, sampled by BAL, can be used to determine cpB by reversed phase high performance liquid chromatography with fluorescence detection. Continuous propofol infusion and PEEP ventilation may have an effect on cpB. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The antioxidative property of propofol offers the opportunity for lung protection in risk patients compared to volatile anaesthetics [1–5]. Propofol is exhaled by the lungs and, compared to the time-course of its plasma concentrations, appears after a time-interval in breath gas within 41.8 s [6,7]. Recent studies demonstrated that online procedures
☆ Financial support for the work: Martin Grossherr, Hartmut Gehring, Leif Dibbelt, Barbara Wollenberg, Peter Schmucker, and Torsten Meier are employees of the University of Luebeck, Germany. Andreas Hengstenberg is an employee of Draegerwerk AG & Co. KGaA, Luebeck, Schleswig-Holstein, Germany. ☆☆ Parts of the work were presented at the 2007 annual meeting of the American Society of Anesthesiologists (ASA), October, 13–17, 2007, San Francisco, California. ⁎ Corresponding author. Department of Anaesthesiology, University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany. Tel.: +49 451 5003467; fax: +49 451 5003510. E-mail address: [email protected] (M. Grossherr). 0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.10.005
using mass spectrometry and electrochemical sensors offer the chance to measure breath gas concentration directly and in real-time [8–12]. However, it is not yet known whether propofol, a volatile phenol, can be measured in the epithelial line fluid of the alveolar space during propofol anaesthesia by taking a bronchoalveolar lavage sample (BAL). It is also unclear if the determination of propofol concentration in BAL (cpB) can be used as a surrogate for the measurement of propofol in epithelial line fluid. Lastly, it is not well known how the blood gas barrier itself is influenced by the applied airway pressure during mechanical ventilation and how alveolar pressure elevation influences the passage of propofol into the alveolar compartment during anaesthesia. The samples in this study had been obtained during an earlier study where the pulmonary cytokine response in healthy lungs was investigated during mechanical ventilation [13]. We therefore investigated as a proof of principle whether cpB could be measured during anaesthesia and compared these values with propofol concentrations measured in plasma (cpPl) at the end of propofol application. Also, we analysed the influence of ventilation without a PEEP (positive end-expiratory pressure) and with a PEEP of 10 cm H2O on cpB at the end of anaesthesia.
M. Grossherr et al. / Clinica Chimica Acta 412 (2011) 190–193
level for all BALs, study patients with a positive microbiology test, a recovery rate of ≤30% or a macrophage ratio of ≤78% in the differential cell count were excluded.
2. Materials and methods 2.1. Study protocol After having obtained approval from the local ethical committee as well as written informed consent, 76 patients scheduled for elective otorhinolaryngology surgery with expected minor systemic trauma (e.g. rhinoplastic surgery and total auricular reconstruction surgery) were enrolled in a prospective, randomized controlled study in the period between February 2002 and November 2003. Patients were eligible if they were aged between 18 and 70 years, classified with ASA status I/II, had an expected surgery time N90 min and a non-smoking history (minimum N1 year). Exclusion criteria included a body mass index (BMI) N35, inflammatory disease or elevation of leukocyte counts, signs of pulmonary disease (pneumonia, COPD, etc.), malignant disease, immunomodulation or -suppression therapy (e.g. steroid medication) and a history of bronchoscopy, BAL or ventilation therapy within the preceding two months. Before induction of anaesthesia, patients were randomized into three groups: the induction group (group I), the total intravenous anaesthesia (TIVA) group ventilated without PEEP (group Z), or the TIVA group ventilated with a PEEP of 10 cm H2O (group P). After premedication, anaesthesia was induced using propofol (2 mg kg− 1 i.v., Fa. Astra Zeneca, Hamburg, Germany), sufentanil (0.5 μg kg − 1 ; Fa. Janssen, Neuss, Germany) and rocuronium (0.5 mg kg− 1, Fa. Schering-Plough, Oberschleissheim, Germany) for muscle relaxation. For maintenance of anaesthesia, propofol was continuously applied (4–6 mg kg− 1 h−1) and further sufentanil (0.2 μg kg− 1) was given as deemed necessary. Mechanical ventilation was performed in a volume controlled mode with a tidal volume (VT) of 8 ml kg− 1 ideal bodyweight, an inspiratory/ expiratory ratio (I:E) of 1:2, a fraction of inspired oxygen (FiO2) of 0.5, and a respiratory rate adjusted to maintain paCO2 between 35 and 45 mm Hg with a standard anaesthesia ventilator (Primus, Draeger Medical, Luebeck, Germany; fresh gas flow: 1 l min−1). 