Animal Care

All animals were domestic shorthair, purpose-bred cats. 27 were from a commercial vendor (Liberty Research, Inc, Waverly, NY). The remainder were bred from a high-responder asthmatic cat colony (Comparative Internal Medicine Laboratory, University of Missouri, Columbia MO). There were 39 males and 14 females. The care of the 53 cats in the study followed the NIH Guide for the Care and Use of Laboratory Animals. The University of Missouri Animal Care and Use Committee approved the study design (ACUC protocols #6912 and 7891). A commercial mixture of kitten and adult maintenance dry kibble diet was fed ad libitum. The research cats were housed in groups in large runs, with a variety of enrichment toys (e.g, hanging hammocks, elevated platforms to climb, baby toys, toy mice, balls with bells, etc.). Additionally, the cats were socialized and monitored at least daily by members of the research team, in addition to being monitored by members of the Office of Laboratory Animal Care. None of the cats became ill or died during this study. All of the cats were adopted to private homes after the study.

Induction of Allergic Asthma

In order to induce allergic asthma, on day 0, cats < 1 year of age were administered 12 μg of Bermuda grass allergen (BGA) in 10 mg of alum and 100 ng Bordetella pertussis toxin (both subcutaneously). On day 14, intranasal BGA (75 μg of BGA in 200 μL of phosphate-buffered saline) was given. On day 21, 12 μg of BGA in 10 mg alum was injected subcutaneously. On day 28, the formation of wheals on an intradermal test confirmed sensitization to BGA. For the next 2 weeks, intensive aerosol challenges were administered to all cats with BGA (500 μg of BGA in 4 mL of phosphate-buffered saline) over 10 min in awake and unrestrained cats in a sealed plastic chamber. A nebulizer (Acorn nebulizer, model 646, Devilbis Health Care, Somerset, PA) delivered an aerosol of the BGA solution at an air flow rate of 9.3 l /min. An air compressor (Easy Air 15, Precision Medical, Inc., Northampton, PA) supplied the compressed air flow at a pressure of 2.93 kg/cm². At week 6, samples were collected 24 h after challenge with BGA aerosol (see below).

Collection of Body Fluids and Evaluation of Airway Eosinophilia

In all cats, EBC and BALF were collected before (day 0) and after (week 6) sensitization to BGA. The EBC was collected non-invasively just prior to BALF collection by placing cats in a 25 L plexiglass chamber for 20 to 30 min according to a previously published design with modifications [39]. BALF was collected in a blind fashion under anesthesia using an 8 French red rubber catheter passed through an endotracheal tube according to a previously described protocol [40]. The percentage of eosinophils in each BALF modified Wright’s stained cytospin was determined by counting 200 nucleated cells, with an asthmatic phenotype defined as >17% eosinophils. The EBC and the supernatant of centrifuged BALF remaining were promptly stored at -80°C until further analysis. NMR spectra of the EBC were measured for use in training and evaluating statistical approaches and biomarkers.

Collection of NMR Spectra of EBC

To maximize the sensitivity and resolution of NMR measurements of inherently dilute EBC specimens, we collected their spectra with a Bruker Avance III 800 MHz spectrometer with 5 mm TCI cryogenic probe. EBC samples were prepared with 7% D₂O and 20 μM trimethylsilyl propanoic acid as 0 ppm reference standard. 1D ¹H NMR spectra were collected using W5-WATERGATE water suppression [41] supplemented by presaturation of the water resonance at minimal power. 32,768 transients were normally averaged. After data collection, the ¹H NMR free induction decays were zero filled to 32,768 points, apodized with 2 Hz exponential broadening to enhance sensitivity, and Fourier transformed into spectra spanning from -2 to 12 ppm, with correction of phases and baseline using Bruker Topspin 3.1.

