Recommendations and pitfalls in measurements of compounds within Exhaled Breath Condensate

2. Recommendations and pitfalls in measurements of compounds within Exhaled Breath Condensate John Hunt, MD Associate Professor of Pediatrics Pulmonology, Allergy & Immunology University of Virginia, USA For the ERS/ATS Joint Task Force on Exhaled Breath Condensate

3. Conflicts of Interest • Founder of Respiratory Research,Inc., which manufactures the RTube EBC collector. • Fan of American „Football“ and European FOOTBALL both of which my son is supposed to play each Saturday AM. Major Conflict.

4. Image stolen without permission from: The Sixth Sense starring Bruce Willis Exhaled Breath Condensate EBC NH3 NO3- NO2- H+ IL-X cysLT 8-isoPGF2α

5. EBC Assays: So easy, a caveman can do it. Cavemen are smart!!!!

6. Because EBC analysis is difficult...

7. EBC--Key Concept #1 • EBC is not a biomarker equivalent to exhaled nitric oxide. • It is not a biomarker at all • EBC is a matrix in which biomarkers with highly diverse chemical properties can be identified. • In this regard, EBC is equivalent to blood, urine, sweat, tears and other body fluids.

8. Key Concept #2 • EBC biomarkers are not clones of each other. • They each have individualized considerations pertaining to their assay.

9. Key Concept #3 • VALIDATE, VALIDATE, VALIDATE • EBC biomarker assays will lie to you if you don’t: • Call their bluff (false positives) • Check out their alibi (false negatives)

11. Storage of EBC prior to assay • Confirm storage stability of your biomarkers of interest in EBC in a pilot study. • To avoid cyclic freeze-thaws, aliquot samples into several tubes when many discrete assays are planned • Immediate storage at < - 70° or colder • Assay as rapidly as your mediator requires • Take your time: Nitrate, pH, DNA, cations(?) • Moderate rush: Nitrite, isoprostanes, LTs, conductivity, proteins. • Hurry as fast as you can: H2O2 • Just forget about it: RNA

12. And….Extremely rapid assay is required for EBC when seeking: • Quarks, Leptons, Neutrinos or • Weapons of Mass Destruction in Iraq

13. Consider: • Opportunities to stabilize your biomarker (or a reaction product) by adding a relevant analyte before freezing. • Assays for Thiobarbiturate reaction products and hydrogen peroxide specifically • Addition of purified albumin to protect protein biomarkers. • Addition of RNA’se inhibitor to condenser prior to collection. • VALIDATE, VALIDATE, VALIDATE

14. Why are you measuring your biomarker(s)?? • Good reasons • To learn about pathologic processes in lung disease • To learn about composition of the airway lining fluid • To validate them as biomarkers of lung disease potentially useful for diagnosis and prognosis, or as objective outcome measures in clinical studies. • Bad reasons: • To get an abstract accepted • To get a paper published • To fulfill research training requirements. • To do something stupid like starting a company

15. H2O2 • Volatile constituent • Reported by multiple laboratories. Dozens of papers. • Reported range generally < 1 uM • 45% intrasubject coefficient of variation • Assay rapidly (how rapidly??) • Colorimetric • Fluorometric • Rapid Biosensor assay (ECoCheck, Viasys)

16. Decay of unstable compounds [uranium 232] Time 15 millenia 30 millenia 60 millenia

17. Decompositoin of unstable compounds [H2O2] Time 15 min 30 min 60 min

18. Nitrogen Oxides • Nitrite, Nitrate, NO2-Tyrosine,S-nitrosothiols • Referred to collectively as “NOx”, “HiNOx”: • Higher Oxides of Nitrogen • Consider each of these separately. • Nitrite and nitrate are likely in part derived from oxidation of absorbed gaseous NO ex vivo. • Nitrite and nitrate are ubiquitous on all lab surfaces, pipette tips, latex gloves, fingers, microcentrifuge tubes, and EBC collection equipment and the inside of your nose.

20. Nitrite (NO2-) • Found at < 200 nM to 6 uM (higher levels highly associated with salivary contamination) • Commonly below detection limit of colorimetric assays. • Borderline usefulness of ion chromatography. • Fully within range of reductive chemilumiscence. • Slow decomposition is pH-dependent • Endotracheally collected samples have substantially lower nitrite levels than oral collections.

21. Nitrate (NO3-) • Found at 1 uM to 20 uM range. Higher suggests salivary contamination. • May be complementary to eNO, but not generally considered to associate with eNO. • No significant difference in levels between matched oral and intubated collections.

