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Detection of pulmonary amylase activity in exhaled breath condensate
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2013 J. Breath Res. 7 046007 (http://iopscience.iop.org/1752-7163/7/4/046007) View the table of contents for this issue, or go to the journal homepage for more
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IOP PUBLISHING
JOURNAL OF BREATH RESEARCH
doi:10.1088/1752-7155/7/4/046007
J. Breath Res. 7 (2013) 046007 (8pp)
Detection of pulmonary amylase activity in exhaled breath condensate M Zweifel, T Rechsteiner, M Hofer and A Boehler Division of Pulmonology, University Hospital Z¨urich, R¨amistrasse 100, 8091 Z¨urich, Switzerland E-mail: [email protected]
Received 7 March 2013 Accepted for publication 25 September 2013 Published 28 November 2013 Online at stacks.iop.org/JBR/7/046007 Abstract Amylase activity in exhaled breath condensate (EBC) is usually interpreted as an indication of oropharyngeal contamination despite the fact that amylase can be found in pulmonary excretions. The aim of this study was to recruit and refine an amylase assay in order to detect amylase activity in any EBC sample and to develop a method to identify EBC samples containing amylase of pulmonary origin. EBC was collected from 40 volunteers with an EcoScreen condenser. Amylase assays and methods to discriminate between oropharyngeal and pulmonary proteins were tested and developed using matched EBC and saliva samples. Our refined 2-chloro-4-nitrophenyl-α-D-maltotriosid (CNP-G3) assay was 40-fold more sensitive than the most sensitive commercial assay and allowed detection of amylase activity in 30 μl of EBC. We developed a dot-blot assay which allowed detection of salivary protein in saliva diluted up to 150 000-fold. By plotting amylase activity against staining intensity we identified a few EBC samples with high amylase activity which were aligned with diluted saliva. We believe that EBC samples aligned with diluted saliva contain amylase activity introduced during EBC collection and that all other EBC samples contain amylase activity of pulmonary origin and are basically free of oropharyngeal protein contamination. (Some figures may appear in colour only in the online journal)
1. Introduction
However, the majority of investigators believe saliva contamination to be a low priority problem which is taken care of by a saliva trap or the design of the EBC collection device. This latter view is also held by the 2005 ATS/ERS task force on EBC, even though ‘more sensitive assays to exclude oropharyngeal contamination’ were put on the wish list of future research (Horv´ath et al 2005). Commercial amylase activity assays based on the cleavage of the chromogenic substrate ethylidene-blocked 4-nitrophenylmaltoheptaoside (EPS) which are routinely used to measure amylase activity in serum or urine allow detection of amylase activity in saliva diluted up to 30 000-fold as inferred from published data (Husz´ar et al 2002, Rohleder et al 2004). Since the average total protein concentration is about 200-fold higher in saliva than in EBC, the lowest salivary protein contamination of EBC detectable with EPS-assays lies at around 0.7% of EBC total protein. This lower limit of detection (LLD) is confirmed by two reports presenting values of EBC amylase activity measured with the EPS-method (Husz´ar et al 2002, Effros et al 2002). From the two reports,
Exhaled breath condensate (EBC) contains a plethora of compounds which in their composition and abundance depend on the pulmonary and systemic state of health. So far, primarily low molecular weight analytes, such as inorganic ions, nitrogen oxides, ammonia, nucleosides, arachidonic acid derivatives, and reactive oxygen induced compounds were in the focus of clinical investigation because their analysis requires only minor adjustment of existing laboratory methods. In contrast, EBC proteins can usually only be quantitated in concentrated form and after methodological refinement. Furthermore, contaminating proteins from the oral cavity can complicate drawing meaningful conclusions from EBC protein analysis (Davis et al 2012). To exclude contamination with oropharyngeal constituents some researchers test their EBC samples for activity of the most abundant salivary protein, salivary α-amylase, usually to observe that the samples do not contain amylase at a level ‘measurable with the assay used’ (Gessner et al 2005). 1752-7155/13/046007+08$33.00
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J. Breath Res. 7 (2013) 046007
M Zweifel et al
relative EBC salivary protein contaminations amounting to 1.6% (Husz´ar et al 2002) or 1.7% (Effros et al 2002) can be calculated. However, according to both reports amylase activity was undetectable in part of the samples, which can be understood as absence of amylase in these samples but also must be expected for statistical reasons as the values are close to the LLD of the applied method. To further complicate matters, amylase as well as other salivary proteins can also be found in the respiratory tract (Schenkels et al 1995, Griese et al 2002). Amylase activity detected in tracheobronchial secretions can originate in biosynthesis by pulmonary tissue (Hayashi et al 1986), aspiration of saliva (Clarke et al 1981, Nandapalan et al 1995, Weiss et al 2013), or aspiration of gastric fluid (Kosanam et al 2012). Therefore, demonstrating amylase activity in EBC not necessarily indicates salivary contamination. In this study we (i) compare three common amylase activity assays and increase the sensitivity of the most sensitive method to allow detection of amylase activity in any EBC sample, (ii) search for methods to identify EBC samples containing traces of saliva without measuring amylase activity, and (iii) present a procedure to identify EBC samples containing amylase activity of pulmonary origin.
of 2-chloro-4-nitrophenyl-α-D-maltotriosid (CNP-G3) by amylase (Winn-Deen et al 1988, Foo and Bais 1998). 2.3.1. Iodine-starch method. 48 μl of 0.17% potato starch (order no. S2004, Sigma-Aldrich, St. Louis MO, USA) in 83.3 mM sodium phosphate buffer pH 7 was added to 32 μl of sample prepared in the wells of a microplate. After loading, the plate was sealed and incubated at 50◦ while shacking at 1000 RPM. Condensate forming on the sealing foil was collected by centrifugation every 30 min. After 90 min the reaction was stopped by addition of 20 μl 1M HCl and 100 μl iodine reagent (5 mM I2, 5 mM KI) was added. 100 μl of the reaction mixes were transferred to a new microplate and the absorbance was measured at 595 nm. 2.3.2. Blue-starch method. 100 μl of a 20% (w/v) suspension of Remazol brilliant blue R dyed starch (order no. 85641, Sigma-Aldrich) in a buffer containing 20 mM sodium phosphate buffer pH 7 and 50 mM NaCl was added to 50 μl sample prepared in the wells of microplate. After loading, the plate was sealed and incubated at 45 ◦ C while shaking at 1000 RPM. Condensate was collected by centrifugation every 30 min. After 90 min the reaction was stopped by addition of 50 μl 0.1M acetic acid. Insoluble starch was pelleted by centrifugation at 3000 RPM for 15 min. 150 μl supernatant was transferred to a fresh microplate and the absorbance was measured at 595 nm.
2. Materials and methods 2.1. EBC and saliva collection EBC was collected with an EcoScreen I condenser during exactly 10 min from 40 healthy volunteers, who were instructed to wear a nose clip and to breathe freely while EBC was collected. The volume of inhaled ambient air was monitored with a flow meter. All EBC samples were frozen immediately after collection and stored at −60 ◦ C. Saliva was collected from ten volunteers by passive drooling immediately after collection of EBC and frozen at −60 ◦ C.
2.3.3. CNP-G3 method. The reaction mix contained 2.25 mM CNP-G3 (order no. 93834, Sigma-Aldrich), 300 mM NaCl, 5 mM CaCl2, 0.9M KSCN, 50 mM MES buffer pH 6, and 100 μg ml−1 BSA. The reaction was started by adding 50 μl 2x concentrated or 70 μl 1.43x concentrated reaction mix to 50 μl or 30 μl of sample prepared in the wells of a microplate. The plate was centrifuged for 5 s, shaken for 10 s at 1000 RPM and immediately read at 415 nm. After exactly 30, 60, 120, 240, and 480 min incubation at room temperature (22 ◦ C) the absorbance at 415 nm was measured again. Standards consisted of 1x reaction buffer (without CNPG3) and 0, 5, 10, 20, 40, 80, 160, 320, and 640 μM 2-chloro4-nitrophenol (order no. C61208, Sigma-Aldrich), the colored cleavage product of CNP-G3, and were always set up in the same microplate as the samples.
2.2. Protein concentration assay Total protein concentration was determined with the bicinchoninic acid method (Smith et al 1985). 200 μl of EBC, diluted saliva, or bovine serum albumin (BSA) standard solutions were mixed with 200 μl Micro-BCA reagent solution (order no. 23235, Thermo Fisher Scientific, Waltham MA, USA) in 2 ml polypropylene tubes. The tubes were incubated at 60 ◦ C for 60 min. 350 μl aliquots were transferred to a 96well microplate and the absorbance was measured at 550 nm in a microplate reader (Model 550, BioRad, Hercules CA, USA).