Spectral Preprocessing

About 30,000 points from 0.02 to 10 pm were retained for analyses, while the region around the suppressed water peak from 4.5 to 5.38 ppm was omitted. Preprocessing steps were conducted with Topspin and Origin. To accommodate the large variability in overall concentrations of biomolecules condensed in EBC, the amplitudes of the NMR spectra were normalized to the mean spectrum by probabilistic quotient normalization (PQN) [31]. The first step of the PQN was unit normalization of the integral of each spectrum to 1.0. The median quotient was calculated from the quotients of all the spectral positions (variables) to those of the mean spectrum [31]. Then all variables of each original spectrum were divided by the median quotient of that spectrum. The NMR spectra of EBC specimens were next binned into segments to accommodate slight inequities in NMR peak positions and line shapes as recommended [26]. (EBC spectra are sparse enough to avoid the greater overlap within buckets of spectra of serum and urine [24]. Collecting the spectra at 800 MHz resolved the overlap of peaks enough to improve the reliability of binning. Shortcomings of bins have been discussed [30]). Each spectrum was divided into 455 bins each 0.02 ppm (16 Hz) wide and was then scaled with either Pareto scaling or glog transformation for comparison. Pareto scaling used the following expression where is the scaled peak height [29]: (1) Scaling by glog transformation instead proved much better at increasing the weighting of the smaller variances. It uses the relationship [42]: (2) where λis the glog transformation parameter. λwas obtained by dividing the same sample into eight replicates (aliquots). Each replicate was handled independently for the NMR analysis. The spectra of the replicates were preprocessed identically to ensure that the variances from the replicates arise solely from “technical” variations. In order to avoid the scaling effect caused by the transformation, the modified Jacobian of the glog function proposed [42] was applied. λwas then calibrated using the Maximum likelihood criterion and Nelder-Mead minimization algorithm in MATLAB, using the equation and script provided in ref [42]. Data were mean-centered at the outset of multivariate statistical analysis.

Multivariate Statistical Analyses and Validation

PCA decomposes the spectra (data matrix X) into PCs, which are a linear combination of weighted variables observed in the spectra. The optimized weights generate a loading plot. Each point of the score plot represents a spectrum’s projection onto the PC. The PCs were computed using eigenvectors by singular value decomposition [43]. PCA was performed using the MATLAB Statistics Toolbox. (The R-script princomp is also suitable; see http://www.inside-r.org/r-doc/stats/princomp).

PLS generated components instead using multiple linear regression, not only to model the spectral data matrix X, but also to calculate the projection matrix that maximizes the covariance between X and the class matrix Y of “response variables” [21, 26] or asthmatic phenotypes. Discriminant analysis (DA) was then applied to seek separation between the categories [21, 26] of absence and presence of allergic asthma. PLS-DA was performed with MATLAB using libraries of H. Li available from http://www.mathworks.com/matlabcentral/fileexchange/47767-libpls-1-95-zip. (R script plsda is also suitable: http://www.inside-r.org/packages/cran/mixOmics/docs/plsda). The collection of EBC before and after allergic induction of asthma was exploited in order to evaluate the multilevel PLS-DA approach to this crossover design in the data [35]. Multilevel PLS-DA was implemented in MATLAB with M-files developed at the Univ. of Amsterdam [44] and available at: http://www.bdagroup.nl/content/Downloads/software/software.php

Preprocessing by filtering out one OSC component to remove the largest variance of X orthogonal to Y (again, presence or absence of asthma) [36–38] was evaluated as well. OSC-PLS-DA calculations used the R script at: https://gist.github.com/dgrapov/5166570. To avoid overfitting, leave-one-out cross-validation was performed to select the optimal number of the components for use in model prediction. The prediction power of the statistical modeling was evaluated and validated by intensive Monte Carlo cross-validation and permutation testing.

