Mass-Spectrometric Detection of Omega-Oxidation Products of

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Mass-spectrometric detection of omega-oxidation products of aliphatic fatty acids in exhaled breath Martin Thomas Gaugg, Tobias Bruderer, Nora Nowak, Lara Eiffert, Pablo Martinez-Lozano Sinues, Malcolm Kohler, and Renato Zenobi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02092 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on August 31, 2017

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Analytical Chemistry

Mass-spectrometric detection of omega-oxidation products of aliphatic fatty acids in exhaled breath Martin Thomas Gaugg1, Tobias Bruderer1,2, Nora Nowak1, Lara Eiffert1, Pablo Martinez-Lozano Sinues1,+, Malcolm Kohler3,4,5, Renato Zenobi1,* 1

Department of Chemistry and Applied Biosciences, Federal Institute of Technology, Zurich, Switzerland

2

Division of Respiratory Medicine, University Children’s Hospital Zurich and Children’s Research Center Zurich

3

Department of Pulmonology, University Hospital Zurich, Zurich, Switzerland

4

Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland

5

Zurich Center for Interdisciplinary Sleep Research, University of Zurich, Switzerland

* ETH Zurich, HCI E 329, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland, Phone: +41 44 632 43 76, [email protected] +

Present address: University Children’s Hospital Basel, University of Basel, Basel, Switzerland

Abstract Omega-oxidation is a fatty acid degradation pathway that can occur alternatively to the dominant β-oxidation. The dysregulation of fatty acid oxidation has been related with a variety of diseases, termed fatty acid oxidation disorders. This work shows evidence for real-time detection in exhaled breath of the complete series of saturated linear ω-hydroxyalkanoic acids, ω-oxoalkanoic acids and alkanedioic acids with carbon chain lengths of 5-15.

We present a comprehensive analytical

workflow using on-line and subsequent off-line methods: secondary electrospray ionization mass spectrometry of exhaled breath and UHPLC-HRMS/MS experiments using exhaled breath condensate, respectively. By analyzing on-line breath measurements of 146 healthy individuals, we were able to obtain strong evidence for the correlation of these metabolite families. This enabled us to monitor the full ω-oxidation pathway in human exhaled breath. We could unambiguously identify these compounds, many of which have never been reported in breath so far. This comprehensive study on breath metabolites reinforces the notion of breath as a valuable source of information, which is underexploited in metabolomics.

Introduction It has long been known that exhaled breath contains crucial information about the current metabolic state of a person.1 This led to the proposal that breath analysis could be used to diagnose a variety of diseases non-invasively. Several methods have emerged over the last decades to analyze the composition of breath. The most prominent ones are gas chromatography mass spectrometry (GC/MS) for off-line analysis 2, and proton-transfer-reaction mass spectrometry (PTR-MS) selected ion flow tube mass spectrometry (SIFT-MS)

4

3

and

for on-line analysis of exhaled breath.

1 Environment ACS Paragon Plus


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Nonetheless, only very few exhaled biomarkers are used in clinical practice to date. Reasons for this include difficulties in sample preparation when working with off-line methods and limited sensitivity for higher-molecular weight metabolites in on-line analysis.5 Many metabolites of interest are present in very low abundance in exhaled breath (ppb to ppt levels) in an incredibly complex mixture of endogenous and exogenous compounds 6. Secondary electrospray ionization mass spectrometry (SESI-MS) was proposed as a way of detecting polar, high molecular weight species in breath in an on-line fashion, with an unprecedented sensitivity and selectivity 7. Nonetheless, like all current online methods, it has the inherent limitation that unambiguous compound identification is difficult, due to the lack of orthogonal separation methods such as liquid chromatography. The use of UHPLCMS measurements of exhaled breath condensate (EBC) as a complementary method for identifying the metabolites detected on-line with SESI-MS combines the strengths of both methods. Through this approach, a variety of new compounds has been detected on-line in exhaled breath, including different aldehydes 8, linear aliphatic fatty acids

9

and more

10,11

. However, previous reports have

focused solely on the identification of these structures in breath. In this work, we additionally use a large number (N = 146) of on-line breath measurements to investigate correlations between the targeted metabolites. This provides crucial information about the metabolic origin of these compounds.