2.4. Electrophoretic assay Electrophoretic runs of EBC and saliva samples were performed on an Agilent BioAnalyzer 2100 microfluidic platform with High Sensitivity 250 Protein Chips (order no. 5057-1575, Agilent Technologies Inc.). Following the vendor’s standard protocol 4 μl sample was labeled with an amine specific fluorescent dye for 30 min on ice and quenched with ethanolamine for 10 min (end volume 5.5 μl). 1 μl of the reaction mix was diluted 200-fold with distilled water to reduce the background of free fluorescent dye. 4 μl of the diluted reaction mix was mixed with 2 μl loading buffer and boiled for 5 min. After a short spin to collect condensate the samples (6 μl each) were transferred to the wells of a microchip
2.3. Amylase activity assays Salivary α-amylase activity was measured by three different methods which were all set up in 96-well microplates, i.e. the iodine-starch method, an amyloclastic method based on dying starch with Lugol’s iodine (Xiao et al 2006); the bluestarch method, an indirect chromogenic method based on amylase dependent liberation of soluble dye from insoluble stained starch (Rinderknecht et al 1967); and the CNP-G3 method, a direct chromogenic method based on the cleavage 2
J. Breath Res. 7 (2013) 046007
M Zweifel et al
and analyzed in parallel to a sizing and concentration standard. To increase the methods sensitivity we followed protocols of the vendor (Agilent application notes 5990-4097EN and 59903703EN), concentrated EBC protein by precipitation with acetone (400 μl acetone per 100 μl EBC) before labeling, or concentrated labeled protein and removed free dye by acetone precipitation (400 μl acetone per 100 μl reaction mix) or by microfiltration (Amicon Ultra-0.5 Device 30 K cutoff, order no. UFC503024, Millipore Corp., Billerica MA, USA).
(A)
2.5. Dot-blot assay A sheet of nitrocellulose membrane of 0.2 μm pore size (order no. 77012, Thermo Fisher Scientific) was soaked in bidistilled water for 15 min and clamped in a 96-well dot-blot apparatus (Pierce ELIFA-system, Thermo Fisher Scientific). Excess water was carefully removed before loading 100 μl EBC sample, diluted saliva, or diluted BSA per well. All samples and standard solutions were adjusted to 20 mM NaOH before loading to maximize protein binding. The samples were drawn through the membrane by negative pressure provided by a peristaltic pump (Reglo Digital, Ismatec, Glattbrugg, Switzerland). Loaded membranes were soaked 3 × 15 min in 20 mM Tris/HCl pH 7.5, 500 mM NaCl, 0.3% (v/v) Tween-20 and 3 × 5 min in bidistilled water. The blot was stained 30 to 60 min in 50 ml colloidal gold (order no. 170-6527, BioRad). When the staining was judged sufficient the membrane was rinsed with tap water and left to air dry on a paper towel. Dry blots were scanned with an office flat bed scanner (CanoScan 8800F, Canon, Tokyo, Japan) and dot intensities were measured with graphic software (Adobe Photoshop CS5, Park Avenue CA, USA). Net staining intensities were obtained by subtracting the integrated gray scale value of an area containing a dot from the value of an equally sized unstained area.
(B )
(C )
Figure 1. Comparison of amylase activity assays, refinement of the CNP-G3 assay, and amylase activities in saliva and EBC. (A) The amylase activity in a dilution series of saliva was measured with the starch iodine method (), the blue-starch method (), or the CNP-G3 method (◦). The activity is depicted in the form of raw absorbance data to allow direct comparison of sensitivity. (B) The amylase activity in diluted saliva samples of ten individuals was measured with the CNP-G3 method in reaction buffer with (◦) or without (•) 100 μg ml−1 BSA over the course of 48 h or 24 h, respectively. In reaction buffer with BSA the saliva samples were measured after 100 000-fold dilution, in buffer without BSA after 10 000-fold dilution. (–) indicates the arithmetic mean of the values measured under the two conditions. (C) Scatter plots of amylase activities found in EBC (◦) and saliva (•) against the respective total protein concentrations. The cross indicates the arithmetic means of the two parameters. Amylase activity of all samples was measured in the presence 100 μg ml−1 BSA. EBC samples were measured undiluted. Saliva samples were measured after 10 000-fold dilution.
2.6. Calculations and statistical analysis The relative salivary protein contamination of EBC was calculated according to (1), with A standing for amylase activity, P standing for total protein concentration, and the subscripts E and S standing for EBC or saliva, respectively: PS × AE . (1) AS × PE When specific values for PS and AS were missing, we used average (median, arithmetic or geometric mean, as specified) values. Note that under the assumption that EBC amylase activity is of salivary origin exclusively or in excessive proportion, dilutional indicators for normalization of AE are unnecessary. All statistical calculations were performed in Microsoft Excel using the functions included in the program or in Microsoft Excel spreadsheets linked to the ‘Handbook of Biological Statistics’ (McDonald 2008). Standard curves for the Micro-BCA and CNP-G3 assays were calculated and plotted with a curve fitting program (FindGraph 2.331,www.uniphiz.com). % salivary protein contamination = 100 ×
3. Results 3.1. 40-fold enhancement of amylase activity assay sensitivity In figure 1(A) we present raw absorbance data from measurements of amylase activity in serial dilutions of saliva according to three published methods, the starchiodine method (Xiao et al 2006), the blue-starch method 3
J. Breath Res. 7 (2013) 046007
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Table 1. Sensitivity of amylase assays. Assay name (manufacturer) Salivary α-amylase assay kit (Salimetrics) ESP 1491 300 (B¨ohringer) α-amylase EPS sys (Roche) EnzChek ultra amylase assay kit (Life technologies) Starch-iodine method (this work) Blue-starch method (this work) CNP-G3 method (this work)
Substrate b
CNP-G3 EPSc EPSc DQ-starch Starch Blue starch CNP-G3b
Activity in salivaa
LLDa
Maximum dilution
92.4 (n = 75) 438 (n = 5) 163 (n = 75) 111 (n = 11)d n.d. (n = 10) n.d. (n = 10) 40.5 (n = 10)
3.28 1.8 0.005 0.002 – – 0.000 017
28 2.4 3.3 5.6 1.0 1.0 2.4
× × × × × ×
102 104 104 103e 104e 106
a
Unit definition of ‘activity in saliva’ and ‘LLD’ was identical for a given assay, but differed between assays. All values were as provided by the manufacturer product sheets except for α-Amylase EPS Sys, where the values were taken from Rohleder et al 2004. b CNP-G3 stands for 2-chloro-4-nitrophenyl-a-D-maltotriosid. c EPS stands for ethylidene-blocked paranitrophenylmaltoheptaoside. d Taken from Effros et al 2005. e Determined from plots of activity against dilution factor.
(Rinderknecht et al 1967), and the CNP-G3 method (WinnDeen et al 1988, Foo and Bais 1998). The two amyloclastic methods were inferior to the chromogenic CNP-G3 method with respect to both, maximal absorbance and sensitivity, and only allowed trustworthy measurement of amylase in saliva diluted up to about 1000 or 10 000-fold, respectively. We therefore maximized the sensitivity of the CNP-G3 method. As shown in figure 1(B) the reaction time in the CNPG3-assay could be extended up to 480 min without loss of apparent activity provided that the reaction buffer contained 100 μg ml−1 BSA (◦). In reaction buffer without BSA the average apparent amylase activity was about five-fold lower to start with and the enzymes half-life was 2 instead of 38 h (•). Extending the reaction time to 480 min and adding 100 μg ml−1 BSA to the reaction buffer led to a more than 40-fold increase in average sensitivity compared to the most sensitive commercially available amylase assay (table 1). In figure 1(C) we show scatter plots of amylase activity against the corresponding total protein concentration of our EBC and saliva samples. Both, amylase activity and total protein concentration varied considerably with coefficients of variation amounting to 325% and 27% in EBC, and 60% and 22% in saliva, and correlated only very weakly with Spearman’s rho values of 0.405 for the EBC samples and 0.285 for the saliva samples. Of note, amylase activity was measurable in every single EBC sample we collected and all measurements were performed in triplicate consuming 90 μl sample in total, which accounts for only about 5% of an average EBC sample (collection time 10 min).
