# Effects of Workers Exposure to Nanoparticles Studied by NMR Metabolomics
## 3 Results and Discussion
### Analysis of Blood Plasma  
Using the Chenomx reference library, 58 metabolites were identified and quantified in each \({}^{1}\)H NMR spectrum of blood plasma samples. The concentration data of all quantified metabolites were used as an input for both multivariate and univariate statistical analyses to reveal important features of each group. The homogeneity of the groups was tested by principal component analysis (PCA) as an unsupervised statistical method. According to PCA, no sample was found significantly outlying. Nevertheless, group discrimination was not achieved (Figure S4, Supplementary Materials). Subsequently, a supervised statistical method (OPLS-DA) was employed to pre-shift and control samples. A very good separation between these two groups was achieved using three components. The model was characterized by 88.2% sensitivity, 73.2% specificity and 80.7% accuracy after Monte Carlo cross-validation (Figure 5a). Figure 4: Boxplots of selected metabolites affected by NPs exposure. The nonparametric Wilcoxon rank-sum test was used to reveal statistically significant compounds that should reflect the effect of chronic exposure to NPs. Only acetone was found under the threshold for statistical significance (adjusted \(p\)-value \(\leq\) 0.05). Four other metabolites were close to this threshold, specifically glutamate, glutamine, cystine and hypoxanthine (Table S3 and Figure S5 in Supplementary Materials). Levels of acetone, glutamine and cystine were found increased in the control group, whereas glutamate and hypoxanthine show higher levels in the pre-shift group. OPLS-DA was also used for differentiation between the post-shift subjects and the healthy controls. A very good separation of the two groups was obtained using a six-component model with an accuracy of 86.0%, sensitivity of 86.4% and specificity of 85.7% after Monte Carlo cross-validation (Figure 5b). The nonparametric Wilcoxon rank-sum test was used to reveal statistically significant compounds (Table S3 and Figure S6 in Supplementary Materials). Seven metabolites were found under the threshold for statistical significance (adjusted \(p\)-value \(\leq\) 0.05). Levels of propylene glycol, glutamate and pyruvate were found increased in the post-shift group, whereas acetone, mannose, 2-oxoisocaproate and \(O\)-acetylcarnitine showed higher levels in the control group. Analogically, the acute effect of NPs exposure was also studied on plasma samples of the pre- and post-shift groups using OPLS-DA (Figure 6a). This discrimination analysis showed a certain potential to distinguish between the two groups with a model based on eight components characterized by 75.4% accuracy, 74.0% sensitivity and 76.9% specificity. Subsequently, mPLS was performed with a remarkable discrimination of the two groups using five components with 89.0% accuracy after Monte Carlo cross-validation (Figure 6b). In this case, mPLS reflects the intra-individual differences within each subject; therefore, it provides better group separation than discrimination analysis based on OPLS. Subsequent analysis by the nonparametric pairwise Wilcoxon signed-rank test revealed 11 statistically significant compounds (Table S3 and Figure S7 in Supplementary Materials). Only compounds with adjusted \(p\)-value \(\leq\) 0.05 after Benjamini-Hochberg correction were deemed statistically significant. Out of the 11 statistically significant compounds, increased levels for eight metabolites were found in the pre-shift group, specifically isobutyrate, 2-hydroxybutyrate, 2-oxoisocaproate, lactate, 3-hydroxybutyrate, isopropanol, tryptophan and 3-methyl-2-oxovalerate. Levels of three statistically significant metabolites were elevated in post-shift groups, namely propylene glycol, glycolate and myo-inositol. The stress induced by NPs exposure is well documented by increased levels of certain metabolites in post-shift samples when compared to the pre-shift samples or controls. In particular, increased levels were found for propylene glycol, glycolate, myo-inositol, pyruvate and glutamate (Figure 7).