# Effects of Workers Exposure to Nanoparticles Studied by NMR Metabolomics
## 3 Results and Discussion
### Analysis of Blood Plasma
Since the concentration of 2-hydroxybutyrate is decreased in the acute state, 2-ketobutyrate is probably transformed into other metabolites, including propionylCoA [59], which is also associated with degradation of branched-chain amino acids, as discussed below. The concentrations of two ketone bodies metabolites, 3-hydroxybutyrate and acetone, were found altered. Both compounds are closely connected to acetoacetate, another ketone body, which was, however, found unaltered. Nevertheless, decreased concentrations of 3-hydroxybutyrate and acetone after exposure to NPs suggest that the metabolic pathway of ketone body metabolism is affected. This was also revealed in the metabolic pathway analysis via MetaboAnalyst (Figure S8b in Supplementary Materials). 3-Hydroxybutyrate is also involved in butanoate metabolism, and it is also a degradation product of branched-chain amino acids, mainly of leucine [60]. The decreased concentrations of 3-hydroxybutyrate and acetone after NPs exposure are in contrast with other studies, which reported elevated levels of 3-hydroxybutyrate [21; 23; 61]. However, these studies were performed on rats exposed to high NPs doses. 3-Hydroxybutyrate is also an end product of \(\beta\)-oxidation of fatty acids [21]. The impairment of this metabolic pathway is also reflected in a decreased concentration of \(O\)-acetyl carnitine. This molecule serves as a carrier of acetyl from acetyl-CoA derived from fatty acids to mitochondria [62], thus taking part in energy metabolism. Moreover, decreased levels of \(O\)-acetyl carnitine can also be associated with oxidative stress. Similarly to this study, decreased levels of \(O\)-acetyl carnitine were also observed in zebrafish and mice embryos after Fe\({}_{2}\)O\({}_{3}\) NPs exposure [62]. Accordingly, workers exposed to NPs during iron oxide pigment production showed elevated markers of lipid, nucleic acid, and protein oxidation in their EBC [63]. Decreased levels of 3-methyl-2-oxovalerate and 2-oxoisocaproate (4-methyl-2-oxovalerate) were found in the post-shift group. These compounds are produced as direct metabolites of isoleucine and leucine during their degradation by branched-chain amino acid aminotransferase [64]. Since the concentrations of leucine and isoleucine were found almost unaffected, leucine and isoleucine are involved in other pathways or processes, and the direct degradation pathway of these amino acids is inhibited. Isobutyrate is another metabolite associated with the metabolism of branched-chain amino acids, mainly of valine [65]. The decreased concentration of isobutyrate in the post-shift group also indicates that branched-chain amino acids' degradation is inhibited in the acute state. However, this inhibition was not manifested in the metabolic pathway analysis performed in MetaboAnalyst. Hypoxanthine is an important part of purine metabolism [66], thus the elevated levels of hypoxanthine found mainly in pre-shift plasma samples indicate alterations in this metabolism. Such an observation is complementary to the findings of a previous study performed on the same cohort, where the markers of nucleic acids' oxidation (8-hydroxyguanosine and 8-hydroxy-2-deoxyguanosine) were identified in pre-shift EBCsamples. Similar markers were also found in other toxicological studies of occupational exposure to different NPs, indicating a general effect of chronic NPs exposure [12; 54; 55]. The metabolic pathway analysis performed mainly indicates that the induced oxidative stress activates anti-oxidative pathways, and antioxidants, such as glutathione, are extensively consumed. Higher demands in the supplement of the consumed antioxidants can be observed in decreasing levels of their intermediates, in particular glutamine and cystine. The decreased tryptophan levels may be related to the production of its metabolites like kynure acid, which have protective effects against oxidative stress and lung inflammation. Moreover, alterations of several other metabolic pathways were observed.