NMR Metabolomic Analysis of Exhaled Breath Condensate of Asthmatic Patients at Two Different Temperatures

Exhaled breath condensate (EBC) collection is a noninvasive method to investigate lung diseases. EBC is usually collected with commercial/custom-made condensers, but the optimal condensing temperature is often unknown. As such, the physical and chemical properties of exhaled metabolites should be considered when setting the temperature, therefore requiring validation and standardization of the collecting procedure. EBC is frequently used in nuclear magnetic resonance (NMR)-based metabolomics, which unambiguously recognizes different pulmonary pathological states. Here we applied NMR-based metabolomics to asthmatic and healthy EBC samples collected with two commercial condensers operating at −27.3 and −4.8 °C. Thirty-five mild asthmatic patients and 35 healthy subjects were included in the study, while blind validation was obtained from 20 asthmatic and 20 healthy different subjects not included in the primary analysis. We initially analyzed the samples separately and assessed the within-day, between-day, and technical repeatabilities. Next, samples were interchanged, and, finally, all samples were analyzed together, disregarding the condensing temperature. Partial least-squares discriminant analysis of NMR spectra correctly classified samples, without any influence from the temperature. Input variables were either integral bucket areas (spectral bucketing) or metabolite concentrations (targeted profiling). We always obtained strong regression models (95%), with high average-quality parameters for spectral profiling (<i>R</i>² = 0.84 and <i>Q</i>² = 0.78) and targeted profiling (<i>R</i>² = 0.91 and <i>Q</i>² = 0.87). In particular, although targeted profiling clustering is better than spectral profiling, all models reproduced the relative metabolite variations responsible for class differentiation. This warrants that cross comparisons are reliable and that NMR-based metabolomics could attenuate some specific problems linked to standardization of EBC collection

Serum Levels of Acyl-Carnitines along the Continuum from Normal to Alzheimer's Dementia

<div><p>This study aimed to determine the serum levels of free L-carnitine, acetyl-L-carnitine and 34 acyl-L-carnitine in healthy subjects and in patients with or at risk of Alzheimer’s disease. Twenty-nine patients with probable Alzheimer’s disease, 18 with mild cognitive impairment of the amnestic type, 24 with subjective memory complaint and 46 healthy subjects were enrolled in the study, and the levels of carnitine and acyl-carnitines were measured by tandem mass spectrometry. The concentrations of acetyl-L-carnitine progressively decreased passing from healthy subjects group (mean±SD, 5.6±1.3 μmol/L) to subjective memory complaint (4.3±0.9 μmol/L), mild cognitive impairment (4.0±0.53 μmol/L), up to Alzheimer’s disease (3.5±0.6 μmol/L) group (p<0.001). The differences were significant for the comparisons: healthy subjects vs. subjective memory complaint, mild cognitive impairment or Alzheimer’s disease group; and subjective memory complaint vs. Alzheimer’s disease group. Other acyl-carnitines, such as malonyl-, 3-hydroxyisovaleryl-, hexenoyl-, decanoyl-, dodecanoyl-, dodecenoyl-, myristoyl-, tetradecenoyl-, hexadecenoyl-, stearoyl-, oleyl- and linoleyl-L-carnitine, showed a similar decreasing trend, passing from healthy subjects to patients at risk of or with Alzheimer’s disease. These results suggest that serum acetyl-L-carnitine and other acyl-L-carnitine levels decrease along the continuum from healthy subjects to subjective memory complaint and mild cognitive impairment subjects, up to patients with Alzheimer’s disease, and that the metabolism of some acyl-carnitines is finely connected among them. These findings also suggest that the serum levels of acetyl-L-carnitine and other acyl-L-carnitines could help to identify the patients before the phenotype conversion to Alzheimer’s disease and the patients who would benefit from the treatment with acetyl-L-carnitine. However, further validation on a larger number of samples in a longitudinal study is needed before application to clinical practice.</p></div

Serum levels (μmol/L) of free L-carnitine and acyl-carnitines in the four studied groups.

<p>Serum levels (μmol/L) of free L-carnitine and acyl-carnitines in the four studied groups.</p

Serum levels of acyl-carnitines classified as VIP by PLS-DA.

<p>Box-plots show median (horizontal line in the box), 25th and 75th percentiles (edges of box), maximum and minimum values (whiskers) and outliers (°,*) of acyl-carnitines concentrations (μmol/L) in the 4 groups of subjects. AD, Alzheimer’s disease; MCI, mild cognitive impairment; SMC subjective memory complaint; HS, healthy subjects; (†) Significantly different from HS groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155694#pone.0155694.t002" target="_blank">Table 2</a> for details).</p

Important features identified by PLS-DA.

<p>Twelve metabolites and 3 molar ratios show a VIP score > 1.3.The colored boxes indicate the relative concentrations of the corresponding metabolite or the relative value of ratios in each group.</p

Score plots for the PLS-DA model discriminating the 4 groups of subjects.

<p>Plots of the first two (A) and first three (B) components that explain, respectively, the 52% and the 56.3% of model variance. AD (red) samples were well separated from HS (light blue), while MCI (blue) and SMC (green) clustered in an intermediate zone.</p

ROC curves of selected acyl-carnitines.

<p>The plots show the optimal value of cutoff (•), the value of full AUC with the 95% confidence intervals and the best delimitation of AUC (black solid line) for each metabolite.</p

Demographic and clinical characteristics of study groups.

<p>Demographic and clinical characteristics of study groups.</p