Identification of Pertinent Metabolites and Implications

For identifying dilute small molecules trapped in EBC, high sensitivity was obtained using the 800 MHZ NMR system with cryoprobe and overnight signal averaging of sensitivity-enhanced ¹³C heteronuclear single quantum coherence (HSQC) [45–47]. For broad bandwidth, the HSQC used chirp adiabatic inversion and refocusing pulses [48]. Reference NMR spectra were found at the Human Metabolome Database (HMDB) [49, 50] and Madison-Qingdao Metabolomics Consortium Database (MMCD) [51]. The database and server named Complex Mixture Analysis by NMR (COLMAR) ¹³C-¹H HSQC [52] was used in semi-automated comparisons of these databases’ reference spectra with the natural abundance ¹³C HSQC in order to identify multiple carboxylic acids detected.

Statistical total correlation spectroscopy (STOCSY) was proposed for identifying biomarkers by calculating the correlation matrix of the NMR data sets [53]. A STOCSY correlation map was prepared from the 106 NMR spectra once preprocessed, segmented into 0.02 ppm bins, and glog-transformed. A ¹H-¹H correlation matrix (455X455 points) was generated from these preprocessed spectra. Covariance with a correlation coefficient r ≥ 0.7 was considered as significant and plotted in the STOCSY correlation map.

Variable Importance in Projection (VIP) from the OSC-PLS-DA and OSC-corrected multi-level analyses was calculated in order to evaluate the diagnostic significance of each spectral bin. Spectral bins with VIP value larger than 1.0 are considered to discriminate between groups [54]. Potential pathways affected by induction of the early stage of allergic asthma were anticipated by submitting the biomarker candidates to the MetaboAnalyst 3.0 server [55] at: http://www.metaboanalyst.ca/faces/ModuleView.xhtml

Preprocessing of NMR Spectra of EBC for Statistical Comparisons

In pursuit of metabolomics discrimination and biomarkers of early allergic asthma, 1D NMR spectra were acquired for the EBC specimens of each of the 53 before and after inducing the asthma. Overall concentrations of solutes in EBC samples varied over a 5-fold range. To remove the impact of this large variation in overall metabolite concentrations among the samples, the peak heights in the NMR spectra were normalized by PQN [31] (S1 Fig). Normalization is part of the spectral binning (untargeted) strategy [26] and enables statistical comparison of the proportions that the NMR peaks represent in the samples (in contrast to absolute concentrations of selected metabolites). PQN suppresses artifacts introduced by normalizing to the integral [31]. This improves the reliability of statistical comparisons of EBC specimens collected from patients with potential variations in exhaled volume, pulmonary gas exchange, and ambient humidity in the condensation chamber. The untargeted approach was also motivated by the biomarkers being unknown during the undertaking and development of the project. Since scaling methods influence statistical results [56], the effects of Pareto scaling (Eq 1) and glog transformation (Eq 2) [42] were compared. Without scaling, the bins with highest intensities (which tend to have higher variances) dominate the total variances (S2 Fig) and biased the statistics towards the tallest NMR peaks. Pareto scaling improved the situation but still resulted in a large range of variance among the bins (S2 Fig). The glog transformation gives greater weight to small variances that most likely result from small peak heights (S1 and S2 Figs). Preprocessing with PQN and glog transformation proved essential to discriminating asthma from health by enabling many spectral regions besides those of concentrated lactate to be considered by multivariate statistical analyses.

Principal Component Analysis of the NMR Spectra

PCA was applied to the 106 preprocessed NMR spectra. The first two PCs account for about 38% of the total variance among the spectra (Fig 1A). The loading plot of PC1 and PC2 identifies many spectral bins with large variances among the 106 spectra. Metabolites represented by some of these spectral bins, identified by efforts described below, are labeled on the loading plots (Fig 1A–1C). The 3D plot of scores using the first three PCs does not adequately separate the healthy and asthmatic states of the cats (Fig 1D). This is due to unsupervised PCA placing high weights on the high variance of NMR peaks that are uncorrelated with the disease state of the cats.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Comparison of loading and score plots generated from preprocessed NMR spectra of EBC from 53 cats before (black) and after induction of early allergic asthma (red), using multivariate statistics.