et al 2012, immune precipitation experiments not shown). A few bands of smaller molecular weight (35, 22, and 15 kD) also appeared prominent in the majority of saliva samples. However, there was no universal pattern of bands which would allow the definition of an electrophoretic ‘saliva footprint’. As shown in figure 2(B), the sensitivity of the electrophoretic method is insufficient at present, except for analyzing strongly contaminated EBC. In the left panel we show overlaid electropherograms and gel images of the analysis of a saliva dilution series. The LLD for this pair of corresponding saliva and EBC samples was 10.9% salivary protein in EBC. The right panel shows the electrophoretic analysis of saliva (200x diluted, black electropherogram, lane 1) and the corresponding EBC sample (blue electropherogram, lane 2), which contained 12.3% saliva protein, provided that the 63 kD peak was of salivary origin. Since the sensitivity of the applied electrophoresis method critically depends on the removal or dilution of unreacted fluorescent dye, we explored various ways to maximize the signal to background ratio, such as concentrating EBC total protein by acetone precipitation before labeling, or fluorescent protein labeling in larger sample volumes followed by immune precipitation, microfiltration, or acetone precipitation. Although we obtained interpretable electropherograms of saliva diluted up to 100 000-fold with some approaches (not shown), the sensitivity, reproducibility, cost, and hands-on time unfavorably compared to the dot-blot method discussed below. In figure 2(C) we show examples of colloidal gold stained dot-blots of EBC samples and serial dilutions of BSA and saliva. All samples were adjusted to 20 mM sodium hydroxide, as preliminary experiments had shown, that under basic conditions the binding of salivary protein and BSA to nitrocellulose filters was maximized (not shown). BSA (dot A1 to dot A5), was detected to at least 0.2 μg ml−1 (dot A5), while salivary protein (dot B5 to dot D7) stained to at least 0.04 μg ml−1 (dot D1) as judged by visual inspection. Quantitative analysis of digital images allowed detection of salivary protein down to 0.01 μg ml−1 (dot D3), which corresponded to a dilution of 164 000-fold. Dot-blots of EBC samples #1 to #24 (dots E1 to G8) showed that EBC samples, despite very similar total protein concentration, strongly varied with respect to their colloidal gold staining intensity, and
3.2. Methods to detect salivary protein in EBC without measuring amylase To estimate salivary protein content in EBC without measuring amylase, we tested an electrophoretic method and a dot-blot assay. In figure 2(A) we show the electrophoretic analysis of ten saliva samples run on a microfluidics-based platform. Most electrophoretic patterns were dominated by one or two strong bands around an apparent molecular weight of 63 kD, which represent salivary amylase isoforms (Kaufman et al 1970, Beeley et al 1991, Vitorino et al 2004, Hirtz et al 2005, Bailey 4
J. Breath Res. 7 (2013) 046007
M Zweifel et al
(A)
(B )
(C )
Figure 2. Electrophoretic and dot-blot analysis of saliva and EBC. (A) Gel images of a microfluidic electrophoretic analysis of ten saliva samples run on an Agilent High Sensitivity 250 Protein Chip. (B) The left panel shows overlaid electropherograms and gel images of a dilution series of sample #40 of panel A. The dilutions were ten-fold (lane 1) to 2560-fold (lane 5) with four-fold dilution per step. The right panel shows electropherograms and gel images of sample #40 (200-fold diluted, lane 1) and of the corresponding EBC (undiluted, lane 2). (C) 100 μl of the following samples were applied to nitrocellulose filters and stained with colloidal gold: a dilution series of BSA (A1, 3.2 μg ml−1 to A5, 0.2 μg ml−1, two-fold dilution each step), a dilution series of saliva in EBC (A7, 15 μg ml−1 to B3, 0.0015 μg ml−1, ten-fold dilution each step), a dilution series of saliva in water (B5, 150 μg ml−1 to D7, 0.0006 μg ml−1, two-fold dilution each step; H1, 30 μg ml−1 to H7, 0.0073 μg ml−1, four-fold dilution each step), EBC (B4, E1 to G8), and water (A6, D8, H8).
that at least half of the samples stained well above the level of saliva diluted 12800-fold (dot H5), as judged by visual inspection. After scanning and digital image analysis of triplicate experiments all dots except E7 and G8 were found to stain above the blank (dot H8). At first instance it was striking to see that EBC samples with very similar total protein concentration varied so extensively with respect to their staining intensity, but we soon realized, that perhaps the variation in staining intensity was due to different levels of salivary protein contamination. Indeed, a dilution series of saliva set up in a weakly staining EBC sample (dot A7 to dot B3) produced dots staining above the background (dot B4) up to a dilution of at least 100 000fold. Furthermore, all saliva samples we collected (n = 10) contained protein which bound to nitrocellulose and was stained by colloidal gold up to a dilution of at least 50 000-fold (not shown).
3.3. Identification of EBC samples containing amylase activity of pulmonary origin Following the idea that colloidal-gold staining on dot-blots may indicate EBC samples contaminated with salivary protein we expected that colloidal gold staining and amylase activity of EBC samples should correlate. To our surprise, plotting EBC amylase activity against the corresponding colloidal gold staining intensity revealed two groups of EBC samples. A large group (•) was distributed over the whole range of staining intensities at low to intermediate amylase activity, while another group of five samples (•∗ ) with intermediate to high amylase activity was aligned with diluted saliva samples (◦). The two groups had rank correlation coefficients (Spearman’s rho) of 0.93 (•, p < 0.0001) or 0.95 (•∗ ,◦, p < 0.0001), respectively, which in our view justified to assign our EBC samples to a non-contaminated group with 5
J. Breath Res. 7 (2013) 046007
M Zweifel et al
Table 2. Estimates of amylase activities, total protein concentrations and relative EBC orophyryngeal protein content. Amylase activity (nmol min–1∗ ml) EBC, all samples (n = 40) EBC, contaminated (n = 5) EBC, non-contaminated (n = 35) EBC, non-contaminated (n = 18)b Saliva (n = 10)
Arithmetic mean Median Geometric meana 1.9 ± 6.01 (0.02–37.9) 0.3 (0.02–37.9) 0.3 ∗ / 6.0 (0.02–37.9) 11.1 ± 15.0 (2.5–37.9) 4.8 (2.5–37.9) 6.5 ∗ / 2.9 (2.5–37.9) 0.5 ± 0.7 (0.02–2.3) 0.2 (0.02–2.3) 0.2 ∗ / 4.2 (0.02–2.3) 0.9 ± 0.8 (0.07–2.3) 0.6 (0.07–2.3) 0.6 ∗ / 2.9 (0.07–2.3) 40.5 ± 24 (14.5–88.6) 31.0 (14.5–88.6) 34.8 ∗ / 1.8 (14.5–88.6) Total protein concentration (μg ml−1)
EBC, all samples (n = 40) EBC, contaminated (n = 5) EBC, non-contaminated (n = 35) EBC, non-contaminated (n = 18)b Saliva (n = 10)
Arithmetic mean Median Geometric meana 8.4 ± 2.2 (5.1–14.5) 7.6 (5.1–14.5) 8.1 ∗ / 1.3 (5.1–14.5) 9.8 ± 2.3 (6.1–11.8) 9.9 (6.1–11.8) 9.5 ∗ / 1.3 (6.1–11.8) 8.2 ± 2.2 (5.1–14.5) 7.3 (5.1–14.5) 7.9 ∗ / 1.3 (5.1–14.5) 8.6 ± 2.2 (5.5–14.5) 8.6 (5.5–14.5) 8.3 ∗ / 1.3 (5.5–14.5) 1.7 ± 0.4 (1.2–2.4) 1.6 (1.2–2.4) 1.6 ∗ / 1.2 (1.2–2.4) EBC oropharyngeal protein content (%)
EBC, contaminated (n = 5) EBC, all samples (n = 40)
Arithmetic mean 4.7 ± 6.3 (1.3–15.8) 0.8 ± 2.5 (0.01–15.8)
Median 2.2 (1.7–20.0) 0.1 (0.01–20.0)
Geometric meana 3.2 ∗ / 2.7 (1.5–18.0) 0.2 ∗ / 5.4 (0.01–18.0)
Minimal (xmin) and maximal value (xmax) in brackets, range R = xmax − xmin. a Notation (∗ /) and calculation of multiplicative standard deviation according to Limpert et al 2001. b EBC samples with colloidal gold staining units smaller than 1.0 × 105 excluded (see text and figure 3 for justification).