22. Nitrotyrosine • Marker of nitrative/oxidative activity in the airways • Assay most commonly used is a sandwich immunoassay • 2 antibody binding sites • Not going to happen on one little amino acid • Sandwich ELISA identifies NITRATED PROTEINS, not the isolated nitrated amino acid. • Assay detects down to 4 ng/ml (of what?) • Identified range < 14 ng/ml (of what?) • Mass spectroscopy assay may be sensitive to pM range. • Few data are available.

23. Nitrosothiols • Relevant endogenous bronchodilators found in airway lining fluid. • Commercial colorimetric assay probably not as sensitive as claimed (25 nM). • Chemiluminescence assay available (20 nM sensitivity) • Uncertain stability in EBC. • Few data are available

24. Adenosine • One of the most technically validated biomarkers in EBC (primarily one group) • Assay—HPLC based, reproducible • Intraassay variability of <10% • Range of levels 0-20 nM • Biomarker with active effects in respiratory system.

25. Prostaglandins and Thromboxanes • Several independent groups have demonstrated these compounds in EBC using discrete assays. • Assays by competitive ELISA and RIA • HPLC based techniques • Specificity of antibodies always a potential issue. • Low pg/ml range • Reportedly small effect size

26. Leukotrienes • A lung biomarker in which there is strong interest because of potential clinical relevance • Identified in various assays by various groups in low pg/ml range • EIA is Competitive ELISA. Intrasample repeat assay correlation is acceptable. Interassay (different assay techniques) may reveal marked discrepancies however. Specificity a concern. • Beware if you are new to this assay!

27. 8-isoprostane • Same issues as for LT’s • A lung biomarker in which there is strong interest because it serves as strong evidence of excessive oxidative processes • Identified in various assays by various groups in low pg/ml range • Assay is Competitive EIA. Intrasample repeat assay correlation is acceptable. Interassay (different assay techniques) may reveal marked discrepancies however. Specificity a concern. • At least one group has clearly validated their EIA against LCMS. • Beware if new to this assay!

28. Lipid peroxidation products • Aldehydes, nexanal, others • Higher levels probably represent injurious oxidative activity • Assay by LC/MS • Levels reported: 40 nM to 1 uM range • Few labs reporting.

29. EBC pH • EBC pH determined by captured volatiles • Acids and bases in the respiratory fluid have differential volatility depending on pH of source fluid • Gas standardization/deaeration is commonly used • Although CO2 is relevant to airway fluid pH, CO2 has no significant differential volatility based on pH of source fluid, and most (but not all) investigators consider it a source of noise in EBC. • gas standardizing to a known CO2 concentration

30. EBC pH • Colorimetric assays, various probes • Validation and reference ranges fairly well established • Intrasubject CV in health: between 3 and 12% • Within range of available assays always • (But this may be a trick!) • Oral ammonia contaminates EBC, does not lead to acidification, but at higher concentrations may decrease the sensitivity of EBC to a lower airway acidity. • Beware of freezing and sublimation (removal of acid from the EBC) • Open storage tubes only after thaw and shaking • Not all pH probes are effective for EBC pH • Consider Ross-type pH electrodes. • Or probes with high fluid leak across reference electrode.

32. Ammonia (NH3/ NH4+) • Volatile constituent • Dominant source is oropharyngeal (80%) • Avoided in intubated subjects • Tells little about lungs. May decrease sensitivity of EBC pH assays to detect true lower airway acidification.

33. Cytokines, chemokines (and proteins in general) • High interest to immune researchers, pharmaceutical companies • Protein compounds. Nonvolatile. • Assay trouble has confounded literature • ELISA usually used.. • Levels at or below detection limit • “Matrix effect” may contribute to false positives

34. Cytokines 2 • Opportunities • Concentration: Lyophilization/resuspension. • Multiplex bead arrays for assay • Potential for thousands of assays on one non-invasive sample • Non-invasive proteomics

35. Assessing salivary contamination • Amylase activity assay • Remember, it will be highly diluted. • Assays needs to be able to identify presence of amylase at 20,000 fold dilution of time-matched saliva samples. • Validate Validate Validate. • What is the stability of Amylase in your freezer? • Can amylase be formed in diseased peripheral lung tissue? • Etc..

36. Measurement of dilution • Quite important, unless • You are measuring volatiles (dilution is irrelevant in that setting) • You are measuring ratios of related compounds • Nitrate/nitrite ratio • IFN gamma/IL-4 ratio • GSH/GSSG ratio • Multiple methods, none well validated yet • Dilution factor assessment requires validation just like any other measurement in EBC before conclusions should be made.

37. Conclusion re: EBC assays • Validate, Validate, Validate • Validate again. • EBC is EASY to Collect. But Hard to Assay! • The biomarkers are there if you look hard enough. • Don’t expect more from an exhaled biomarker than you would from any clinical test. • Clinical tests have tons of problems too! • So we need to continue to use our brains. Like the Cavemen do.

38. Seeking those neutrinos in exhaled breath!