Circulatory Glutamine/Glucose ratio for evaluating disease activity in Takayasu arteritis: A NMR based serum metabolomics study

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Highlights

  • Quantitative assessment of disease activity is important for effective care of patients with Takayasu arteritis (TA).

  • Activated glutaminolysis and reduced glycolytic-flux is the hallmark of active inflammatory condition.

  • The study demonstrated that Glutamine/Glucose ratio (QGR) can serve as an indicant of active-inflammation in TA.

Abstract

Quantitative assessment of disease activity is important for effective care of patients with Takayasu arteritis (TA). Activated glutaminolysis and reduced glycolytic flux is the hallmark of active inflammation. Based on this, we hypothesize that the circulatory Glutamine/Glucose ratio (QGR) can serve as an indicant of active inflammation in TA. To probe this hypothesis, the serum samples were collected from 45 active and 53 inactive TA patients fulfilling American College of Rheumatology (ACR) criteria and assessed for disease activity according to Indian Takayasu Clinical Activity Score (ITAS) using acute phase reactant–erythrocyte sedimentation rate [ITAS-A (ESR)]. The quantitative profiles of circulatory metabolites implicated in glutaminolysis (Glutamine and Glutamate) and those which estimate glycolytic flux (i.e. glucose and lactate) were measured using high field (800 MHz) NMR spectroscopy. The recorded spectra were analyzed using CHENOMX NMR Suite and the estimated concentration profiles were compared and evaluated for their diagnostic potential using Metaboanalyst. Compared to inactive-TA patients, the sera of active-TA patients were characterized by significantly decreased serum levels of glutamine and lactate suggesting that these patients exhibit activated glutaminolysis and reduced glycolytic activity. This is further supported by significantly decreased QGR and lactate to glucose ratio (LGR) levels in active compared to inactive TA patients. The receiver operating characteristic (ROC) curve analysis revealed satisfactory accuracy, sensitivity and specificity for QGR [with area under ROC curve (AUROC) = 0.76 and 95% confidence interval (CI) = 0.66–0.84) compared to that for LGR (with AUROC = 0.67 and CI = 0.561-0.77). Therefore, we believe that the circulatory QGR has the potential to serve as surrogate marker for the assessment of disease activity in TA patients. However, the use of this ratio in clinical settings will require future studies on large patient cohorts and procedural optimization as well to improve accuracy.

Introduction

Quantitative assessment of disease activity is important for effective care of patients with Takayasu arteritis (TA) [1,2]. TA is a relatively rare autoimmune inflammatory disease that involves aorta, its major branches, and the pulmonary vessels [3,4]. However, so far, there are no reliable and effective quantitative measures to monitor disease activity in TA [[2], [3], [4]]. Non-specific tests based on inflammatory markers, such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), did not perform well in differentiating active from inactive TA patients [[2], [3], [4]]. About 30–40% of patients with active vascular inflammation have normal ESR while 40% without active inflammation have elevated ESR. Therefore, clinical activity scores (such as Indian Takayasu Clinical Activity Score (ITAS) have been developed as surrogate markers to assess the disease activity and has been improvised further as ITAS-ESR and ITAS-CRP considering circulatory ESR and CRP parameters, respectively [5]. However, assessment of disease activity based on such empirical scores too often comes down to physician’s judgment, which is too unreliable for clinical trials and monitoring the treatment response. Thus, there is an unmet need to discover biomarkers in TA for reliable screening of active and inactive TA patients.