pulmonary amylase activity (•) or a contaminated group with oropharyngeal amylase activity introduced during EBC collection (•∗ ). For comparison, the overall rank correlation coefficient (Spearman’s rho) including all samples was 0.74 (p < 0.0001). On the left-hand side of the plot, below about 1 × 105 colloidal gold staining units, the two groups converged. EBC samples with colloidal gold staining of less than 1 × 105 units could therefore contain minute amounts of oropharyngeal proteins brought in during EBC collection. But since the mean relative salivary protein contamination of these samples was calculated to be in the order of 0.05%, we did not subdivide the non-contaminated EBC group, except for the purpose of calculating an estimate of pulmonary amylase activity (see below). In table 2 we present some statistics of the two groups of EBC samples together with values obtained following the common practice of interpreting EBC amylase activity as an indication of oropharyngeal contamination during EBC collection. Clearly, the contaminated and non-contaminated groups strongly differed in their mean amylase activity and varied only little in their total protein concentration. As the distribution of amylase activities in EBC was strongly skewed to the right, the median or the geometric mean are clearly more suitable to obtain estimates (Limpert et al 2001, Haeckel and Wosniok 2010). Due to the small number of samples (n = 5) the average amylase activity of the contaminated group could not be reliably estimated. On the other hand, our estimates of 0.2 ∗ / 4.5 nmol min−1 (n = 35) and 0.6 ∗ / 2.9 nmol min−1 (n = 18, EBC samples with potential minor oropharyngeal contamination excluded) amylase activity per ml non-contaminated EBC, are based on an adequate sample size. To account for varying dilution during EBC collection we calculated the specific amylase activity and found geometric
Figure 3. Identification of contaminated EBC. Scatter plot of amylase activity against colloidal gold staining units. (•) EBC, (•∗ ) EBC with salivary protein contamination, (◦) diluted saliva samples. The amylase activity of the saliva samples depicted was calculated by dividing the mean amylase activity of ten saliva samples (40.5 μmol min−1∗ ml) by known dilution factors (figure 2(C) and dot-blot (not shown)). In the inset, the ordinate (amylase activity) is scaled up five-fold and all values above 8 nmol min−1∗ ml are omitted for better visibility of the two groups converging.
means of 0.03 ∗ / 3.9 nmol min−1∗ μg (n = 35) or 0.07 ∗ / 2.9 nmol min−1∗ μg (n = 18), respectively. Most important, the average relative oropharyngeal protein contamination of EBC samples judged contaminated according to figure 3 was more than ten times higher than the contamination calculated under the paradigm that any amylase activity detectable in EBC indicates oropharyngeal contamination, namely 2.2% versus 0.1% (based on medians) or 3.2% versus 0.2% (based on geometric means), respectively. 6
J. Breath Res. 7 (2013) 046007
M Zweifel et al
4. Discussion
has to remain open, but it seems clear that EBC amylase activity not necessarily indicates oropharyngeal contamination due to careless EBC collection. Following the common practice of interpreting any amylase activity found in EBC as oropharyngeal contamination relative oropharyngeal protein contents of EBC of 1.7%, 1.6%, and 0.05% can be calculated from published data on amylase activity in EBC of healthy volunteers (Effros et al 2002, 2005, Husz´ar et al 2002). On the background of our study the divergent values can be easily reconciled. For our experimentally defined contaminated group we found 2.2% oropharyngeal protein content (median), which lies just above the values of Effros et al 2002 and Husz´ar et al 2002, who found amylase activity only in part of their EBC samples since a relatively insensitive amylase assay was applied. The majority of their samples was therefore excluded from the calculation for technical rather than experimentally justified reasons. If a more sensitive amylase assay is used, amylase activity was detected in all EBC-samples and a relative oropharyngeal protein content of 0.05% results (Effros et al 2005). This lower value is of the same order of magnitude as our value of 0.1% (median) which we calculated including all samples. Clearly, the most interesting and useful finding of our study is that EBC samples containing amylase activity of pulmonary origin may perhaps be identified by plotting EBC amylase activity against colloidal gold staining intensity on dot-blots. However, further studies investigating the identity of pulmonary amylase with antibodies against salivary or pancreatic amylase and proper validation and standardization of our method are necessary before it may eventually be applied to detect oropharyngeal or gastric aspiration in EBC of mechanically ventilated or lung transplant patients.
In our search for a more sensitive amylase assay we first compared three amylase assays and chose to increase the sensitivity of the CNP-G3 amylase assay by extending the reaction time. We found that adding BSA to the reaction mix raised the apparent activity of salivary amylase five-fold and increased the half-life of enzyme activity from 2 to 38 h. With our improved CNP-G3 activity assay we were able to detect amylase activity well above the assays lower detection limit in as little as 30 μl of every single EBC-sample we analyzed (n = 40). In stark contrast, other authors found amylase activity only in a fraction of their EBC samples (Husz´ar et al 2002, Effros et al 2002, Gaber et al 2006) or had to concentrate them (Effros et al 2005). After proper validation and standardization our enhanced CNP-G3 assay may therefore find application in breath research and other fields of inquiry where a highly sensitive amylase assay is essential. While testing buffer components to maximize protein binding to nitrocellulose filters we observed that EBC samples, despite similar total protein concentration, strongly varied in their content of proteins binding to nitrocellulose as judged by colloidal gold staining intensity. This prompted us to develop a dot-blot assay for salivary contamination since we had also observed that saliva diluted up to 150 000-fold still produced stainable dots on nitrocellulose filters. However, a scatter plot of EBC amylase activity against EBC colloidal gold staining intensity showed a very peculiar pattern: The samples with the five topmost amylase activities had only intermediate staining intensities and correlated with results from highly diluted saliva, while the majority of EBC samples in clear separation scattered over the whole range of colloidal gold staining intensities at low to intermediate amylase activity. At very low amylase activity and low staining intensity the two groups converged. We hypothesize that EBC samples aligned with diluted saliva contained amylase activity introduced during EBC collection and that the amylase activity found in all other EBC samples was of pulmonary origin. That amylase indeed could be a component of airway lining fluid can be inferred from the analysis of bronchoalveolar lavage (BAL), although the presence of amylase in BAL, similar to EBC amylase activity, could be an experimental artifact. Amylase activity was found in BAL of mechanically ventilated patients (Clarke et al 1981, Nandapalan et al 1995, Weiss et al 2013) and salivary as well as pancreatic amylase were found in BAL of lung transplant patients in a proteomic study (Kosana et al 2012). Histological evidence for pulmonary amylase expression comes from analysis of surgically resected specimens from lobar and segmental bronchi (Hayashi et al 1986). Both, immunohistological and activity based histochemical staining of amylase was found primarily in serous cells of bronchial glands and with less intensity in the apical part of ciliated epithelial cells. Although the antibody used in this study was raised against salivary amylase, it cross-reacted with the pancreatic isoform. In conclusion, whether and in which proportion amylase present in BAL originates from pulmonary expression or aspiration of oropharyngeal or gastric material
References Bailey U M, Punyadeera C, Cooper-White J J and Schulz B L 2012 Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 911 21–26 Beeley J A, Sweeney D, Lindsay J C, Buchanan M L, Sarna L and Khoo K S 1991 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of human parotid salivary proteins Electrophoresis 12 1032–41 Clarke P D, Bain B C, Davies A, Levin G E and Lambert H P 1981 Aspiration in seriously ill patients: a study of amylase in bronchial secretions J. Clin. Pathol. 34 803–5 Davis M D, Montpetit A and Hunt J 2012 Exhaled breath condensate: an overview Immunol. Allergy Clin. North. Am. 32 363–75 Effros R M, Hoagland K W, Bosbous M, Castillo D, Foss B, Dunning M, Gare M, Lin W and Sun F 2002 Dilution of respiratory solutes in exhaled condensates Am. J. Respir. Crit. Care Med. 165 663–9 Effros R M, Peterson B, Casaburi R, Su J, Dunning M, Torday J, Biller J and Shaker R 2005 Epithelial lining fluid solute concentrations in chronic obstructive lung disease patients and normal subjects J. Appl. Physiol. 99 1286–92 Foo A Y and Bais R 1998 Amylase measurement with 2-chloro-4-nitrophenyl maltotrioside as substrate Clin. Chim. Acta 272 137–47 7