2.2. Bronchoalveolar lavage protocol For the study, each BAL was performed by the same investigator (T.M.). A fractionated BAL was performed immediately, which was on average 4 min after induction of anaesthesia (group I), or at the end of anaesthesia, ventilated without a PEEP (group Z) or with a PEEP of 10 cm H2O (group P). Bronchoscopy was performed via the endotracheal tube with the end of the scope wedged into the right middle lobe. Six 20-ml-aliquots of sterile saline solution were instilled and gently aspirated. The first recovered BAL-aliquot, which contained the bronchiolar fraction, was not included in the analysis. 2.3. Preparation of BAL samples for the determination of propofol, BAL cell count and differentiation The alveolar cells were separated from the BAL fluids by centrifugation (150 ×g, 10 min). The supernatant was then stored at −80 °C until further analysis. Following this procedure approximately 105 cells ml−1 were separated from the cell pellet for cell counting and Pappenheim's staining. In order to achieve a comparable quality
Table 1 Demographic data (mean ± standard deviation (SD)).
Age (year) Gender, f/m BMI (kg/m2) ASA, I/II
191
Group I (n = 15)
Group Z (n = 15)
Group P (n = 15)
35 (± 10.0) 8/7 25.2 (± 3.5) 13/2
35 (± 12.4) 7/8 24.2 (± 3.5) 12/3
39 (± 15.3) 6/9 24.2 (± 3.7) 13/2
Body mass index (BMI), ASA (American Society of Anesthesiologists).
2.4. Measured plasma propofol concentration At the end of surgery, venous blood samples were collected alongside the BAL in groups Z and P so that cpPl could be measured. 2.5. Measurement of cpB and cpPl After thawing, 200 μl plasma were mixed with a twofold volume of methanol containing the internal standard (2,4-di-t-butylphenol). Following centrifugation, propofol was determined in the supernatant (10 μl) by reversed phase high performance liquid chromatography with fluorimetric detection as described in more detail elsewhere [14]. The assay range extended from 10 to 6000 μg l− 1. Using this method, the obtained intra-assay coefficients of variation (CV) were 2.6, 0.5 and 0.7% at mean propofol concentrations of 40, 490 and 2850 μg l− 1 while the inter-assay CV values were 8.7 and 4.5% at mean concentrations of 50 and 1000 μg l− 1, respectively; the mean recovery of propofol added to blank plasma samples ranged between 91 and 115%. In order to quantify propofol in the BAL samples, the method described above was slightly modified: 200 μl BAL fluid was mixed with 50 μl methanol containing the internal standard before the mixture was centrifuged, and 50 μl was injected for chromatographic analysis as cited above. The lower limit of detection for this modified method was 2 μg l− 1, while the linear range extended to 1500 μg l− 1. At mean propofol concentrations of 48.9 and 982 μg l− 1 the intra-assay CV values were 2.9 and 3.1% and the inter-assay CV values were 3.6 and 5.6%, respectively; recovery of added propofol ranged between 97 and 105%. 2.6. Statistical analysis The non-parametric Mann–Whitney U-test was used to compare the data of the three groups. Data are presented as median and as 25 and 75% quartiles. Wherever a significant difference between groups is stated, the corresponding test statistic produced a p-value of less than 5%. For each sampling point for cpB and cpPl, measured values were compared by regression analysis. 3. Results Samples of 45 patients were included for the evaluation of propofol concentration (group I, n = 15; group Z, n = 15; group P, n = 15), because of the exclusion criteria and limited available reserves. Table 1 presents the demographic data of the patients. There were no significant differences in the distribution of age, gender, body mass index and ASA status between the randomized patients. The BAL recovery rates were comparable for group I with 70.5 (56/75) ml, group Z with 75 (69/80) ml and group P with 73 (61/77) ml, here presented as median and 25%/75% quartiles. Between groups Z and P no differences in the length of the anaesthesia were observed (group Z: median 147.5 min, range 90–320 min; group P: median 155.2 min, range 95–252 min). Propofol was detected in every BAL and serum sample, while for group P one blood sample could not be analysed for technical reasons (Table 2).
