Targeted Compound Analysis with Breath Biopsy · enzymes in vivo, as well as the function of...
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• After five decades of breath research focussing on the identification of endogenous VOCs as disease biomarkers, no breath test using VOCs is routinely used in clinic. • Hypothesis-driven administration of exogenous compounds could provide a means to investigate the analysis of selected compounds in breath. • We propose a novel approach that uses exogenous VOC (EVOC) probes to measure the activity of metabolic enzymes in vivo, as well as the function of organs, through breath analysis. Introduction • Ingestion of an EVOC probe resulted in a marked increase in concentration of terpenes in breath after 30 minutes, compared to baseline levels (Figure 1). • No increase was observed for endogenous compounds not contained in the EVOC probe, such as acetone and isoprene. Terpenes as EVOC Probes • EVOC Probes hold great potential for development of specific disease biomarkers – by building on understanding of biological pathways, specific metabolic targets can be identified and exploited to reveal the presence of disease. • EVOC Probe strategies could increase signal-to-noise ratio, and help to overcome challenges associated with intra- and inter-individual variation in biomarker measurements. • EVOC Probes could herald a new wave of substrate-based breath tests, translating breath biopsy to clinical applications. Conclusions VOCs are low molecular weight metabolites that are excreted in breath as a result of metabolic processes in the body, and can be of endogenous and/or exogenous origin. Origins of Endogenous and Exogenous VOCs in Breath The kinetics of metabolism and subsequent breath excretion of the EVOC probe, or of its products, could enable in vivo assessment of the activity of specific metabolic enzymes and organ function. EVOC Probes for In Vivo Metabolic Phenotyping Almost fifty years after the first studies in breath research [1], while many biomarkers have been proposed in proof of concept studies, there are no VOC-based tests routinely applied in the clinic. Why? These challenges cause research results to gravitate to the exhaled VOCs occurring in the widest range of subjects at the highest concentrations. Consequently, certain abundant VOCs, e.g. acetone and isoprene, are overrepresented in the literature, and are reported to be associated with a wide range of health states. Historical Challenges in Breath Research Reproducible fold changes were observed in longitudinal study over 5 weeks (Figure 2). High Reproducibility in Breath Measurement of EVOC Probes EVOC Probes Overcome Challenges Associated with Biological Variability EVOC probes resulted in a significant increase in limonene levels, generating a separated distribution of breath limonene concentrations (Figure 3). [1] Pauling L, Robinson A B, Teranishi R and Cary P 1971 Quantitative analysis of urine vapor and breath by gas-liquid partition chromatography Proc. Natl. Acad. Sci. U. S. A. 68 2374–6. [2] Broadhurst D I and Kell D B 2006 Statistical strategies for avoiding false discoveries in metabolomics and related experiments Metabolomics 2 171–96. References owlstonemedical.com Targeted Compound Analysis with Breath Biopsy Aditya Malkar, Edoardo Gaude, Stefano Patassini, Jasper Boschmans, Morad K Nakhleh, Max Allsworth, Billy Boyle, Marc van der Schee Owlstone Medical Ltd, 183 Cambridge Science Park, Cambridge CB4 0GJ, UK GENOMICS TRANSCRIPTOMICS PROTEOMICS METABOLOMICS mRNA protein metabolites gene ENVIRONMENT MICROBIOME EXOGENOUS VOC SOURCES ENDOGENOUS VOC SOURCES VOCs produced from internally available metabolic substrates - part of physiological and pathological mechanisms VOCs originate from metabolism and distribution of external compounds that are introduced in the body Number of subjects Number of features < 1 -8 0 5 r = 0.64 r = 0.69 10 15 20 25 -6 -4 -2 3 8 6 4 0 Acetone isoprene Found to be associated with many unrelated diseases • Lung cancer • Cystic fibrosis • Asthma • Malaria • Pneumonia • Influenza • Renal disease • Liver disease • etc….. SOME PUZZLING QUESTIONS 1 2 SOME HISTORICAL CHALLENGES WITHOUT SOLVING THESE YOU CAN'T HAVE CONFIDENCE IN BIOMARKER DISCOVERY AND VALIDATION Why is there very little agreement in identified biomarkers within a disease? Why is breath testing not used routinely in clinical setting? • Typically small-size cross-sectional case-control studies screening as many potential biomarkers as possible • A small ratio of subjects to biomarkers (typically 1:5 - 1:10) • High risk of overfitting, resulting in falsely identified ‘voodoo’ correlations [2] • Risk of obscuring true biomarkers LIMITATIONS OF 'OMICS STUDIES A 'MEASURE-ALL' UNTARGETED APPROACH CAN BE CHALLENGING WHEN SUBJECT NUMBERS ARE RELATIVELY SMALL COMPARED TO THE MEASURED FEATURES • EVOC probes are volatile compounds that, administered to a subject through various routes, undergo metabolism and distribution in the body and are excreted via breath. • Virtually any exogenous VOC that is metabolized by the human body can offer a readout of metabolic enzymes/organs. Targeted Breath Analysis using EVOC Probes Absorption Exogenous VOCs and volatile metabolic products Valuable, specific, quantifiable, characteristic metabolic information • Targeted approach facilitates rigorous method development • Enables high performance for a target analyte • Ensures any trends in the data can be attributed to biology rather than technical variability α-Pinene β-Pinene Limonene Eucalyptol p-Methan-3-one Acetone Isoprene 0.0 15.0 25.0 35.0 45.0 20.0 30.0 40.0 50.0 5.0 10.0 CTRL CTRL 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 PEAK AREA FOLD CHANGE vs. CONTROL TIME AFTER INGESTION OF PEPPERMINT CAPSULE (HOURS) Figure 1: Washout curves of acetone, isoprene, and different terpenes/terpenoids from one healthy subject at baseline (Ctrl) and after ingestion of the EVOC probe (peppermint oil capsule). Data were normalized on average baseline levels, error bars represents SD of 2 breath samples obtained simultaneously by the ReCIVA Breath Sampler at each time point. Figure 2: Boxplots of breath levels of acetone, isoprene and different terpenes at baseline (before EVOC), peak (45 min after EVOC) and 3 hours after EVOC, acquired across 5 weeks in one healthy subject. *, **, and *** represent paired t-student p-value < 0.05, 0.01, 0.001, respectively. n.s. = not significant. Figure 3: Comparison of background levels of limonene and EVOC-induced limonene changes. Breath concentrations of limonene were measured in 136 subjects (blue circles) and compared with limonene levels after EVOC probe administration (orange dots). 0.0 2.5 7.5 5.0 10.0 BASELINE PEAK 3 HOURS FOLD CHANGE vs. BASELINE Acetone 0.0 2.5 7.5 5.0 10.0 BASELINE PEAK 3 HOURS 0 10 20 30 BASELINE PEAK 3 HOURS 0 10 20 30 40 BASELINE PEAK 3 HOURS Isoprene α-pinene β-pinene 0.0 2.5 7.5 5.0 10.0 BASELINE PEAK 3 HOURS`` FOLD CHANGE vs. BASELINE Limonene 0.0 2.5 7.5 5.0 10.0 BASELINE PEAK 3 HOURS 0.0 2.5 5.0 10.0 7.5 12.5 BASELINE PEAK 3 HOURS 0 20 40 60 80 BASELINE PEAK 3 HOURS Eucalyptol Menthol p-Menthan-3-one—2- n.s. n.s. n.s. ** ** ** * *** 0 0.4 0.8 0.6 1.0 0.2 Concentration (ppb) Limonene • Modern breath testing commenced in 1971, with the work of Nobel Prize winner Linus Pauling • Hundreds of scientific papers published suggesting the presence of VOC biomarkers across a range of diseases • High end, expensive spectrometer vs. low performance eNose • Different analytical techniques required in biomarker discovery and clinical translation • Study design and size - small patient numbers in pilot studies and lack of blinded validation studies • Maturity of breath sampling hardware and protocols for robust, repeatable sampling COMMON ENDOGENOUS VOCS LIKELY TO BE HIGHLY UNSPECIFIC BIOMARKERS EVOC probe EVOC probe • Stable isotope tests are based on administration of 13 C labelled compounds, biotransformation of the substrate to generate 13 CO 2 , followed by isotope enrichment analysis in breath • A powerful strategy for assessing metabolic phenotypes and organ functions in vivo • All probes lead to breath secretion of 13 CO 2 - can only assess one target at a time • Only a handful of products approved for commercialization – perhaps because if invasive intravenous administration of the probe is required, this impacts on regulatory approval Stable Isotope Probes – Proven to Enable Highly Accurate Breath Tests 13 C-labelled Substrate 13 CO 2 13 CO 2 • Breath clearance of the EVOC probe can be monitored in breath, as well as multiple products that can derive from metabolism of the EVOC probe by specific enzymes • Can administer a cocktail of probes – test multiple targets • Completely non-invasive • Targeted hypothesis-driven approach EVOC product(s) TIME VOCs EVOC Product(s)
Transcript of Targeted Compound Analysis with Breath Biopsy · enzymes in vivo, as well as the function of...
• After five decades of breath research focussing on the identification of endogenous VOCs as disease biomarkers, no breath test using VOCs is routinely used in clinic.