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Gaber F, Acevedo F, Delin I, Sundblad B M, Palmberg L, Larsson K, Kumlin M and Dahl´en S E 2006 Saliva is one likely source of leukotriene B4 in exhaled breath condensate Eur. Respir. J. 28 1229–35 Gessner C, Scheibe R, W¨otzel M, Hammerschmidt S, Kuhn H, Engelmann L, Hoheisel G, Gillissen A, Sack U and Wirtz H 2005 Exhaled breath condensate cytokine patterns in chronic obstructive pulmonary disease Respir. Med. 99 1229–40 Griese M, Noss J and von Bredow C 2002 Protein pattern of exhaled breath condensate and saliva Proteomics 2 690–6 Haeckel R and Wosniok W 2010 Observed, unknown distributions of clinical chemical quantities should be considered to be log-normal: a proposal Clin. Chem. Lab. Med. 48 1393–6 Hayashi Y, Fukayama M, Koike M and Nakayama T 1986 Amylase in human lungs and the female genital tract. Histochemical and immunohistochemical localization Histochemistry 85 491–6 Hirtz C, Chevalier F, Centeno D, Rofidal V, Egea J C, Rossignol M, Sommerer N and Deville de P´eri`ere D 2005 MS characterization of multiple forms of alpha-amylase in human saliva Proteomics 5 4597–607 Horv´ath I et al 2005 Exhaled breath condensate: methodological recommendations and unresolved questions Eur. Respir. J. 26 523–48 Husz´ar E, Vass G, Vizi E, Csoma Z, Bar´at E, Moln´ar Vil´agos G, Herjavecz I and Horv´ath I 2002 Adenosine in exhaled breath condensate in healthy volunteers and in patients with asthma Eur. Respir. J. 20 1393–8139 Kauffman D L, Zager N I, Cohen E and Keller P J 1970 The isoenzymes of human parotid amylase Arch. Biochem. Biophys. 137 325–39 Kosanam H, Sato M, Batruch I, Smith C, Keshavjee S, Liu M and Diamandis E P 2012 Differential proteomic analysis of bronchoalveolar lavage fluid from lung transplant patients with
and without chronic graft dysfunction Clin. Biochem. 45 223–30 Limpert E, Stahel W A and Abbt M 2001 Log-normal distributions across the sciences: keys and clues BioScience 51 341–52 McDonald J H 2008 Handbook of Biological Statistics (Baltimore, MD: Sparky House Publishing) Nandapalan V, McIlwain J C and Hamilton J 1995 A study of alpha-amylase activity in tracheobronchial secretions of seriously ill patients with tracheostomies J. Laryngol. Otol. 109 640–3 Rinderknecht H, Wilding P and Haverback B J 1967 A new method for the determination of alpha-amylase Experientia 23 805 Rohleder N, Nater U M, Wolf J M, Ehlert U and Kirschbaum C 2004 Psychosocial stress-induced activation of salivary alpha-amylase: an indicator of sympathetic activity? Ann. NY Acad. Sci. 1032 258–63 Schenkels L C, Veerman E C and Nieuw Amerongen A V 1995 Biochemical composition of human saliva in relation to other mucosal fluids Crit. Rev. Oral. Biol. Med. 6 161–75 Smith P K et al 1985 Measurement of protein using bicinchoninic acid Anal. Biochem. 150 76–85 Vitorino R, Lobo M J, Ferrer-Correira A J, Dubin J R, Tomer K B, Domingues P M and Amado F M 2004 Identification of human whole saliva protein components using proteomics Proteomics 4 1109–15 Weiss C H, Moazed F, DiBardino D, Swaroop M and Wunderink R G 2013 Bronchoalveolar lavage amylase is associated with risk factors for aspiration and predicts bacterial pneumonia Crit. Care Med. 41 765–73 Winn-Deen E S, David H, Sigler G and Chavez R 1988 Development of a direct assay for alpha-amylase Clin. Chem. 34 2005–8 Xiao Z, Storms R and Tsang A 2006 A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities Anal. Biochem. 351 146–8
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Detection of pulmonary amylase activity in exhaled breath condensate
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IOP PUBLISHING
JOURNAL OF BREATH RESEARCH
doi:10.1088/1752-7155/7/4/046007
J. Breath Res. 7 (2013) 046007 (8pp)
Detection of pulmonary amylase activity in exhaled breath condensate M Zweifel, T Rechsteiner, M Hofer and A Boehler Division of Pulmonology, University Hospital Z¨urich, R¨amistrasse 100, 8091 Z¨urich, Switzerland E-mail: [email protected]
Received 7 March 2013 Accepted for publication 25 September 2013 Published 28 November 2013 Online at stacks.iop.org/JBR/7/046007 Abstract Amylase activity in exhaled breath condensate (EBC) is usually interpreted as an indication of oropharyngeal contamination despite the fact that amylase can be found in pulmonary excretions. The aim of this study was to recruit and refine an amylase assay in order to detect amylase activity in any EBC sample and to develop a method to identify EBC samples containing amylase of pulmonary origin. EBC was collected from 40 volunteers with an EcoScreen condenser. Amylase assays and methods to discriminate between oropharyngeal and pulmonary proteins were tested and developed using matched EBC and saliva samples. Our refined 2-chloro-4-nitrophenyl-α-D-maltotriosid (CNP-G3) assay was 40-fold more sensitive than the most sensitive commercial assay and allowed detection of amylase activity in 30 μl of EBC. We developed a dot-blot assay which allowed detection of salivary protein in saliva diluted up to 150 000-fold. By plotting amylase activity against staining intensity we identified a few EBC samples with high amylase activity which were aligned with diluted saliva. We believe that EBC samples aligned with diluted saliva contain amylase activity introduced during EBC collection and that all other EBC samples contain amylase activity of pulmonary origin and are basically free of oropharyngeal protein contamination. (Some figures may appear in colour only in the online journal)
1. Introduction
However, the majority of investigators believe saliva contamination to be a low priority problem which is taken care of by a saliva trap or the design of the EBC collection device. This latter view is also held by the 2005 ATS/ERS task force on EBC, even though ‘more sensitive assays to exclude oropharyngeal contamination’ were put on the wish list of future research (Horv´ath et al 2005). Commercial amylase activity assays based on the cleavage of the chromogenic substrate ethylidene-blocked 4-nitrophenylmaltoheptaoside (EPS) which are routinely used to measure amylase activity in serum or urine allow detection of amylase activity in saliva diluted up to 30 000-fold as inferred from published data (Husz´ar et al 2002, Rohleder et al 2004). Since the average total protein concentration is about 200-fold higher in saliva than in EBC, the lowest salivary protein contamination of EBC detectable with EPS-assays lies at around 0.7% of EBC total protein. This lower limit of detection (LLD) is confirmed by two reports presenting values of EBC amylase activity measured with the EPS-method (Husz´ar et al 2002, Effros et al 2002). From the two reports,
Exhaled breath condensate (EBC) contains a plethora of compounds which in their composition and abundance depend on the pulmonary and systemic state of health. So far, primarily low molecular weight analytes, such as inorganic ions, nitrogen oxides, ammonia, nucleosides, arachidonic acid derivatives, and reactive oxygen induced compounds were in the focus of clinical investigation because their analysis requires only minor adjustment of existing laboratory methods. In contrast, EBC proteins can usually only be quantitated in concentrated form and after methodological refinement. Furthermore, contaminating proteins from the oral cavity can complicate drawing meaningful conclusions from EBC protein analysis (Davis et al 2012). To exclude contamination with oropharyngeal constituents some researchers test their EBC samples for activity of the most abundant salivary protein, salivary α-amylase, usually to observe that the samples do not contain amylase at a level ‘measurable with the assay used’ (Gessner et al 2005). 1752-7155/13/046007+08$33.00
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J. Breath Res. 7 (2013) 046007
M Zweifel et al
relative EBC salivary protein contaminations amounting to 1.6% (Husz´ar et al 2002) or 1.7% (Effros et al 2002) can be calculated. However, according to both reports amylase activity was undetectable in part of the samples, which can be understood as absence of amylase in these samples but also must be expected for statistical reasons as the values are close to the LLD of the applied method. To further complicate matters, amylase as well as other salivary proteins can also be found in the respiratory tract (Schenkels et al 1995, Griese et al 2002). Amylase activity detected in tracheobronchial secretions can originate in biosynthesis by pulmonary tissue (Hayashi et al 1986), aspiration of saliva (Clarke et al 1981, Nandapalan et al 1995, Weiss et al 2013), or aspiration of gastric fluid (Kosanam et al 2012). Therefore, demonstrating amylase activity in EBC not necessarily indicates salivary contamination. In this study we (i) compare three common amylase activity assays and increase the sensitivity of the most sensitive method to allow detection of amylase activity in any EBC sample, (ii) search for methods to identify EBC samples containing traces of saliva without measuring amylase activity, and (iii) present a procedure to identify EBC samples containing amylase activity of pulmonary origin.
of 2-chloro-4-nitrophenyl-α-D-maltotriosid (CNP-G3) by amylase (Winn-Deen et al 1988, Foo and Bais 1998). 2.3.1. Iodine-starch method. 48 μl of 0.17% potato starch (order no. S2004, Sigma-Aldrich, St. Louis MO, USA) in 83.3 mM sodium phosphate buffer pH 7 was added to 32 μl of sample prepared in the wells of a microplate. After loading, the plate was sealed and incubated at 50◦ while shacking at 1000 RPM. Condensate forming on the sealing foil was collected by centrifugation every 30 min. After 90 min the reaction was stopped by addition of 20 μl 1M HCl and 100 μl iodine reagent (5 mM I2, 5 mM KI) was added. 100 μl of the reaction mixes were transferred to a new microplate and the absorbance was measured at 595 nm. 2.3.2. Blue-starch method. 100 μl of a 20% (w/v) suspension of Remazol brilliant blue R dyed starch (order no. 85641, Sigma-Aldrich) in a buffer containing 20 mM sodium phosphate buffer pH 7 and 50 mM NaCl was added to 50 μl sample prepared in the wells of microplate. After loading, the plate was sealed and incubated at 45 ◦ C while shaking at 1000 RPM. Condensate was collected by centrifugation every 30 min. After 90 min the reaction was stopped by addition of 50 μl 0.1M acetic acid. Insoluble starch was pelleted by centrifugation at 3000 RPM for 15 min. 150 μl supernatant was transferred to a fresh microplate and the absorbance was measured at 595 nm.
