# Untargeted 1H-NMR Urine Metabolomic Analysis of Preterm Infants with Neonatal Sepsis
## 4 Conclusions  
The findings of our study indicate a discrete metabolic profile of septic neonates. NMR based metabolomic approach revealed the relation among septic and control neonates, highlighting gluconate, myo-inositol, hippurate, taurine, \(N\), \(N\)-Dimethylglycine, betaine, creatinine, glucose and lactose as significant metabolites. Our study reported, for the first time, altered urinary amounts of betaine in EOS and LOS neonates and \(N\), \(N\)-Dimethylglycine in LOS neonates. Differentiated concentration levels of taurine and hippurate via LC-MS analysis have been previously reported by [PERSON] et al., and through UPLC-MS by [PERSON] et al. [28; 29]; however, our study was the first to detect them in urine samples of septic neonates via NMR. The utilization of the two control groups and their discrete analysis, based on the NICU hospitalization, showed that NICU treatment has a significant impact on neonates' urine metabolome. The observed spectral pattern indicative for the most of the NICU neonates, suggests that it is related to endogenous or exogenous metabolites of the personalized nutrition or medical treatment. Additionally, the impact of nutrition is confirmed from the greatly elevated levels of gluconate for the septic group and lactose for neonates fed from their mothers. Differentiation between EOS and LOS, and the adaptation of the fetus to neonate during the first days of extrauterine life that occur in parallel are reflected to the metabolism. Changes through the first days of life, associated with EOS and LOS, highlight the necessity for chronological coupled sampling with the onset and specific time of the disease progression. This research builds on the power of NMR metabolomic analysis to determine the status of an entire organism by a small amount of non-invasive collected biological sample. The establishment of NMR analysis of metabolome for clinical research in the field of neonatology, leading to large-scale multicenter studies, gives new and promising perspectives for its incorporation into the clinical daily routine and the validation of new combined diagnostic biomarkers. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12041932/s1](https://www.mdpi.com/article/10.3390/app12041932/s1)]([https://www.mdpi.com/article/10.3390/app12041932/s1](https://www.mdpi.com/article/10.3390/app12041932/s1)); Figure S1: Scores and loadings plot of PCA for NMR data belonging to neonates diagnosed with EOS (pink circles) and healthy neonates without need for NICU hospitalization (blue circles). (a) PCA scores plot of the EOS and healthy non-NICU preterms. (b) Loadings plot of PC1 and PC2; Figure S2: 1D-STOCY pseudo-NMR spectrum of correlation coefficients to the other signals in the median urine NMR spectrum and maximum intensity correlation peaks are color encoded and projected into statistical difference spectra: \"driver peak\" was set the one at 1.41 ppm; Figure S3: Scores and loadings plot of PCA for NMR data belonging to neonates diagnosed with EOS (orange circles) and neonates hospitalized in NICU without EOS (purple circles). (a) PCA scores plot of the EOS and control group. (b) Loadings plot of PC1 and PC2; Figure S4: Scores and loadings plot of PCA for NMR data belonging to urine samples of neonates diagnosed with EOS the first (green circles) and the third day (blue circles) of their life. (a) PCA scores plot of the first- and third-day's samples. (b) Loadings plot of PC1 and PC2; Figure S5: Scores and loadings plot of PCA for NMR data belonging to neonates diagnosed with LOS (pink circles) and control neonates with-out need for hospitalization (blue circles). (a) PCA scores plot of the LOS and healthy non-NICU preterms without need for hospitalization. (b) Loadings plot of PC1 and PC2; Figure S6: Scores and loadings plot of PCA for NMR data belonging to neonates diagnosed with LOS (orange circles) and control neonates of NICU (purple circles). (a) PCA scores plot of the LOS and control group.