Background and hypothesis Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by fibrosis of the skin and internal organs, as well as vascular damage. Recent research has suggested that abnormal metabolism of certain sugars, such as mannose and myo-inositol, may contribute to the development and progression of SSc. Clinical studies have found that SSc patients have increased mannose and decreased myoinositol levels in their blood plasma/serum samples compared to those of healthy controls, suggesting that abnormal mannose and myoinositol metabolism may contribute to SSc pathogenesis. However, further research on patient cohorts of different ethnicity is imperative to validate these findings and understanding the mechanisms underlying these metabolic abnormalities and to develop new therapies that target these pathways. The present hypothesis-free NMR based clinical metabolomics study is an effort in this direction to compare the circulatory levels of mannose, myo-inositol and other endogenous metabolites in the sera of scleroderma patients and control subjects so that to validate the proposed abnormalities in mannose and myo-inositol metabolic pathways and association between them. The selected metabolic features and the metabolic ratio i.e. myo-inositol to mannose ratio (MMR) were further evaluated for their clinical potential in diagnostic and prognostic screening. Methods: The serum sample from 83 SSc patients meeting ACR 1980 criteria for Systemic Sclerosis, and 43 age and sex matched normal controls, were analyzed using one dimensional (1D) 1H NMR spectroscopy coupled with multivariate statistical analysis such as Partial Least Square-Discriminate Analysis (PLS-DA). The NMR spectra were analysed using NMR suite of commercial software CHENOMX (www.chenomx.com/) and the metabolic concentrations were measured with respect to endogenous metabolite formate (as an internal calibration standard and concentration was set to 30 µM). For evaluating serum metabolic disparity between the study groups, the machine learning model was generated using random forest (RF) classification method and the Mean decrease accuracy (MDA) scores were used to identify the distinctive metabolic abnormalities in SSc. Univariate receiver operator characteristic (ROC) curve analysis was used to evaluate the diagnostic potential of the selected metabolic features. Results: There was clear distinction between SSc and healthy controls on the PLS-DA score plots [R2= 0.98] and aberrant metabolic changes were evident in the sera of SSc patients. The sera of SSc patients were characterized by decreased serum levels of alanine, valine, myoinositol, creatinine, pyruvate, and lactate; whereas the serum levels of, acetate, and 3-hydroxybutyrate were found to be significantly elevated. Contrary to expectation, the serum levels of mannose found to be insignificantly different (SSc: 46.0 µM and NC: 43.8 µM). The majority of these metabolic alterations found to be well consistence with those reported previously in other clinical metabolomics studies [1,2]. The mean values for circulatory levels of Myoinositol in SSC patients and NC subjects were 29.2 µM and 67.4 µM, respectively. Further, the circulatory MMR levels were estimated as [Myo-inositol/ Mannose] and compared between the study groups. Like myo-inositol, the MMR levels were also found to be significantly decreased in SSc patients (mean value =0.71 ± 0.32 compared to 1.85 ± 0.73 as observed for NC subjects. Conclusion: The altered levels of myo-inositol and other endogenous metabolites in the sera of SSc patients suggested abnormalities in myo-inositol metabolism in SSc patients and future studies are warranted to underscore its role in the pathobiology of scleroderma. References: [1] Federica Murgia, Silvia Svegliati, Simone Poddighe, Milena Lussu, Aldo Manzin, Tatiana Spadoni, Colomba Fischetti, Armando Gabrielli, and Luigi Atzori. "Metabolomic profile of systemic sclerosis patients." Scientific Reports 8, no. 1 (2018): 7626. [2]. Thomas Bögl, Franz Mlynek, Markus Himmelsbach, Norbert Sepp, Wolfgang Buchberger, and Marija Geroldinger-Simić. "Plasma metabolomic profiling reveals four possibly disrupted mechanisms in systemic sclerosis." Biomedicines 10, no. 3 (2022): 607.

{"references": ["Durga P Misra, Mohit K Rai, Sakir Ahmed, Durgesh Dubey, Atul Rawat, Dinesh Kumar and Vikas Agarwal \"Nitrosodimethylamine and methylamine, novel metabolites in scleroderma, may drive dual pathogenic processes of endothelial cell apoptosis and fibrosis\" Rheumatology (April 2019), 58 (Supplement 3) (DOI: 10.1093/rheumatology/kez107.045). (Impact Factor:5.245; ISSN: 1462-0324; eISSN 1462-0332; Publisher: Oxford Academic)", "Mohit Kumar Rai, Sakir Ahmed, Durgesh Dubey, Atul Rawat, Durga P Misra, Dinesh Kumar, Vikas Agarwal, \"Methylamine, a novel metabolite, may be a potential biomarker and player in the pathogenesis of scleroderma\" Indian J Rheumatol (2018), vol 13 (Issue 6) Suppl S2:79-92 (Conference Paper selected for Oral Presentation in IRACON 2018: OPB0008) (DOI: 10.4103/0973-3698.247335) (Impact Factor: 0.271; ISSN: 0973-3698; Publisher: Wolters Kluwer \u2013 Medknow)", "Sakir Ahmed, Mohit Kumar Rai, Durgesh Dubey, Atul Rawat, Dinesh Kumar, Durga Prasanna Misra, and Vikas Agarwal, \"Nuclear Magnetic Resonance Based Metabolomics Study Identifies Highly Discriminatory Metabolites in 87 Systemic Sclerosis Patients\", Arthritis and Rheumatology (2017), 69 (S10), ID:1723. (DOI:10.1002/art.40321 (PDF Version) ACR Journal page. Impact Factor: 9.002; Online ISSN:2326-5205; Publisher: Wiley Online Library (An Offical Journal of The American College of Rheumatology)"]}

This is a proof-of-concept study demonstrating the potential of NMR based Clinical metabolomics analysis for unraveling the metabolic pathway alterations in Systemic Sclerosis (SSc). Metabolic ratios can be useful as diagnostic markers in certain clinical conditions and in this clinical metabolomics study, we proposed that myoinositol-to-mannose ratio may serve as a surrogate marker for improving the clinical diagnosis and disease management strategies in SSc. However, future studies on independent patient cohorts are required to underscore its role in the pathobiology of SSc.