A range of diseases has been shown to be connected with abnormal fatty acid degradation, making the associated metabolites of great interest for medical research

12-14

. The main degradation

pathways are β-oxidation, α-oxidation and ω-oxidation, with the first being the dominant route. The latter is still poorly understood and considered to be a “rescue pathway” in case of disturbed βoxidation 15. It was also found to be the primary catabolic pathway of leukotriene B4, which plays a key role in regulation of inflammation

16-19

. The first step of the ω-oxidation pathway involves the

introduction of a hydroxyl group in the ω-position of the fatty acid. Therefore, compounds that are unsuitable substrates for β-oxidation (i.e. 3-methyl-substituted fatty acids), can also be oxidized. The primary alcohol can then be further oxidized to the aldehyde and carboxylic acid, which is excreted or subjected to further β-oxidation

20

. Since real-time breath analysis can give an insight into the

metabolism of a person quickly and noninvasively, detection of these compounds might be of great diagnostic value. While products of β- and α-oxidation have been reported in exhaled breath, apart from a few isolated molecules, ω-oxidation products were largely lacking

6,21,22

. Here we report the

detection of a whole series of ω-oxidation products of unbranched medium-chain fatty acids in exhaled breath, namely ω-hydroxyalkanoic acids, ω-oxoalkanoic acids and alkanedioic acids with carbon chain lengths of 5-15, and also provide evidence for their metabolic connection.


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Materials and methods Chemicals and solvents. Solvents used for UHPLC-MS measurements were water (LC/MS Grade, Merck Millipore), methanol (LC/MS grade, Fisher Chemical) and formic acid (≥ 98%, Aldrich Fine Chemicals). Purchased standards were 8-hydroxyoctanoic acid (≥ 98.5%, Fluka Chemie AG), 2-hydroxyoctanoic acid (> 98%, AlfaAesar), 10-hydroxydecanedioic acid (Apollo Scientific Ltd), 11-hydroxy-undecanoic acid (96%, Aldrich Fine Chemicals), pentanedioic acid (99%, Aldrich Fine Chemicals), heptanedioic acid (≥99%, Fluka Chemie AG), octanedioic acid (≥ 98%, Fluka Chemie AG), decanedioic acid (≥ 95%, Fluka Chemie AG), undecanedioic acid (> 97%, Tokyo Chemical Industry), dodecanedioic acid (99%, Acros Organics) and tetradecanedioic acid (99%, Aldrich Fine Chemicals). Since none of the ω-oxoalkanoic acids were commercially available, 10-oxodecanoic acid was synthesized in our lab as described in the literature, and the identity of the product was confirmed by NMR 23,24 (see supporting information for experimental details). Exhaled breath condensate sampling. EBC was collected using a custom-built sampling device following the guidelines by the ATS/ERS task force 25. Two healthy, non-smoking subjects were asked to exhale through a Teflon tube connected to a glass cold trap (T = -78.5 °C, isopropanol/dry ice) twice for approximately 45 min, yielding 2 x 8 mL each. The collected EBC samples were pooled (total volume of 32 mL), 0.5 mL were extracted and stored at 4 °C and the remainder lyophilized to dryness (24 h, p = 0.05 mbar, T = -40 °C) 21. The residue was then dissolved in a mixture of 142.5 µL of the previously aliquoted EBC, 7.5 µL of methanol, and acidified with 0.1% formic acid, and directly subjected to UHPLC-HRMS/MS analysis. UHPLC-HRMS/MS analysis. UHPLC separation was done on an ACQUITY UPLC system (I-Class, Waters, MA, USA) with an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 x 50 mm, Waters) with precolumn filter. The flow rate was set to 500 µL min-1 using a binary mixture of solvent A (water with 0.1% formic acid) and solvent B (methanol with 0.1% formic acid). The following gradient was used: 5% B (1 min), 5-95% B (3 min), 100% B (1 min), 5% B (2 min). The column was kept at 30°C and the sampler at 5°C. All standards were dissolved in methanol/water = 5/95 (v/v) with 0.1% formic acid at concentrations of 0.1 - 10 µg mL-1, and analyzed with an injection volume of 10 µl. A commercial quadrupole time-of-flight mass spectrometer (AB Sciex, Triple TOF 5600+, Concord, ON, Canada) was used to measure a mass range of m/z = 40 - 500 in negative ion mode, with 1 - 5 ppm mass accuracy. Product ion scans (PIS) and data dependent acquisitions (DDA) were used for fragment spectra acquisition. The total cycle time was kept at 450 ms to obtain at least 12 points/peak (minimal LC peak width = 6 s) with 80 ms for TOF MS and 320 ms for 1 - 4 product ion scans acquired with a collision energy CE = 10/40/70 eV.