2. Materials and methods 2.1. EBC and saliva collection EBC was collected with an EcoScreen I condenser during exactly 10 min from 40 healthy volunteers, who were instructed to wear a nose clip and to breathe freely while EBC was collected. The volume of inhaled ambient air was monitored with a flow meter. All EBC samples were frozen immediately after collection and stored at −60 ◦ C. Saliva was collected from ten volunteers by passive drooling immediately after collection of EBC and frozen at −60 ◦ C.
2.3.3. CNP-G3 method. The reaction mix contained 2.25 mM CNP-G3 (order no. 93834, Sigma-Aldrich), 300 mM NaCl, 5 mM CaCl2, 0.9M KSCN, 50 mM MES buffer pH 6, and 100 μg ml−1 BSA. The reaction was started by adding 50 μl 2x concentrated or 70 μl 1.43x concentrated reaction mix to 50 μl or 30 μl of sample prepared in the wells of a microplate. The plate was centrifuged for 5 s, shaken for 10 s at 1000 RPM and immediately read at 415 nm. After exactly 30, 60, 120, 240, and 480 min incubation at room temperature (22 ◦ C) the absorbance at 415 nm was measured again. Standards consisted of 1x reaction buffer (without CNPG3) and 0, 5, 10, 20, 40, 80, 160, 320, and 640 μM 2-chloro4-nitrophenol (order no. C61208, Sigma-Aldrich), the colored cleavage product of CNP-G3, and were always set up in the same microplate as the samples.
2.2. Protein concentration assay Total protein concentration was determined with the bicinchoninic acid method (Smith et al 1985). 200 μl of EBC, diluted saliva, or bovine serum albumin (BSA) standard solutions were mixed with 200 μl Micro-BCA reagent solution (order no. 23235, Thermo Fisher Scientific, Waltham MA, USA) in 2 ml polypropylene tubes. The tubes were incubated at 60 ◦ C for 60 min. 350 μl aliquots were transferred to a 96well microplate and the absorbance was measured at 550 nm in a microplate reader (Model 550, BioRad, Hercules CA, USA).
2.4. Electrophoretic assay Electrophoretic runs of EBC and saliva samples were performed on an Agilent BioAnalyzer 2100 microfluidic platform with High Sensitivity 250 Protein Chips (order no. 5057-1575, Agilent Technologies Inc.). Following the vendor’s standard protocol 4 μl sample was labeled with an amine specific fluorescent dye for 30 min on ice and quenched with ethanolamine for 10 min (end volume 5.5 μl). 1 μl of the reaction mix was diluted 200-fold with distilled water to reduce the background of free fluorescent dye. 4 μl of the diluted reaction mix was mixed with 2 μl loading buffer and boiled for 5 min. After a short spin to collect condensate the samples (6 μl each) were transferred to the wells of a microchip
2.3. Amylase activity assays Salivary α-amylase activity was measured by three different methods which were all set up in 96-well microplates, i.e. the iodine-starch method, an amyloclastic method based on dying starch with Lugol’s iodine (Xiao et al 2006); the bluestarch method, an indirect chromogenic method based on amylase dependent liberation of soluble dye from insoluble stained starch (Rinderknecht et al 1967); and the CNP-G3 method, a direct chromogenic method based on the cleavage 2
J. Breath Res. 7 (2013) 046007
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and analyzed in parallel to a sizing and concentration standard. To increase the methods sensitivity we followed protocols of the vendor (Agilent application notes 5990-4097EN and 59903703EN), concentrated EBC protein by precipitation with acetone (400 μl acetone per 100 μl EBC) before labeling, or concentrated labeled protein and removed free dye by acetone precipitation (400 μl acetone per 100 μl reaction mix) or by microfiltration (Amicon Ultra-0.5 Device 30 K cutoff, order no. UFC503024, Millipore Corp., Billerica MA, USA).
(A)
2.5. Dot-blot assay A sheet of nitrocellulose membrane of 0.2 μm pore size (order no. 77012, Thermo Fisher Scientific) was soaked in bidistilled water for 15 min and clamped in a 96-well dot-blot apparatus (Pierce ELIFA-system, Thermo Fisher Scientific). Excess water was carefully removed before loading 100 μl EBC sample, diluted saliva, or diluted BSA per well. All samples and standard solutions were adjusted to 20 mM NaOH before loading to maximize protein binding. The samples were drawn through the membrane by negative pressure provided by a peristaltic pump (Reglo Digital, Ismatec, Glattbrugg, Switzerland). Loaded membranes were soaked 3 × 15 min in 20 mM Tris/HCl pH 7.5, 500 mM NaCl, 0.3% (v/v) Tween-20 and 3 × 5 min in bidistilled water. The blot was stained 30 to 60 min in 50 ml colloidal gold (order no. 170-6527, BioRad). When the staining was judged sufficient the membrane was rinsed with tap water and left to air dry on a paper towel. Dry blots were scanned with an office flat bed scanner (CanoScan 8800F, Canon, Tokyo, Japan) and dot intensities were measured with graphic software (Adobe Photoshop CS5, Park Avenue CA, USA). Net staining intensities were obtained by subtracting the integrated gray scale value of an area containing a dot from the value of an equally sized unstained area.
(B )
(C )
Figure 1. Comparison of amylase activity assays, refinement of the CNP-G3 assay, and amylase activities in saliva and EBC. (A) The amylase activity in a dilution series of saliva was measured with the starch iodine method (), the blue-starch method (), or the CNP-G3 method (◦). The activity is depicted in the form of raw absorbance data to allow direct comparison of sensitivity. (B) The amylase activity in diluted saliva samples of ten individuals was measured with the CNP-G3 method in reaction buffer with (◦) or without (•) 100 μg ml−1 BSA over the course of 48 h or 24 h, respectively. In reaction buffer with BSA the saliva samples were measured after 100 000-fold dilution, in buffer without BSA after 10 000-fold dilution. (–) indicates the arithmetic mean of the values measured under the two conditions. (C) Scatter plots of amylase activities found in EBC (◦) and saliva (•) against the respective total protein concentrations. The cross indicates the arithmetic means of the two parameters. Amylase activity of all samples was measured in the presence 100 μg ml−1 BSA. EBC samples were measured undiluted. Saliva samples were measured after 10 000-fold dilution.
2.6. Calculations and statistical analysis The relative salivary protein contamination of EBC was calculated according to (1), with A standing for amylase activity, P standing for total protein concentration, and the subscripts E and S standing for EBC or saliva, respectively: PS × AE . (1) AS × PE When specific values for PS and AS were missing, we used average (median, arithmetic or geometric mean, as specified) values. Note that under the assumption that EBC amylase activity is of salivary origin exclusively or in excessive proportion, dilutional indicators for normalization of AE are unnecessary. All statistical calculations were performed in Microsoft Excel using the functions included in the program or in Microsoft Excel spreadsheets linked to the ‘Handbook of Biological Statistics’ (McDonald 2008). Standard curves for the Micro-BCA and CNP-G3 assays were calculated and plotted with a curve fitting program (FindGraph 2.331,www.uniphiz.com). % salivary protein contamination = 100 ×
3. Results 3.1. 40-fold enhancement of amylase activity assay sensitivity In figure 1(A) we present raw absorbance data from measurements of amylase activity in serial dilutions of saliva according to three published methods, the starchiodine method (Xiao et al 2006), the blue-starch method 3
J. Breath Res. 7 (2013) 046007
M Zweifel et al
Table 1. Sensitivity of amylase assays. Assay name (manufacturer) Salivary α-amylase assay kit (Salimetrics) ESP 1491 300 (B¨ohringer) α-amylase EPS sys (Roche) EnzChek ultra amylase assay kit (Life technologies) Starch-iodine method (this work) Blue-starch method (this work) CNP-G3 method (this work)
Substrate b
CNP-G3 EPSc EPSc DQ-starch Starch Blue starch CNP-G3b
Activity in salivaa
LLDa
Maximum dilution
92.4 (n = 75) 438 (n = 5) 163 (n = 75) 111 (n = 11)d n.d. (n = 10) n.d. (n = 10) 40.5 (n = 10)
3.28 1.8 0.005 0.002 – – 0.000 017
28 2.4 3.3 5.6 1.0 1.0 2.4
× × × × × ×
102 104 104 103e 104e 106
a
Unit definition of ‘activity in saliva’ and ‘LLD’ was identical for a given assay, but differed between assays. All values were as provided by the manufacturer product sheets except for α-Amylase EPS Sys, where the values were taken from Rohleder et al 2004. b CNP-G3 stands for 2-chloro-4-nitrophenyl-a-D-maltotriosid. c EPS stands for ethylidene-blocked paranitrophenylmaltoheptaoside. d Taken from Effros et al 2005. e Determined from plots of activity against dilution factor.
