1H NMR based targeted metabolite profiling for understanding the complex relationship connecting oxidative stress with endometriosis

Abstract

Accumulating evidence indicates the active role of oxidative stress in the development of endometriosis; however, the mechanism of reactive oxygen species generation is poorly understood. Metabonomics/metabolomics is a scientific discipline that can be used to study changes in metabolite ensembles associated with disease pathophysiology. The present study focuses on the use of proton nuclear magnetic resonance spectroscopy based targeted metabolite profiling approach to explore dysregulation in metabolites expression in women with endometriosis. Further, association of oxidative stress with the metabolite ensembles, if any, is investigated. Using multivariate statistics, partial least square discriminant analysis model was generated which could classify endometriosis patients with sensitivity and specificity of 92.83% and 100%, respectively, and with a classification rate of 96.4%. In conjunction with increased glucose metabolism, citrate and succinate were found to be elevated in endometriosis patients. Higher levels of reactive oxygen species, lipid peroxidation, and advanced oxidation protein products and lower levels of total antioxidant capacity, superoxide dismutase, catalase, and glutathione were also observed. Increased glucose metabolism and defects in the mitochondrial respiratory system are suggested to be the possible sources of excessive reactive oxygen species generation in endometriosis.

Figure 1

Scores scatter plot of (a) PC1 versus PC2 and (b) component 1 versus component 2 resulting from PCA and PLS-DA, respectively, applied to 1H NMR spectra of serum of endometriosis patients (black diamond) and controls (white diamond). Validation of the PLS-DA model was performed by (c) receiver operating characteristic (ROC) analysis where area under the curve (AUC) was found to be 0.99 (d) Permutation test statistics at 1000 permutations with observed statistic at P < 0.001.

Figure 2

Figure depicting the role of metabolites in generation of free radicals. Lower levels of glucose along with elevated levels of pyruvate, citrate, and succinate indicate enhanced glucose metabolism along with impaired mitochondrial respiration. I, II, and III represent complex I, II, and III, respectively, of electron transport chain. Complex I and III are the primary sites of free radical generation. Elevated levels of succinate indicate complex III to be the most probable site for ROS generation in endometriosis. These free radicals are further removed from the system by various ROS scavengers such as SOD and catalase. An imbalance in the rate of formation and removal of free radicals results in oxidative stress. In endometriosis, lower levels of these enzymes lead to continued prevalence of oxidative stress.