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Real-time SESI-HRMS breath analysis. Real-time breath analysis was performed using a commercial low-flow SESI-ion source 26,27 coupled to the TripleTOF 5600+ mass spectrometer that recorded spectra from 50-350 Da in negative ion mode. To gain additional certainty about compound identity, on-line MS2 spectra were recorded for selected compounds in breath. The heated breath sampling tube (130°C) was equipped with a low-∆p mass flow controller (Bronkhorst, Switzerland), which was set to 200 mL min-1. To enable sampling of end-tidal breath, a flow-splitter was installed front-end. A database of on-line breath measurements of 146 people with no symptoms of lung diseases, which were measured in a similar fashion on a different SESI-source (see SI), was used to analyze correlations between the metabolites. The peak intensities of this control dataset were batch corrected used a moving-median single point correction (spanning = 20 samples, measurement date as covariant) for each m/z value, to reduce confounding time drifts 28. All participants gave written and informed consent to participate. The measurements were approved by the Ethics Committee of the Canton Zurich (PB_2016-00421) and the study was registered on www.ClinicalTrials.gov (NCT02595632).

Results and discussion On-line detection of ω-oxidation metabolites in exhaled breath. A key strength of on-line analysis of exhaled breath is the direct detection of vapors without any sample preparation, which reduces confounding influences. It is therefore a promising tool to get insight into metabolism in a rapid, noninvasive and painless fashion. Using the SESI setup described above, we were able to detect real-time signals of ω-hydroxyalkanoic acids (CxH2xO3), ω-oxoalkanoic acids (CxH2x-2O3) and alkanedioic acids (CxH2x-2O4) with carbon chain-lengths of 5-15 in exhaled breath. The detected molecules thereby cover all transformation steps in the degradation of linear aliphatic fatty acids via the ω-oxidation pathway, as shown in figure 1a. Figure 1b shows the time-traces of these three families of compounds over the course of three exhalations of a healthy subject in negative ion mode. The main factors that determine breath concentration of metabolites are systemic and lining fluid concentrations as well as compound volatility 29. Li et al. were able to monitor the pharmacokinetics of ketamine (injected i.v.) in mouse breath using SESI-MS 30, which clearly shows that SESI-MS is capable of detecting vapors of blood metabolites with very low vapor pressure. Despite the low volatility of the compounds studied, a comparison of the estimated vapor pressure at 37°C of ketamine (v.p. = 2.84x10-4 mmHg) with the compounds in this study (e.g. 8-oxooctanoic acid: v.p. = 3.63x10-4 mmHg) supports that these also reflect systemic metabolites (vapor pressures calculated with SPARC, ARChem). It is visible that the