Panels A and D show PCA results. Panels B and E plot PLS-DA results. Panels C and F report multi-level PLS-DA results. Components 1 and 2 are plotted in the loading plots (A–C). Components 1, 2, and 3 are plotted in the score plots (D–F). Biomarkers that increased in the experimental asthma are labeled in red. A marker that decreased in the experimental asthma is labeled in blue. A peak too weak to be useful is labeled in gray. Efforts to identify the markers are described below.

https://doi.org/10.1371/journal.pone.0164394.g001

Partial Least Squares Improves Separation of Asthma from Health

Supervised PLS-DA methods were applied in a quest for better separation of asthmatic and healthy states of the cats and to recognize diagnostic spectral features. Five PLS components optimized the performance and reliability of the separation of the cats when healthy or asthmatic, as judged by the decrease of the root mean square error of the prediction (RMSEP) and the increase of the Q² value (S3 Fig) from leave-one-out cross-validation. PLS-DA achieved much better separation than PCA in the score plot between cats before and after developing early allergic asthma (Fig 1D and 1E), presumably due to increased weights on variables that distinguish the health of the cats.

Multi-level PLS-DA was introduced to paired data sets to separate between-subject variation (e.g., due to genetic background) and within-subject variation (effect of “treatment”) in the data [35]. The crossover design of our data set (before and after induction of asthma) allowed us to test if multi-level PLS-DA could improve performance. After removing the between-subject variation, the loading plot is better dispersed than that of PLS-DA (Fig 1B and 1C). The score plot from multi-level PLS-DA better separates the groups, provided that the PQN type of normalization has been used (Fig 1F).

The spectra were preprocessed further using orthogonal signal correction (OSC) that removed two OSC components. This filtered out variances between spectra unrelated to separation between health and asthma in subsequent PLS-DA and multi-level PLS-DA calculations. Plots of RMSEP and Q² generated by leave-one-out cross-validation showed that OSC-PLS-DA and multi-level PLS-DA perform clearly better in predictions than PLS-DA (S3 Fig). Multi-level OSC-PLS-DA performed the best of all by these criteria (S3 Fig). OSC-PLS-DA and multi-level PLS-DA reduced the RMSEP and increased Q² more significantly than did PLS-DA when using the same number of components (S3 Fig). The first three to four components are enough to optimize the performance OSC-PLS-DA, which is a simplifying advantage over PLS-DA (S3 Fig). Two to three components suffice for multi-level OSC-PLS-DA to offer superior predictive power (S3 Fig).

The score plot from OSC-PLS-DA using the first three components shows group separation (Fig 2A) that is much improved over PCA and PLS-DA (Fig 1D and 1E). In the 2D score plot form OSC-PLS-DA, around three spectra of EBC from asthma are not distinguished from health (Fig 2A). Multi-level OSC-PLS-DA fully resolves the specimens from heath and asthma in the score plot (Fig 2C), and needs only two components to do so (S4 Fig). In order to test the prediction power of the model, two-thirds of the 106 samples were randomly selected as the training set, and the remaining third was used as the test set. Monte Carlo cross-validation suggests that the OSC-PLS-DA model with four components has very good predictive power with Q² = 0.70, contrasting the negative control of random permutation testing with Q² = -0.07 (Table 1). Cross-validation suggests even better predictive power from the multi-level OSC-PLS-DA model with three components where Q² = 0.84 and the randomly permuted negative control has Q² = -0.15 (Table 1). The prediction for the training set (a third of the specimens) using the OSC-PLS-DA model with four components has sensitivity of 94.1% and specificity of 94.1% (Table 1). The multi-level OSC-PLS-DA separation of the specimens into healthy and asthmatic classes is annotated with the names of the cats on a 2D score plot in S4 Fig. The prediction from multi-level OSC-PLS-DA model using three components has sensitivity of 94.1% and specificity of 100% (Table 1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Orthogonal signal-corrected statistical results for the preprocessed spectra of EBC from 53 cats before and after induction of allergic asthma.