Table 2 Propofol concentration in plasma at the end of anaesthesia.
CpPl [μg l− 1]
Group Z (n = 15)
Group P (n = 14)
3000 (2800; 4200)
3500 (3100; 3600)
CpPl: propofol concentration in plasma. Data are presented as medians (25%; 75% quartiles). The difference of CpPl between groups Z and P was not statistically significant.
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M. Grossherr et al. / Clinica Chimica Acta 412 (2011) 190–193 Table 3 Correlation between propofol concentration in BAL (cpB) and plasma (cpPl) for the groups Z and P.
Z: cpB/cpPl P: cpB/cpPl
n
r
p
15 14
−0.157 0.379
0.576 0.182
r: correlation coefficient.
Fig. 1. Propofol concentration in bronchoalveolar lavage performed immediately after intubation (group I, n = 15) and at the end of anaesthesia without PEEP ventilation (group Z, PEEP = 0 cm H2O, n = 15) or with PEEP ventilation (group P, PEEP = 10 cm H2O, n = 15). Differences between groups I and P are demonstrated (*: p = 0.03).
CpB for all three groups ranged between 23 and 167 μg l− 1. At the end of anaesthesia, patients ventilated with PEEP (group P) showed significantly higher cpB (median 74.5 μg l− 1) compared to those measured immediately after induction of anaesthesia (median 42.0 μg l− 1) (group I) (p: 0.03). At the end of anaesthesia, patients in group Z (median 52.5 μg l− 1) however showed no difference compared to groups I (p: 0.14) and P (p: 0.16) (Fig. 1). Only a poor correlation between the cpB and measured cpPl could be observed (Table 3).
4. Discussion In the presented study, the cpB was successfully determined by reversed phase high performance liquid chromatography. CpB ranged between 23 and 167 μg l− 1. Compared to the corresponding cpPls, cpBs were lower by a factor of 50. Because of different dilution factors in the BAL, the real concentration in the epithelial line fluid of the alveolus is probably higher. Our results demonstrate that propofol can be measured in the BAL with the same method for the analysis of propofol concentration in plasma. A significantly increased cpB was found at the end of anaesthesia with PEEP ventilation compared to the time point immediately after induction. For ventilation without PEEP during anaesthesia (group Z), no difference was found for cpB compared to alveolar propofol concentration after induction of anaesthesia (group I) or at the end of surgery in patients ventilated with PEEP (group P). CpPl could not be determined after induction in this investigation. Nevertheless, the timeeffect of ongoing propofol infusion on propofol concentration in the lung tissue itself should be investigated in further studies. For group P a higher variability in the cpB was found. It can be assumed that PEEP ventilation increases the proportion of open, nonatelectatic lung tissue to a different extent [15–17]. The individual lung mechanics of the patient lead to an individual response to the PEEP ventilation and therefore more lung tissue can be reached by BAL fluids. So, in some patients a higher cpB can be detected. Consequently, in the whole study group ventilated with PEEP a wider range of cpB can be identified. This may also explain that Meier et al. observed an increased mediator level with a wider range under PEEP ventilation [13]. The correlation between cpB and measured cpPl is rather low due to the diluting effect of the BAL (Table 2). Besides the distribution and redistribution of propofol between plasma and lung tissue [18–22] and the exhalation of propofol [6–12,23] the lung protective effect of propofol has been discussed because of the possible antioxidative capacity [1,2].