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signal intensity decreases with increasing chain length, which is in agreement with decreasing vapor pressure and perhaps decreasing systemic abundance. Dicarboxylic acids and ω-oxoacids have been reported to be constituents of environmental aerosols coming from a variety of sources (natural and anthropogenic), which were extensively reviewed recently by Kawamura and Bikkina 31. Therefore, an unknown fraction of these compounds could be of exogenous origin. Additionally, some compounds (predominately dicarboxylic acids) can also be generated by other metabolic pathways (i.e. glutaric acid as an intermediate in lysine and tryptophan metabolism 32,33). To enforce our hypothesis that these compounds are connected by a metabolic transformation cascade, we performed a correlation analysis of these peaks using a database of online SESI breath measurements of 146 subjects that were recorded as part of other ongoing medical case-control studies in our group. As expected, a significant correlation between and within these families was observed. Figure 1c shows the normalized intensities of 11-hydroxyundecanoic acid, 11-oxoundecanoic acid and undecanedioic acid plotted against each other. Pearson correlation coefficients of 0.91 (11-hydroxyundecanoic acid vs. 11-oxoundecanoic acid), 0.85 (11-hydroxyundecanoic acid vs. undecanedioic acid) and 0.86 (11-oxoundecanoic acid vs. undecanedioic acid) underline the strong connection between these compounds. The same picture holds true for all studied compounds (see SI), supporting the hypothesis that these molecules indeed reflect the ω-oxidation pathway. Interestingly, correlations of short-chain fatty acid metabolites appear weaker than those of longer chain metabolites. We attribute this to confounding exogenous influences by background concentrations of these compounds. The abundance of dicarboxylic acids and ω-oxoacids in environmental aerosols correlates negatively with their chain length 34. Therefore, a stronger confounding effect on the correlation is expected for compounds of lower molecular weight, due to their higher abundance in environmental air. Off-line identification of ω-oxidation metabolites in exhaled breath condensate. Since on-line analysis lacks a separation step, compound identification is difficult and isomers cannot be distinguished. We therefore used UHPLC-MS analysis of EBC as a complementary method to confirm the identity of the compounds. Due to confounding dilution effects by water in EBC, UHPLC-MS experiments were used only to obtain qualitative information about the structures. Table 1 gives an overview of all compounds detected in this work. We were able to observe chromatographic peaks for all diacids (C5-15), ω-oxoalkanoic acids (C5-15) and ω-hydroxyalkanoic acids (C5-15). To confirm the identity of the compounds, retention times and fragment spectra were compared to commercial standards where available. In total, seven alkanedioic acids, one ω-oxoalkanoic acid, and three ωhydroxyalkanoic acids were confirmed in this manner, and 23 compounds showed sufficient signal intensity to obtain MS2 fragmentation spectra. The full data (chromatograms and MS2 spectra with



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assigned neutral losses) is presented in supporting information. Figure 2 shows the extracted ion chromatograms of the [M-H]- ions of the linear alkanedioic acids in an UHPLC-MS run of EBC (a) as well as of the measured commercial standards (b). Figure 2c shows the head-to-tail plot of the MS2 spectra of octanedioic acid from EBC and the standard. To gain further evidence that this compound is also detected in the real-time analysis, we compared the fragments in an on-line MS2 spectrum of exhaled breath to the standard (figure 2d). Due to the lack of chromatographic separation in on-line analysis, multiple isobaric precursors are selected (precursor selection: 173.1 ± 0.35 Da), leading to a very complex fragment spectrum. Because of the high mass resolution of the TOF analyzer (R ≈ 30,000 at m/z = 173), we were nonetheless able to assign the corresponding real-time fragments to the fragments of the standard. While the alkanedioic acids showed strong fragmentation at the conditions used, the ω-hydroxyalkanoic acids and ω-oxoalkanoic acids showed only minor fragments. The most prominent neutral losses of the alkanedioic acids are [M-H2O]- for C10-C15, [M-CO2]- for C5-C7, and [M-H2O-CO2]- for all compounds, which is consistent with their chemical structure. While ω-hydroxyalkanoic acids and ω-oxoalkanoic acids both show neutral losses of 46.005 Da and 18.012 Da, probably corresponding to [M-CH2O2]- and [M-H2O]-, only in case of the ω-oxoalkanoic acids a [M-CO2]- peak could be detected. All measured standards showed a good match in retention time and MS2 spectra when compared to EBC. We used the NIST MS Search 2.0 software to compute matching scores (normal and reverse) for the fragment spectra, which ranged from 851 - 979. It is based on a modified cosine of the angle between the spectral vectors, where 800 - 900 indicates a good and 900 - 999 an excellent match 35. The retention time and fragments of 10-oxodecanoic acid matched well with the synthesized standard (NIST matching score = 851) and the fragmentation patterns of 6-oxohexanoic acid, 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid in EBC all showed a similar fragmentation behavior as that of the 10-oxodecanoic standard (small neutral losses of H2O, CO2 and CH2O2). Although ω-oxidation is considered a minor pathway of fatty acid degradation, this work clearly shows that metabolites produced via ω-oxidation are crucial components of exhaled breath. While alkanedioic acids show only one main chromatographic peak (except for pentanedioic acid), hydroxyalkanoic acids show multiple retention times, probably representing different positions of the hydroxyl group. For hydroxyoctanoic acid (C8H16O3) we could observe three main chromatographic peaks in EBC, with 8-hydroxyoctanoic acid eluting first and 2-hydroxyoctanoic acid last (both confirmed by standard measurements, see figure 3). The middle peak most probably corresponds to 3-hydroxyoctanoic acid as it shows the fragment ion [C2H3O2]-, resulting from a