Panels (A,B) show results from OSC-PLS-DA. Panels (C,D) show results from multi-level OSC-PLS-DA. (A,C) Scores are plotted, using the three largest principal components, for specimens collected before (black) and after induction of asthma (red). (B,D) The variable importance in projection (VIP) plots suggest the spectral features that most distinguish the experimental asthma from the health of the cats. As VIP values rise above 1.0, they imply increasing diagnostic significance [54]. Biomarkers increased by feline asthma are labeled red. A marker decreased by the experimental asthma is labeled blue. The weakness of the tentatively assigned NMR peak of malonate (labeled in gray) diminishes its diagnostic value below that implied by the VIP values.

https://doi.org/10.1371/journal.pone.0164394.g002

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Sensitivity, specificity, and testing of predictions from the orthogonal signal-corrected models.

https://doi.org/10.1371/journal.pone.0164394.t001

NMR Identification of Metabolites in EBC from Cats

Independently from the untargeted statistical process of discriminating asthma from heath, we sought to identify more of the metabolites in feline EBC using two strategies. The more informative strategy proved to be a ¹³C HSQC spectrum of a relatively concentrated EBC sample from a healthy cat (Fig 3). Compounds present in the natural abundance ¹³C HSQC spectrum were analyzed using the COLMAR ¹³C HSQC database and server [51] that matches the spectrum against the MMCD and HMDB databases of NMR spectra of 555 compounds. Additional matching was done manually against reference NMR spectra available in HMDB [48, 49] and MMCD [50] databases. These efforts identified the amino acids alanine, aspartic acid, glycine, isoleucine, leucine, phenylalanine, serine, threonine, tyrosine, and valine (Fig 3). The ¹³C HSQC identified additional metabolically significant carboxylic acids of acetate, lactate, and pyruvate (Fig 3A and 3B). Also present are the dicarboxylic acids phthalate, hexanedioic acid (adipate) and either or both of heptanedioic acid (pimelate) and octanedioic acid (suberate), for which the NMR peaks overlap. Weak peaks for most but not all of the groups of sucrose or a related sugar are also observed (Fig 3A). An anomeric proton doublet at 5.41 ppm in 1D spectra is consistent with the presence of a sucrose-like molecule. A weak peak matching trimethylamine is also observed in the ¹³C HSQC (not shown). Overall, carboxylic acids predominate in NMR spectra of feline EBC.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Multiple carboxylic acids are exhaled by cats.

Regions of a natural abundance ¹³C HSQC NMR spectrum of a comparatively concentrated EBC specimen from a healthy cat are plotted with assignments for (A) the methine (-CH) region, (B) the methylene and methyl regions, and (C) the aromatic region. The sensitivity-enhanced HSQC was acquired at 25°C for 17.5 h at 800 MHz with a cryoprobe. Apodization with a squared sine bell introduced enough line broadening in the ¹H dimension to mask multiplet splittings.

https://doi.org/10.1371/journal.pone.0164394.g003

The second strategy to gain more information to identify and confirm molecules was to ascertain co-variation among the 106 preprocessed 1D NMR spectra and to plot the co-variation as a correlation map known as STOCSY [52] (Fig 4). Two or more co-varying NMR peaks lying away from the diagonal constitute a ‘spin system’ represented by spots along a row or column (Fig 4 and S5 Fig), which better define the identity of a molecule than a single peak can. Comparison of the positions of the correlations in the STOCSY against the database of TOCSY spectra at the COLMAR server [57] identified niacinamide, benzoate, and another aromatic metabolite that probably contains a hydroxyphenyl moiety (Fig 4). The STOCSY also confirmed phthalate and tyrosine. The aliphatic region of the STOCSY and comparison of spectra of EBC from cats with the HMDB database identifies isopropanol, propionate, and butyrate (S5 Fig). (The latter’s potential alternative assignment as methyl butanoate appears unlikely due to the absence from the STOCSY of a correlation to the methyl peak expected in the range of 3.35 to 3.65 ppm. Nor is there a correlation for isobutyrate.) The STOCSY confirms the assignments of lactate and hexanedioate (S5 Fig) based on the ¹³C HSQC (Fig 3A). The butyrate is confirmed by TOCSY spectra and multiplet line shapes in 1D NMR spectra. The propionate and isopropanol are also confirmed by 1D spectra. A number of spin systems evident in the STOCSY (S5 Fig) failed to match the online databases, suggesting the absence from the databases of multiple compounds present in EBC.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Covalent connectivity of metabolites in feline EBC is suggested by co-variation (STOCSY) among the 106 preprocessed NMR spectra.