• Hypothesis-driven administration of exogenous compounds could provide a means to investigate the analysis of selected compounds in breath.
• We propose a novel approach that uses exogenous VOC (EVOC) probes to measure the activity of metabolic enzymes in vivo, as well as the function of organs, through breath analysis.
Introduction
• Ingestion of an EVOC probe resulted in a marked increase in concentration of terpenes in breath after 30 minutes, compared to baseline levels (Figure 1).
• No increase was observed for endogenous compounds not contained in the EVOC probe, such as acetone and isoprene.
Terpenes as EVOC Probes
• EVOC Probes hold great potential for development of specific disease biomarkers – by building on understanding of biological pathways, specific metabolic targets can be identified and exploited to reveal the presence of disease.
• EVOC Probe strategies could increase signal-to-noise ratio, and help to overcome challenges associated with intra- and inter-individual variation in biomarker measurements.
• EVOC Probes could herald a new wave of substrate-based breath tests, translating breath biopsy to clinical applications.
Conclusions
VOCs are low molecular weight metabolites that are excreted in breath as a result of metabolic processes in the body, and can be of endogenous and/or exogenous origin.
Origins of Endogenous and Exogenous VOCs in Breath
The kinetics of metabolism and subsequent breath excretion of the EVOC probe, or of its products, could enable in vivo assessment of the activity of specific metabolic enzymes and organ function.
EVOC Probes for In Vivo Metabolic Phenotyping
Almost fifty years after the first studies in breath research [1], while many biomarkers have been proposed in proof of concept studies, there are no VOC-based tests routinely applied in the clinic. Why?
These challenges cause research results to gravitate to the exhaled VOCs occurring in the widest range of subjects at the highest concentrations. Consequently, certain abundant VOCs, e.g. acetone and isoprene, are overrepresented in the literature, and are reported to be associated with a wide range of health states.
Historical Challenges in Breath Research
Reproducible fold changes were observed in longitudinal study over 5 weeks (Figure 2).
High Reproducibility in Breath Measurement of EVOC Probes
EVOC Probes Overcome Challenges Associatedwith Biological Variability
EVOC probes resulted in a significant increase in limonene levels, generating a separated distribution of breath limonene concentrations (Figure 3).
[1] Pauling L, Robinson A B, Teranishi R and Cary P 1971 Quantitative analysis of urine vapor and breath by gas-liquid partition chromatography Proc. Natl. Acad. Sci. U. S. A. 68 2374–6.
[2] Broadhurst D I and Kell D B 2006 Statistical strategies for avoiding false discoveries in metabolomics and related experiments Metabolomics 2 171–96.
References
owlstonemedical.com
Targeted Compound Analysis with Breath BiopsyAditya Malkar, Edoardo Gaude, Stefano Patassini, Jasper Boschmans, Morad K Nakhleh,
Max Allsworth, Billy Boyle, Marc van der Schee
Owlstone Medical Ltd, 183 Cambridge Science Park, Cambridge CB4 0GJ, UK
GENOMICS TRANSCRIPTOMICS PROTEOMICS METABOLOMICS
mRNA protein metabolitesgene
ENVIRONMENT MICROBIOME
EXOGENOUS VOC SOURCESENDOGENOUS VOC SOURCES
VOCs produced from internally availablemetabolic substrates - part of physiological
and pathological mechanisms
VOCs originate from metabolismand distribution of external
compounds that areintroduced in the body
Number of subjectsNumber of features
< 1
-80 5
r = 0.64
r = 0.69
10 15 20 25
-6
-4
-2
3
8
6
4
0
Acetone
isoprene
Found to be associated with many unrelated diseases• Lung cancer• Cystic fibrosis• Asthma • Malaria• Pneumonia• Influenza• Renal disease• Liver disease• etc…..
SOME PUZZLING QUESTIONS
1 2SOME HISTORICAL CHALLENGES
WITHOUT SOLVING THESE YOU CAN'THAVE CONFIDENCE IN BIOMARKER
DISCOVERY AND VALIDATION
Why is there very little agreement in identified biomarkers within a disease?
Why is breath testing not used routinely in clinical setting?
• Typically small-size cross-sectional case-control studies screening as many potential biomarkers as possible
• A small ratio of subjects to biomarkers (typically 1:5 - 1:10)• High risk of overfitting, resulting in falsely identified ‘voodoo’ correlations [2]
• Risk of obscuring true biomarkers
LIMITATIONS OF 'OMICS STUDIES
A 'MEASURE-ALL' UNTARGETEDAPPROACH CAN BE CHALLENGING
WHEN SUBJECT NUMBERS ARERELATIVELY SMALL COMPARED TO
THE MEASURED FEATURES
• EVOC probes are volatile compounds that, administered to a subject through various routes, undergo metabolism and distribution in the body and are excreted via breath.