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McLafferty-type rearrangement that leads to cleavage of the C(2)-C(3) bond and the formation of a stable enolate anion 36. Several compounds studied in this work have been reported as potential markers of lung cancer 37 and different fatty acid oxidation disorders 12,38,39. All of them have been reported in body fluids, mainly blood, urine, or saliva. While off-line analysis often provides more detailed information about compound identity, several issues with the necessary sample handling have been discussed in literature. These include storage 40, temperature stability 41, dilution effects 42, and others, which can lead to confounding biases when performing global metabolomic studies 43,44. By using on-line SESI-MS analysis of exhaled breath for population screenings, and subsequent targeted UHPLC-MS experiments of EBC, we can circumvent these potentially confounding influences, but are still able to perform unambiguous metabolite identification. Nonetheless, obtaining absolute gas-phase concentrations by SESI-MS remains a challenge. Further work should be conducted in order to develop standard delivery systems that can generate robust gas-phase concentrations of these lowvolatility compounds to enable reliable quantification.

Conclusions We present here a comprehensive analytical workflow, using on-line and subsequent off-line methods together with a correlation analysis using on-line breath measurements of 146 healthy individuals, to obtain information about compound identity as well as metabolic origin. We thereby show that metabolites of ω-oxidation are significant constituents of exhaled breath that can be detected in an on-line fashion with SESI-MS. To the best of our knowledge, this is the first time these complete series of ω-hydroxyalkanoic acids, ω-oxoalkanoic acids and alkanedioic acids with carbon chain lengths of 5 - 15 have been detected by on-line breath analysis, combined with unambiguous compound identification. This not only expands the scope of potential new biomarkers, but also shows that breath analysis with SESI-HRMS/MS in combination with UHPLC-HRMS/MS is a powerful tool to gain insight into the metabolism of humans.



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Tables ω-Hydroxyalkanoic acids

ω-Oxoalkanoic acids

Alkanedioic acids

(CxH2xO3)

(CxH2x-2O3)

(CxH2x-2O4)