Antioxidative properties of propofol can be used for lung protection. Chen et al. described diminished or reversed effects of oleate-induced acute lung injury by pre- or post-treatment with high doses of propofol (30 mg kg− 1) under experimental conditions [1]. These pathological changes depressed Na+-K+-ATPase activity and upregulated inducible nitric oxide synthetase. Allaouchiche et al. observed decreased oxidative markers in plasma and BAL under anaesthesia with propofol, thus revealing a further effect of its oxidative capacity [2]. A quantification of propofol in epithelial line fluid as shown in our study might confirm the existence of antioxidative propofol in the whole lung tissue since it completely traverses the blood gas barrier [3,4]. However, only standard doses were used in our study to induce anaesthesia, and these were only one tenth of the doses applied in the latter experimental study by Allaouchiche et al. In a recent investigation Abou-Elenain et al. demonstrated the antioxidative properties of propofol in comparison to the volatile anaesthetic sevoflurane and asked for more methods to monitor the antioxidative status [5]. Therefore, the measurement of local propofol in the BAL is a further step to verify sufficient propofol concentration in the lung itself. Further studies are required to investigate the effects of various doses on propofol concentrations in BAL. Limitations of the present study were the technical factors and errors in the BAL procedure which may have generated differences in the composition of the epithelial line fluid, which is influenced by various pulmonary physicochemical factors [16]. These factors were not controlled in this study. Also, because of the study protocol employed, no blood samples were available after the induction of anaesthesia. The invasive nature of BAL sampling demanded the evaluation of surplus samples by new methods so that their usefulness could be tested as we did here. In summary, propofol concentrations in BAL can be determined by a modified reversed phase high performance liquid chromatography. Furthermore, differences in applied airway pressure during mechanical ventilation may have an effect on propofol concentration in the epithelial line fluid. Acknowledgement The authors thank Ellen Spies, Technician, Institute of Clinical Chemistry, University of Luebeck, Schleswig-Holstein, Germany for technical support. References [1] Chen HI, Hsieh NK, Kao SJ, Su CF. Protective effects of propofol on acute lung injury induced by oleic acid in conscious rats. Crit Care Med 2008;36:1214–21. [2] Allaouchiche B, Debon R, Goudable J, Chassard D, Duflo F. Oxidative stress status during exposure to propofol, sevoflurane and desflurane. Anesth Analg 2001;93: 981–5. [3] Haitsma JJ, Lachmann B, Papadakos PJ. Additives in intravenous anesthesia modulate pulmonary inflammation in a model of LPS-induced respiratory distress. Acta Anaesthesiol Scand 2009;53:176–82. [4] Chu CH, David Liu D, Hsu YH, Lee KC, Chen HI. Propofol exerts protective effects on the acute lung injury induced by endotoxin in rats. Pulm Pharmacol Ther 2007;20:503–12. [5] Abou-Elenain K. Study of the systemic and pulmonary oxidative stress status during exposure to propofol and sevoflurane anaesthesia during thoracic surgery. Eur J Anaesthesiol 2010;27:566–71. [6] Takita A, Masui K, Kazama T. On-line monitoring of end-tidal propofol concentration in anesthetized patients. Anesthesiology 2007;106:659–64. [7] Grossherr M, Hengstenberg A, Meier T, et al. Propofol concentration in exhaled air and arterial plasma in mechanically ventilated patients undergoing cardiac surgery. Br J Anaesth 2009;102:608–13.
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