(Rinderknecht et al 1967), and the CNP-G3 method (WinnDeen et al 1988, Foo and Bais 1998). The two amyloclastic methods were inferior to the chromogenic CNP-G3 method with respect to both, maximal absorbance and sensitivity, and only allowed trustworthy measurement of amylase in saliva diluted up to about 1000 or 10 000-fold, respectively. We therefore maximized the sensitivity of the CNP-G3 method. As shown in figure 1(B) the reaction time in the CNPG3-assay could be extended up to 480 min without loss of apparent activity provided that the reaction buffer contained 100 μg ml−1 BSA (◦). In reaction buffer without BSA the average apparent amylase activity was about five-fold lower to start with and the enzymes half-life was 2 instead of 38 h (•). Extending the reaction time to 480 min and adding 100 μg ml−1 BSA to the reaction buffer led to a more than 40-fold increase in average sensitivity compared to the most sensitive commercially available amylase assay (table 1). In figure 1(C) we show scatter plots of amylase activity against the corresponding total protein concentration of our EBC and saliva samples. Both, amylase activity and total protein concentration varied considerably with coefficients of variation amounting to 325% and 27% in EBC, and 60% and 22% in saliva, and correlated only very weakly with Spearman’s rho values of 0.405 for the EBC samples and 0.285 for the saliva samples. Of note, amylase activity was measurable in every single EBC sample we collected and all measurements were performed in triplicate consuming 90 μl sample in total, which accounts for only about 5% of an average EBC sample (collection time 10 min).
et al 2012, immune precipitation experiments not shown). A few bands of smaller molecular weight (35, 22, and 15 kD) also appeared prominent in the majority of saliva samples. However, there was no universal pattern of bands which would allow the definition of an electrophoretic ‘saliva footprint’. As shown in figure 2(B), the sensitivity of the electrophoretic method is insufficient at present, except for analyzing strongly contaminated EBC. In the left panel we show overlaid electropherograms and gel images of the analysis of a saliva dilution series. The LLD for this pair of corresponding saliva and EBC samples was 10.9% salivary protein in EBC. The right panel shows the electrophoretic analysis of saliva (200x diluted, black electropherogram, lane 1) and the corresponding EBC sample (blue electropherogram, lane 2), which contained 12.3% saliva protein, provided that the 63 kD peak was of salivary origin. Since the sensitivity of the applied electrophoresis method critically depends on the removal or dilution of unreacted fluorescent dye, we explored various ways to maximize the signal to background ratio, such as concentrating EBC total protein by acetone precipitation before labeling, or fluorescent protein labeling in larger sample volumes followed by immune precipitation, microfiltration, or acetone precipitation. Although we obtained interpretable electropherograms of saliva diluted up to 100 000-fold with some approaches (not shown), the sensitivity, reproducibility, cost, and hands-on time unfavorably compared to the dot-blot method discussed below. In figure 2(C) we show examples of colloidal gold stained dot-blots of EBC samples and serial dilutions of BSA and saliva. All samples were adjusted to 20 mM sodium hydroxide, as preliminary experiments had shown, that under basic conditions the binding of salivary protein and BSA to nitrocellulose filters was maximized (not shown). BSA (dot A1 to dot A5), was detected to at least 0.2 μg ml−1 (dot A5), while salivary protein (dot B5 to dot D7) stained to at least 0.04 μg ml−1 (dot D1) as judged by visual inspection. Quantitative analysis of digital images allowed detection of salivary protein down to 0.01 μg ml−1 (dot D3), which corresponded to a dilution of 164 000-fold. Dot-blots of EBC samples #1 to #24 (dots E1 to G8) showed that EBC samples, despite very similar total protein concentration, strongly varied with respect to their colloidal gold staining intensity, and
3.2. Methods to detect salivary protein in EBC without measuring amylase To estimate salivary protein content in EBC without measuring amylase, we tested an electrophoretic method and a dot-blot assay. In figure 2(A) we show the electrophoretic analysis of ten saliva samples run on a microfluidics-based platform. Most electrophoretic patterns were dominated by one or two strong bands around an apparent molecular weight of 63 kD, which represent salivary amylase isoforms (Kaufman et al 1970, Beeley et al 1991, Vitorino et al 2004, Hirtz et al 2005, Bailey 4
J. Breath Res. 7 (2013) 046007
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(A)
(B )
(C )
Figure 2. Electrophoretic and dot-blot analysis of saliva and EBC. (A) Gel images of a microfluidic electrophoretic analysis of ten saliva samples run on an Agilent High Sensitivity 250 Protein Chip. (B) The left panel shows overlaid electropherograms and gel images of a dilution series of sample #40 of panel A. The dilutions were ten-fold (lane 1) to 2560-fold (lane 5) with four-fold dilution per step. The right panel shows electropherograms and gel images of sample #40 (200-fold diluted, lane 1) and of the corresponding EBC (undiluted, lane 2). (C) 100 μl of the following samples were applied to nitrocellulose filters and stained with colloidal gold: a dilution series of BSA (A1, 3.2 μg ml−1 to A5, 0.2 μg ml−1, two-fold dilution each step), a dilution series of saliva in EBC (A7, 15 μg ml−1 to B3, 0.0015 μg ml−1, ten-fold dilution each step), a dilution series of saliva in water (B5, 150 μg ml−1 to D7, 0.0006 μg ml−1, two-fold dilution each step; H1, 30 μg ml−1 to H7, 0.0073 μg ml−1, four-fold dilution each step), EBC (B4, E1 to G8), and water (A6, D8, H8).
that at least half of the samples stained well above the level of saliva diluted 12800-fold (dot H5), as judged by visual inspection. After scanning and digital image analysis of triplicate experiments all dots except E7 and G8 were found to stain above the blank (dot H8). At first instance it was striking to see that EBC samples with very similar total protein concentration varied so extensively with respect to their staining intensity, but we soon realized, that perhaps the variation in staining intensity was due to different levels of salivary protein contamination. Indeed, a dilution series of saliva set up in a weakly staining EBC sample (dot A7 to dot B3) produced dots staining above the background (dot B4) up to a dilution of at least 100 000fold. Furthermore, all saliva samples we collected (n = 10) contained protein which bound to nitrocellulose and was stained by colloidal gold up to a dilution of at least 50 000-fold (not shown).