The aromatic region is plotted. Off-diagonal spots represent correlations (r ≥ 0.7) of two chemical groups across the spectra, suggesting that they belong to the same metabolite or pathway.

https://doi.org/10.1371/journal.pone.0164394.g004

Metabolites and Pathways Perturbed by Allergic Asthma

The VIP plots from OSC-PLS-DA and multi-level OSC-PLS-DA suggest the spectral features that best distinguish the EBC of cats in states of health and early allergic asthma. The trends in the six best markers in the VIP plots were manually inspected in the 106 PQN-normalized NMR spectra (Table 2). The highest VIP score (Fig 2B and 2D) suggests acetone to be the best biomarker identified. Acetone in EBC is clearly elevated by early allergic asthma in 74% of the cats, in whom acetone was scarcely detectable during health (Table 2, Fig 5A). The unidentified metabolites giving rise to broad, overlapped peaks near 5.8 ppm are elevated by early allergic asthma in 70% of the cats (Fig 5B). The unidentified, probably hydroxyphenyl-containing aromatic metabolite is increased by the experimental asthma in 62% of the cats (Table 2). Isopropanol is increased in 51% of the cats when asthmatic, and niacinamide in 43% of them (Table 2). Leucine levels are not affected by asthma in 73% of the cats, rendering it unreliable as a marker despite VIP scores > 1. Decreased phthalate upon onset of experimental allergic asthma in 60% of the cats (Table 2, Fig 5C) suggests its diagnostic value, despite its origins in environmental sources.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. Changes in the NMR peaks of three biomarkers in several cats before (solid lines) and after development of allergic asthma (dashed lines).

The spectra shown are PQN-normalized. The cats providing these EBC samples are named at right. (A) Acetone is increased in 74% of cats soon after developing allergic asthma, suggesting a shift in central energy metabolism. (B) The unidentified collection of broad, overlapped peaks that increased in 70% of the cats with experimental asthma is plotted. (C) The doublet NMR peak of phthalate is often decreased in the early allergic asthma. The legend names the cats sampled, with a suffix of H denoting health and a suffix of A denoting asthma.

https://doi.org/10.1371/journal.pone.0164394.g005

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Biomarkers of early allergic asthma in cats, ranked in descending order of diagnostic value.

The relative sizes of the NMR peaks were compared across the 106 probabilistic quotient normalized spectra.

https://doi.org/10.1371/journal.pone.0164394.t002

The primary marker of acetone and secondary markers of isopropanol and niacinamide were submitted to metabolic pathway analysis by MetaboAnalyst 3.0 [55, 58]. A P-value of 0.05 suggests that nicotinate and nicotinamide (niacinamide) metabolism could be perturbed in the asthmatic animals that exhale more niacinamide. NAD⁺, NADP⁺, nicotinamide ribotide, and nicotinamide riboside each are precursors to nicotinamide. The frequent elevation of acetone in early allergic asthma in cats implicates synthesis and degradation of ketone bodies with P-value of 0.007. Inclusion of isopropanol in the pathway analysis implies perturbation of propanoate metabolism by the experimental asthma, with the confidence of a P-value of 0.0006. Acetone accumulates in the majority of cats with allergic asthma presumably because of active synthesis of ketone bodies. This can be regarded as belonging to a larger set of pathways of propanoate metabolism. Isopropanol, which appears elevated in half of the cats with experimental asthma, is an immediate precursor to acetone. Acetoacetate is the better known precursor of acetone and lies on the pathway from acetyl-CoA, which is central in metabolism.