• Virtually any exogenous VOC that is metabolized by the human body can o�er a readout of metabolic enzymes/organs.
Targeted Breath Analysis using EVOC Probes
Absorption
ExogenousVOCs and volatile
metabolic products
Valuable, specific,quantifiable, characteristic
metabolicinformation
• Targeted approach facilitates rigorous method development
• Enables high performance for a target analyte• Ensures any trends in the data can be attributed
to biology rather than technical variability
α-Pineneβ-PineneLimoneneEucalyptolp-Methan-3-one
AcetoneIsoprene
0.0
15.0
25.0
35.0
45.0
20.0
30.0
40.0
50.0
5.0
10.0
CTRL CTRL 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
PE
AK
AR
EA
FO
LD C
HA
NG
E vs.
CO
NT
RO
L
TIME AFTER INGESTION OF PEPPERMINT CAPSULE (HOURS)
Figure 1: Washout curves of acetone, isoprene, and different terpenes/terpenoids from one healthy subject at baseline (Ctrl) and after ingestion of the EVOC probe (peppermint oil capsule). Data were normalized on average baseline levels, error bars represents SD of 2 breath samples obtained simultaneously by the ReCIVA Breath Sampler at each time point.
Figure 2: Boxplots of breath levels of acetone, isoprene and different terpenes at baseline (before EVOC), peak (45 min after EVOC) and 3 hours after EVOC, acquired across 5 weeks in one healthy subject. *, **, and *** represent paired t-student p-value < 0.05, 0.01, 0.001, respectively. n.s. = not significant.
Figure 3: Comparison of background levels of limonene and EVOC-induced limonene changes. Breath concentrations of limonene were measured in 136 subjects (blue circles) and compared with limonene levels after EVOC probe administration (orange dots).
0.0
2.5
7.5
5.0
10.0
BASELINE PEAK 3 HOURS
FO
LD C
HA
NG
E vs.
BA
SELI
NE
Acetone
0.0
2.5
7.5
5.0
10.0
BASELINE PEAK 3 HOURS
0
10
20
30
BASELINE PEAK 3 HOURS
0
10
20
30
40
BASELINE PEAK 3 HOURS
Isoprene α-pinene β-pinene
0.0
2.5
7.5
5.0
10.0
BASELINE PEAK 3 HOURS``
FO
LD C
HA
NG
E vs.
BA
SELI
NE
Limonene
0.0
2.5
7.5
5.0
10.0
BASELINE PEAK 3 HOURS
0.0
2.5
5.0
10.0
7.5
12.5
BASELINE PEAK 3 HOURS
0
20
40
60
80
BASELINE PEAK 3 HOURS
Eucalyptol Menthol p-Menthan-3-one—2-
n.s.n.s.
n.s.
** **
******
0
0.4
0.8
0.6
1.0
0.2
Co
ncen
trat
ion
(pp
b)
Limonene
• Modern breath testing commenced in 1971, with the work of Nobel Prize winner Linus Pauling
• Hundreds of scientific papers published suggesting the presence of VOC biomarkers across a range of diseases
• High end, expensive spectrometer vs. low performance eNose
• Di�erent analytical techniques required in biomarker discovery and clinical translation
• Study design and size - small patient numbers in pilot studies and lack of blinded validation studies
• Maturity of breath sampling hardware and protocols for robust, repeatable sampling
COMMON ENDOGENOUS VOCS LIKELY TO BE HIGHLYUNSPECIFIC BIOMARKERS
EVOC probe
EVOC probe
• Stable isotope tests are based on administration of 13C labelled compounds, biotransformation of the substrate to generate 13CO2, followed by isotope enrichment analysis in breath
• A powerful strategy for assessing metabolic phenotypes and organ functions in vivo
• All probes lead to breath secretion of 13CO2 - can only assess one target at a time• Only a handful of products approved for commercialization – perhaps because if invasive
intravenous administration of the probe is required, this impacts on regulatory approval
Stable Isotope Probes – Proven to Enable HighlyAccurate Breath Tests
13C-labelledSubstrate
13CO2
13CO2
• Breath clearance of the EVOC probe can be monitored in breath, as well as multiple products that can derive from metabolism of the EVOC probe by specific enzymes
• Can administer a cocktail of probes – test multiple targets
• Completely non-invasive• Targeted hypothesis-driven approach
EVOC product(s)
TIME
VO
Cs
EVOCProduct(s)