3.3. Identification of EBC samples containing amylase activity of pulmonary origin Following the idea that colloidal-gold staining on dot-blots may indicate EBC samples contaminated with salivary protein we expected that colloidal gold staining and amylase activity of EBC samples should correlate. To our surprise, plotting EBC amylase activity against the corresponding colloidal gold staining intensity revealed two groups of EBC samples. A large group (•) was distributed over the whole range of staining intensities at low to intermediate amylase activity, while another group of five samples (•∗ ) with intermediate to high amylase activity was aligned with diluted saliva samples (◦). The two groups had rank correlation coefficients (Spearman’s rho) of 0.93 (•, p < 0.0001) or 0.95 (•∗ ,◦, p < 0.0001), respectively, which in our view justified to assign our EBC samples to a non-contaminated group with 5
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Table 2. Estimates of amylase activities, total protein concentrations and relative EBC orophyryngeal protein content. Amylase activity (nmol min–1∗ ml) EBC, all samples (n = 40) EBC, contaminated (n = 5) EBC, non-contaminated (n = 35) EBC, non-contaminated (n = 18)b Saliva (n = 10)
Arithmetic mean Median Geometric meana 1.9 ± 6.01 (0.02–37.9) 0.3 (0.02–37.9) 0.3 ∗ / 6.0 (0.02–37.9) 11.1 ± 15.0 (2.5–37.9) 4.8 (2.5–37.9) 6.5 ∗ / 2.9 (2.5–37.9) 0.5 ± 0.7 (0.02–2.3) 0.2 (0.02–2.3) 0.2 ∗ / 4.2 (0.02–2.3) 0.9 ± 0.8 (0.07–2.3) 0.6 (0.07–2.3) 0.6 ∗ / 2.9 (0.07–2.3) 40.5 ± 24 (14.5–88.6) 31.0 (14.5–88.6) 34.8 ∗ / 1.8 (14.5–88.6) Total protein concentration (μg ml−1)
EBC, all samples (n = 40) EBC, contaminated (n = 5) EBC, non-contaminated (n = 35) EBC, non-contaminated (n = 18)b Saliva (n = 10)
Arithmetic mean Median Geometric meana 8.4 ± 2.2 (5.1–14.5) 7.6 (5.1–14.5) 8.1 ∗ / 1.3 (5.1–14.5) 9.8 ± 2.3 (6.1–11.8) 9.9 (6.1–11.8) 9.5 ∗ / 1.3 (6.1–11.8) 8.2 ± 2.2 (5.1–14.5) 7.3 (5.1–14.5) 7.9 ∗ / 1.3 (5.1–14.5) 8.6 ± 2.2 (5.5–14.5) 8.6 (5.5–14.5) 8.3 ∗ / 1.3 (5.5–14.5) 1.7 ± 0.4 (1.2–2.4) 1.6 (1.2–2.4) 1.6 ∗ / 1.2 (1.2–2.4) EBC oropharyngeal protein content (%)
EBC, contaminated (n = 5) EBC, all samples (n = 40)
Arithmetic mean 4.7 ± 6.3 (1.3–15.8) 0.8 ± 2.5 (0.01–15.8)
Median 2.2 (1.7–20.0) 0.1 (0.01–20.0)
Geometric meana 3.2 ∗ / 2.7 (1.5–18.0) 0.2 ∗ / 5.4 (0.01–18.0)
Minimal (xmin) and maximal value (xmax) in brackets, range R = xmax − xmin. a Notation (∗ /) and calculation of multiplicative standard deviation according to Limpert et al 2001. b EBC samples with colloidal gold staining units smaller than 1.0 × 105 excluded (see text and figure 3 for justification).
pulmonary amylase activity (•) or a contaminated group with oropharyngeal amylase activity introduced during EBC collection (•∗ ). For comparison, the overall rank correlation coefficient (Spearman’s rho) including all samples was 0.74 (p < 0.0001). On the left-hand side of the plot, below about 1 × 105 colloidal gold staining units, the two groups converged. EBC samples with colloidal gold staining of less than 1 × 105 units could therefore contain minute amounts of oropharyngeal proteins brought in during EBC collection. But since the mean relative salivary protein contamination of these samples was calculated to be in the order of 0.05%, we did not subdivide the non-contaminated EBC group, except for the purpose of calculating an estimate of pulmonary amylase activity (see below). In table 2 we present some statistics of the two groups of EBC samples together with values obtained following the common practice of interpreting EBC amylase activity as an indication of oropharyngeal contamination during EBC collection. Clearly, the contaminated and non-contaminated groups strongly differed in their mean amylase activity and varied only little in their total protein concentration. As the distribution of amylase activities in EBC was strongly skewed to the right, the median or the geometric mean are clearly more suitable to obtain estimates (Limpert et al 2001, Haeckel and Wosniok 2010). Due to the small number of samples (n = 5) the average amylase activity of the contaminated group could not be reliably estimated. On the other hand, our estimates of 0.2 ∗ / 4.5 nmol min−1 (n = 35) and 0.6 ∗ / 2.9 nmol min−1 (n = 18, EBC samples with potential minor oropharyngeal contamination excluded) amylase activity per ml non-contaminated EBC, are based on an adequate sample size. To account for varying dilution during EBC collection we calculated the specific amylase activity and found geometric
Figure 3. Identification of contaminated EBC. Scatter plot of amylase activity against colloidal gold staining units. (•) EBC, (•∗ ) EBC with salivary protein contamination, (◦) diluted saliva samples. The amylase activity of the saliva samples depicted was calculated by dividing the mean amylase activity of ten saliva samples (40.5 μmol min−1∗ ml) by known dilution factors (figure 2(C) and dot-blot (not shown)). In the inset, the ordinate (amylase activity) is scaled up five-fold and all values above 8 nmol min−1∗ ml are omitted for better visibility of the two groups converging.
means of 0.03 ∗ / 3.9 nmol min−1∗ μg (n = 35) or 0.07 ∗ / 2.9 nmol min−1∗ μg (n = 18), respectively. Most important, the average relative oropharyngeal protein contamination of EBC samples judged contaminated according to figure 3 was more than ten times higher than the contamination calculated under the paradigm that any amylase activity detectable in EBC indicates oropharyngeal contamination, namely 2.2% versus 0.1% (based on medians) or 3.2% versus 0.2% (based on geometric means), respectively. 6
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4. Discussion
has to remain open, but it seems clear that EBC amylase activity not necessarily indicates oropharyngeal contamination due to careless EBC collection. Following the common practice of interpreting any amylase activity found in EBC as oropharyngeal contamination relative oropharyngeal protein contents of EBC of 1.7%, 1.6%, and 0.05% can be calculated from published data on amylase activity in EBC of healthy volunteers (Effros et al 2002, 2005, Husz´ar et al 2002). On the background of our study the divergent values can be easily reconciled. For our experimentally defined contaminated group we found 2.2% oropharyngeal protein content (median), which lies just above the values of Effros et al 2002 and Husz´ar et al 2002, who found amylase activity only in part of their EBC samples since a relatively insensitive amylase assay was applied. The majority of their samples was therefore excluded from the calculation for technical rather than experimentally justified reasons. If a more sensitive amylase assay is used, amylase activity was detected in all EBC-samples and a relative oropharyngeal protein content of 0.05% results (Effros et al 2005). This lower value is of the same order of magnitude as our value of 0.1% (median) which we calculated including all samples. Clearly, the most interesting and useful finding of our study is that EBC samples containing amylase activity of pulmonary origin may perhaps be identified by plotting EBC amylase activity against colloidal gold staining intensity on dot-blots. However, further studies investigating the identity of pulmonary amylase with antibodies against salivary or pancreatic amylase and proper validation and standardization of our method are necessary before it may eventually be applied to detect oropharyngeal or gastric aspiration in EBC of mechanically ventilated or lung transplant patients.
In our search for a more sensitive amylase assay we first compared three amylase assays and chose to increase the sensitivity of the CNP-G3 amylase assay by extending the reaction time. We found that adding BSA to the reaction mix raised the apparent activity of salivary amylase five-fold and increased the half-life of enzyme activity from 2 to 38 h. With our improved CNP-G3 activity assay we were able to detect amylase activity well above the assays lower detection limit in as little as 30 μl of every single EBC-sample we analyzed (n = 40). In stark contrast, other authors found amylase activity only in a fraction of their EBC samples (Husz´ar et al 2002, Effros et al 2002, Gaber et al 2006) or had to concentrate them (Effros et al 2005). After proper validation and standardization our enhanced CNP-G3 assay may therefore find application in breath research and other fields of inquiry where a highly sensitive amylase assay is essential. While testing buffer components to maximize protein binding to nitrocellulose filters we observed that EBC samples, despite similar total protein concentration, strongly varied in their content of proteins binding to nitrocellulose as judged by colloidal gold staining intensity. This prompted us to develop a dot-blot assay for salivary contamination since we had also observed that saliva diluted up to 150 000-fold still produced stainable dots on nitrocellulose filters. However, a scatter plot of EBC amylase activity against EBC colloidal gold staining intensity showed a very peculiar pattern: The samples with the five topmost amylase activities had only intermediate staining intensities and correlated with results from highly diluted saliva, while the majority of EBC samples in clear separation scattered over the whole range of colloidal gold staining intensities at low to intermediate amylase activity. At very low amylase activity and low staining intensity the two groups converged. We hypothesize that EBC samples aligned with diluted saliva contained amylase activity introduced during EBC collection and that the amylase activity found in all other EBC samples was of pulmonary origin. That amylase indeed could be a component of airway lining fluid can be inferred from the analysis of bronchoalveolar lavage (BAL), although the presence of amylase in BAL, similar to EBC amylase activity, could be an experimental artifact. Amylase activity was found in BAL of mechanically ventilated patients (Clarke et al 1981, Nandapalan et al 1995, Weiss et al 2013) and salivary as well as pancreatic amylase were found in BAL of lung transplant patients in a proteomic study (Kosana et al 2012). Histological evidence for pulmonary amylase expression comes from analysis of surgically resected specimens from lobar and segmental bronchi (Hayashi et al 1986). Both, immunohistological and activity based histochemical staining of amylase was found primarily in serous cells of bronchial glands and with less intensity in the apical part of ciliated epithelial cells. Although the antibody used in this study was raised against salivary amylase, it cross-reacted with the pancreatic isoform. In conclusion, whether and in which proportion amylase present in BAL originates from pulmonary expression or aspiration of oropharyngeal or gastric material
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