Ketosis in Allergic Asthma

Clinically relevant ketones in cats produced during states of decreased glucose utilization or negative energy balance include acetone, β-hydroxybutyrate, and acetoacetate. In 74% of the cats of this study, exhaled acetone was increased with induction of allergic asthma, implying increased ketogenesis. The acetone may have been produced locally by microbiota in the lung or emanated from the circulation of these cats. Acetone is one of the serum metabolites that was found to be related to the severity of eosinophilic asthma and airflow limitation in adult humans [62]. In cats, increased serum β-hydroxybutyrate, is characteristic of diabetes [63], diabetic ketoacidosis, hepatic lipidosis, and less frequently other conditions such as chronic kidney disease and hyperthyroidism in which fat is used as an energy source [64, 65]. Ketonemia in cats is typically associated with a shift from glucose utilization to β-oxidation of fatty acids and negative energy balance [64, 66]. No evidence of negative energy balance was noted in the 53 cats of this study however.

Similarly in humans, increases of acetone in serum accompanied decreases of % forced expiratory volume in 1 s (FEV₁%), an important manifestation of asthma [62, 67]. The lower FEV₁% was also correlated with much increased very low and low density lipoprotein (VLDL/LDL), which is strongly suggestive of altered lipid metabolism [62, 67]. The authors suggested the increases of acetone and histamine release result from the elevation of VLDL/LDL [67]. This could be related to the increased exhalation of volatile organic compounds in asthma, which has been attributed to increased peroxidation of lipids [25, 60].

Other Metabolic Biomarkers of Asthma

The decrease of glucose and increase of lactate in serum from adult humans with asthma, detected by NMR-based metabolomics, may be suggestive of hypoxia [67]. Those changes could be consistent with the increase of exhaled acetone and apparent ketogenesis in asthmatic cats. At the early stage of feline allergic asthma, the changes in lactate levels were, however, highly heterogeneous and without clear trend (not shown). This may be attributable to variability in the recent exercise and nutrition of the cats. An NMR-detected study of EBC from adults suggested mild asthma to decrease short-chain fatty acids, valine, formate, hippurate and urocanic acid, as well as to increase proline, propionate, isobutyrate, and phenylalanine [24], Such changes cannot, however, be corroborated in cat EBC upon onset of experimental asthma. The human study normalized the EBC spectra to their spectral area [24], a common practice that can introduce artifacts of some metabolites appearing to decrease [30]. The study of EBC of adult humans made no mention of an increase of acetone by asthma. These differences in prospective biomarkers of asthma suggest the need for caution and limits to generalizing across very different cohorts of asthma patients.

Why was phthalate observed in this feline study? Phthalates are found in many consumer products. Humans consume phthalate esters introduced to the diet by the processing of foods, especially food packaging films and meats, which contain di-2-ethylhexylphthalate [68–74]. The dry kibble diet of the cats was highly processed and contained animal fats, i.e. likely sources of phthalate esters by analogy with the contamination documented in human diets. Perhaps exhalation could be a route of elimination of phthalates that was impaired by the experimental asthma in the cats.

The most serious technical impediment to use of EBC from cats is the inherent large variability, which appears unrelated to the pulmonary disease state. This stymied PCA from distinguishing early experimental asthma from health. Fortunately, probabilistic quotient normalization [31] enabled successful discrimination using supervised statistical methods based on PLS-DA. For the best diagnostic separations, multi-level PLS-DA or preprocessing with orthogonal signal correction suppressed much variability that interfered in diagnostic classification. With OSC-PLS-DA, the predictive model was simplified to only three to four components and attained predictive power of 94% sensitivity and 94% specificity (Table 1). Multi-level suppression of between-subject variability [35] combined in novel fashion with orthogonal signal correction (Fig 2C) notably increased the specificity in predicting asthma to 100% (Table 1). Consequently, the combination of OSC and multi-level enhancements to PLS-DA appears promising for comparative metabolomic and clinical research studies where specimens can be assayed from each subject before and after a treatment or change in clinical status.