5

0.97

117.0559

5.1

0.95

115.0404

3.7

tr /min 0.96

6

1.90

131.0721

5.6

1.81

129.0562

3.5

1.87

#C

tr /min

m/z

ppm

7

2.38

145.0875

5.1

8

2.74

159.1030a,b

3.5

9

3.03

173.1183

3.4

10

3.28

187.1341a,b

3.9

3.49

a,b

6.0

11

201.1500

NIST match

tr /min

m/z

ppm

NIST match

m/z

ppm

131.0354a,b

5.1

145.0513

5.6 a,b

NIST match 947

2.34

143.0717

2.1

2.32

159.0672

5.1

979

2.70

157.0876

3.7

2.68

173.0828a,b

3.5

962

3.00

171.1032

3.3

2.98

187.0983

3.4

911

3.24

185.1189a,b

3.4

3.22

201.1139a,b

3.9

973

908

3.45

199.1347

3.4

3.43

215.1297a,b

6.0

968

a,b

3.6

977

900

851

12

3.68

215.1661

3.6

3.64

213.1502

2.9

3.61

229.1459

13

3.82

229.1818

5.4

3.79

227.1662

4

3.77

243.1611

5.4

14

3.94

243.1974

6.1

3.92

241.1820

4.6

3.90

257.1772a,b

6.1

15

4.06

257.2123

5.1

4.03

255.1972

2.3

4.02

271.1932

5.1 -

Table 1: Families of ω-hydroxyalkanoic acids, ω-oxoalkanoic acids and alkanedioic acids detected as the [M-H] ion in EBC with UHPLC-MS and on-line with SESI-MS. For compounds in bold, signal intensity was sufficient to obtain UHPLC-MS2 spectra. The NIST matching scores were computed using the software NIST MS Search 2.0, where 800-900 indicates a good, a

2

and 900-999 an excellent match. Compound confirmed by a match of retention-time and MS spectrum with the standard. b

2

Fragments of standard also detected in an on-line MS experiment.


955

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Figures

Figure 1: On-line detection of ω-oxidation metabolites in exhaled breath. a) Scheme of the ω-oxidation of linear aliphatic fatty acids, where fatty acids are transformed into ω-hydroxyalkanoic acids, ω-oxoalkanoic acids and finally alkanedioic acids. b) Time-traces of the corresponding metabolites detected on-line over the course of three exhalations of a healthy subject as the [M-H] ions in negative mode (au = arbitrary units). The signal for hydroxypentanoic acid is cut to improve the visibility of the others. c) Normalized intensities of hydroxyundecanoic acid, oxoundecanoic acid and undecanedioic acid for on-line measurements of 146 healthy subjects plotted against each other and the corresponding Pearson linear correlation coefficient. Although there might be a signal contribution of structural isomers, a strong correlation is observed. The same picture holds true for all carbon chain lengths from 5 - 15 (see supporting information for data processing and other chain lengths).



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Figure 2: Extracted ion chromatograms of the [M-H]- ions of linear, saturated dicarboxylic acids with chain lengths of 5-15 in an UHPLC-MS run of exhaled breath condensate (EBC) (a) as well as commercial standards (Std) (b). The compounds were confirmed by matching retention time and MS2 fragments with the standards. c) Head-to-tail plot of the base-peak 2 2 normalized MS spectrum of octanedioic acid in EBC (top) and standard (bottom). d) Base-peak normalized on-line MS spectrum of exhaled breath (precursor: 173.1 ± 0.35 Da). The insert shows a zoom of the precursor mass. As no chromatographic separation is performed, multiple parent masses are selected, explaining the abundance of other fragments. Nonetheless, the high resolution TOF analyzer makes it possible to clearly identify all fragments of octanedioic acid, supporting the presence of this compound in breath.

Figure 3: Top: Extracted ion chromatograms of the mass at 159.1027 Da corresponding to the molecular formula [C8H19O3]in an UHPLC-MS run of EBC after blank subtraction (a). The three main peaks were identified as the [M-H]- ions of 8-hydroxyoctanoic acid, 3-hydroxyoctanoic acid and 2-hydroxyoctanoic acid. Bottom: Head-to-tail plot of the base-peak normalized MS2 spectra of 8-hydroxyoctanoic acid (b) and 2-hydroxyoctanoic acid (d) in EBC and the standards, as well as the MS2 spectrum of putative 3-hydroxyoctanoic acid (c). The fragment [C2H3O2]- is formed by a McLafferty-type rearrangement, supporting the position of the hydroxyl group.


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Supporting Information: Experimental and data treatment details, anthropometric data, correlations of ω-oxidation metabolites, synthesis details of 10-oxodecanoic acid, compound identification (chromatograms and MS2 spectra),

Acknowledgments: We would like to thank the Swiss National Science Foundation for financial support of this project (grant # CR23I2_149617).



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For TOC only


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Mass-Spectrometric Detection of Omega-Oxidation Products of

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