Targeted NMR‐based serum metabolic profiling of serine, glycine and methionine in acute‐on‐chronic liver failure patients: Possible insights into mitochondrial dysfunction

Abstract

Background A recent study based on blood metabolomics analysis revealed inflammation‐associated mitochondrial dysfunction as a potential mechanism underlying acute‐on‐chronic liver failure (ACLF) in cirrhotic patients. Serine, glycine, and methionine serve to maintain a healthy immune system and adequately sustain mitochondrial functionality in hepatocytes for regulating redox homeostasis through the production of antioxidant glutathione (GSH). Based on this, we hypothesized that the circulatory levels of serine, glycine and methionine will be altered in ACLF patients due to acute worsening of hepatic function and may provide novel insights into the mitochondrial dysfunction as well. Methods The circulatory concentrations of serine, glycine, and methionine were estimated in the sera of 40 ACLF patients and 49 normal controls (NC) subject using 1D 1H‐CPMG NMR spectra recorded at 800 MHz NMR spectrometer. The resulting metabolite concentrations were compared using unpaired Student t‐test and p‐value < 0.05 was considered as the criterion of statistical significance. The diagnostic potential and statistical correlations were established using receiver‐operating‐characteristic (ROC) curve analysis and Pearson‐r method, respectively. Results Circulating levels of serine and glycine were significantly decreased in ACLF patients (Ser = 23.06 ± 1.67 µM and Gly = 83.11±7.52 µM) compared to NC subjects (Ser = 55.61 ± 2.28 µM and Gly = 156.9±7.16 µM) with p‐value < 0.0001, whereas those of methionine were significantly increased in ACLF (22.60 ± 2.49 µM) compared to NC subjects (=14.63 ± 0.85 µM) with p‐value < 0.0015. Further, the ROC analysis yielded satisfactory sensitivity and specificity for serine, glycine, and methionine‐to‐glycine ratio (MGR) with area under ROC (AUROC) curve values equal to: 0.95 [95%CI = 0.91‐0.99] for Ser; 0.87 [95%CI = 0.79‐0.95] for Gly; and 0.90 [95%CI = 0.83‐0.97] for MGR. Conclusion Compared to NC subjects, the sera of ACLF patients were characterized by hypermethioninemia and aberrantly decreased levels of serine and glycine suggesting mitochondrial dysfunction as the possible mechanism for disturbed redox homeostasis and therefore depressed immune system in ACLF.

Elevated Circulatory Proline to Glutamine Ratio (PQR) in Endometriosis and Its Potential as a Diagnostic Biomarker

“…Figure A shows the stack plot of the 1D 1 H CPMG NMR spectra recorded on serum samples obtained from EM and NC subjects. The present study involves concentration profiling of proline and glutamine in the serum samples of EM patients and NC subjects following a targeted 1 H NMR based metabolomics approach as described previously. − , For this, first we have identified the NMR signals corresponding to proline and glutamine and assigned by following a chemical shift and peak pattern matching procedure employing the 800 MHz spectral database of metabolites in the NMR suite of CHENOMX software; the selected spectral regions are shown in Figure B (for glutamine) and Figure C (for proline). To minimize the experimental/methodological deviations, the proline and glutamine concentrations were explicitly measured using formate as an internal reference (explicit details are described in the Materials and Method).…”

“…Afterward, the spectrum was opened in the PROCESSOR module of CHENOMX NMR Suite 8.6 software and further better corrected for baseline and calibrated with respect to the formate peak at 8.43 ppm. For concentration profiling, formate was also used as an internal reference and the concentration was set to 10 μM (i.e., nearly close to the detection limit of metabolites in the CPMG NMR spectra of serum samples recorded at 800 MHz NMR spectrometer). , The advantage of selecting formate as an internal reference has already been demonstrated in previous methodological studies , including recent metabolomics studies from our lab. − , Studies have shown that, unlike TSP, formate does not interact with serum proteins/macromolecules − and, therefore, has legitimate potential to serve as an internal reference for quantitative profiling of metabolites from blood serum (and eventually other biological fluids) in normal and diseased conditions not involving disorders of endogenous formate metabolism. After data processing, the spectrum was imported to the PROFILER-Module of CHENOMX and the concentrations of selected metabolites (i.e., proline and glutamine) were estimated in all the serum samples of EM patients and NC subjects.…”

“…58,59 The advantage of selecting formate as an internal reference has already been demonstrated in previous methodological studies 60,61 including recent metabolomics studies from our lab. [16][17][18]20 Studies have shown that, unlike TSP, formate does not interact with serum proteins/macromolecules 60−62 and, therefore, has legitimate potential to serve as an internal reference for quantitative profiling of metabolites from blood serum (and eventually other biological fluids) in normal and diseased conditions not involving disorders of endogenous formate metabolism. After data processing, the spectrum was imported to the PROFILER-Module of CHENOMX and the concentrations of selected metabolites (i.e., proline and glutamine) were estimated in all the serum samples of EM patients and NC subjects.…”

“…Alternatively in our lab, we are using formate as an internal calibration standard for NMR analysis of normal plasma and serum samples, and the resulting concentration profiles (estimated w.r.t. formate) are then used to estimate the metabolic ratios (ratiometric parameters). ,− As compared to conventionally used normalized spectral features, the circulatory metabolic ratios were found to be more reliable in reflecting the pathophysiological state of the patients and correlate well with the clinical parameters or disease severity scores. − The approach has been extended here further for investigating the circulatory levels of proline and glutamine and the proline to glutamine ratio (PQR) in female endometriosis (EM) patients with respect to normal control (NC) female subjects. The purpose of selecting these two specific amino acids (i.e., proline and glutamine) is that EM is often associated with altered inflammatory and immune processes and shares some cancer-like characteristics such as activated glutaminolysis , and increased proline biosynthesis. − Both proline and glutamine are interconvertible metabolically, and studies have shown their roles in cancer cell metabolic reprogramming, redox homeostasis, occurrence/development of cancer (including endometrial carcinoma), and its further progression toward the malignant state. − So based on these pathophysiological hallmarks, we hypothesized that the circulatory proline to glutamine ratio (PQR) would be altered in EM and may serve as an indicative biomarker to improve the clinical diagnosis of EM.…”

Editorial for special issue: Metabolomics in India

“…Another NMR‐based targeted metabolomics study by Arya et al 6 investigated the circulatory levels of serine and glycine in the sera of 40 ACLF patients and 49 normal controls (NC) subject using 1D 1H CPMG NMR spectra that provided insights into the potential mechanisms of acute‐on‐Chronic liver failure (ACLF) in cirrhotic patients. The authors showed that the circulating levels of serine and glycine were significantly decreased in ACLF patients (Ser = 23.06 ± 1.67 and Gly = 83.11 ± 7.52) compared to NC subjects (Ser = 55.61 ± 2.28 and Gly = 156.9 ± 7.16) with p ‐value < 0.0001, indicative of depressed immune system and mitochondrial dysfunction.…”