Clinical metabolomics -which aims to identify disease specific metabolic patterns in patient-derived biofluids- has attracted signifcant attention to support discovery of novel non-invasive biomarkers [6]. Previously, two consecutive NMR based clinical metabolomics studies have been reported from our group on TA [1,2]. In our first study, we identified the serum metabolic disturbances associated with TA and in the second study we demonstrated that the serum metabolic profiling has the potential to aid differential diagnosis of active and inactive TA patients. In both these studies, we compared the normalized spectral features (i.e. integrated spectral bins normalized w.r.t. the total spectral intensity) and revealed that spectral bins dominated by circulatory glutamate and NAG have the significant discriminatory potential and may serve as surrogate biomarkers to assess disease activity in TA [2]. However, normalized spectral features may sometimes lead to contradictory results, particularly, when spectral bins of discriminatory significance contain signals from multiple metabolites or the quantitative change is very small compared to total spectral intensity. To rule out any such possibility and validate some of our previous findings, the present study has been undertaken. As such, dampened glycolysis and augmented glutaminolysis have been found to be prominent immuno-metabolic disturbances associated with autoimmune diseases [1,7,8]. Augmented glutaminolysis is critically important to support activation-induced T-cell proliferation and other immuno-biological functions [9]. Autoimmune diseases including TA often result from abnormal innate and adaptive immune responses that are closely linked to metabolic programming [10,11]. Upon activation, quiescent naive T cells undergo a growth phase followed by rapid proliferation and differentiation [12]. Accumulation of cell biomass during the initial growth and rapid proliferation during the expansion phase is associated with dramatically increased demand of energy and biosynthesis of nucleotides, proteins and fatty acids for various immuno-biological functions [11,12]. To meet with the necessity of these immune functions, glutaminolysis lies in the central position [11]. Glutamine is the most abundant amino acid circulating in human blood/muscles and yields more ATP per O2 in the glutamine to lactate metabolism as compared to glucose to CO2 [13]. It is involved in more metabolic processes than any other amino acid including synthesis of metabolites that maintain mitochondrial metabolism; generation of antioxidants to remove reactive oxygen species; synthesis of most non-essential amino acids (NEAAs) for protein synthesis, purines, pyrimidines, and fatty acids for cellular replication; and activation of cell signaling [7,14,15]. Another potential hallmark of immune-mediated inflammation is dampened glycolysis [1,2] and activation of pro-oxidative pathways (i.e. pentose phosphate pathway (PPP), Polyol pathway, Hexosamine pathway and Diacylglycerol-Protein Kinase-C (DAG-PKC) [11,12,16,17]. The present study has been envisaged to evaluate these conjectures through establishing an association of augmented glutaminolysis with disease activity in TA. For this, the circulatory concentrations of four metabolites (i.e. glutamate, glutamine, glucose, and lactate) and their specific ratios (i.e. glutamine to glutamate ratio (QER), glutamine to glucose ratio (QGR) and lactate to glucose (LGR) were estimated and compared between the active and inactive TA patients using the same serum samples as investigated previously [2].

Section snippets

Sample collection from Takayasu arteritis patients

Serum samples were obtained from 98 patients diagnosed with TA (based on the 1990 American College of Rheumatology (ACR) classification criteria [1,2]) attending the Department of Clinical Immunology at SGPGIMS, Lucknow. The study protocol was approved by the Institutional Research Ethics Committee, SGPGIMS, Lucknow, India and samples were collected with informed patient consent and stored with permission. Demographic parameters, clinical symptoms, and physical examination findings of TA

Results and discussion

A total of 98 TA patients were enrolled and of these, 45 were active (with mean ITAS-ESR of 10.6 ± 5.4) and 53 inactive TA patients (with mean ITAS-ESR of 1.6 ± 1.0). The median age of active and inactive TA groups was 27 [(IQR) 22–35 years] and 27 [(IQR) 23–37 years], respectively, and female to male ratios were 3.5:1 and 4.9:1, respectively. The angiographic classification and clinical features of TA patients have been tabulated in Supplementary material Table S1. The changes in circulatory

Concluding remarks

As hypothesized, the active TA patients were associated with activated glutaminolysis and circulatory Glutamine to Glucose ratio (QGR) showed satisfactory sensitivity and specificity for differentiating active and inactive TA patients. Circulatory QGR profiles showed adequate correlation with both circulatory inflammatory parameters (i.e. CRP and ESR) suggesting that the QGR have the potential to complement the existing markers for the assessment of disease activity and guiding treatment in TA

CRediT authorship contribution statement

Umesh Kumar: Writing - original draft. Avinash Jain: Writing - review & editing. Anupam Guleria: Resources, Software, Visualization, Data curation, Writing - review & editing. Venkatesh Kumar R: Writing - review & editing. Durga P Misra: Writing - review & editing. Ruchika Goel: Writing - review & editing. Debashish Danda: Writing - review & editing. Ramnath Misra: Conceptualization, Methodology, Supervision. Dinesh Kumar: Writing - original draft, Conceptualization, Methodology, Supervision.

Declaration of Competing Interest

On behalf of all authors, the corresponding authors state that there is no conflict of interest.

Acknowledgement

DK acknowledges the Department of Science and Technology, India for financial assistance under SERB EMR Scheme (Ref. No.: EMR/2016/001756). AG acknowledges the Department of Science and Technology (DST), Government of India for financial assistance under DST INSPIRE Faculty Award (Ref. No. DST/Inspire Faculty Award 2014/LSBM-120). We would also like to acknowledge the Department of Medical Education, Govt. of Uttar Pradesh for supporting the High Field NMR Facility at Centre of Biomedical

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