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INTRODUCTION: Diabetes (T3cDM) secondary to chronic pancreatitis (CP) arises due to endocrine dysfunction and metabolic dysregulations. Currently, diagnostic tests are not available to identify patients who may progress from normoglycemia to hyperglycemia in CP. We conducted plasma metabolomic profiling to diagnose glycemic alterations early in the course of disease. METHODS: Liquid chromatography-tandem mass spectrometry was employed to generate untargeted, targeted plasma metabolomic profiles in CP patients, controls (n=445) following TRIPOD guidelines. Patients were stratified based on glucose tolerance tests following ADA guidelines. Multivariate analysis was performed using PLS-DA to assess discriminatory ability of metabolites among stratified groups. COMBIROC, logistic regression were employed to derive biomarker signatures. AI-ML tool(Rapidminer) was employed to verify these preliminary results. RESULTS: Ceramide, lysophosphatidylethanolamine, phosphatidylcholine, lysophosphatidic acid, phosphatidylethanolamine, carnitine and lysophosphatidylcholine discriminated T3cDM CP patients from healthy controls with AUC 93%(95%CI 0.81-0.98, p<0.0001), integration with pancreatic morphology improved AUC to 100%(95%CI 0.93-1.00, p<0.0001). Lysophosphatidic acid, phosphatidylinositol and ceramide discriminated non-diabetic CP with glycemic alterations (pre-diabetic CP);AUC 66% (95% CI 0.55-0.76, p=0.1),integration enhanced AUC to 74%,(95% CI 0.55-0.88,p=0.86). T3cDM was distinguished from pre-diabetic by lysophosphatidic acid, phosphatidylinositol and sphinganine(AUC 70%; 95%CI 0.54-0.83,p=0.08), integration improved AUC to 83% (95%CI 0.68-0.93,p=0.05). CombiROC cutoff identified 75% and 78% prediabetes in validation 1 and 2 cohorts. Random forest algorithm assessed performance of integrated panel demonstrating AUC of 72% in predicting glycemic alterations. DISCUSSION: We report for the first time that a panel of metabolites integrated with pancreatic morphology detects glycemia progression prior to HbA1c in CP patients.
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Acetylation of amino acids is important in the molecular biology and biochemistry because they are part of several metabolic pathways. N-acetyl amino acids can form through degradation of N-acetyl proteins or direct acetylation of amino acids by specific enzymes. Acetylation of α-amino acids can be either on the alpha -NH2 or on the side-chain functional group, where both the acetyl products are isomeric and can show different biological roles. Theoretically, all proteinogenic α-amino acids are expected to undergo acetylation and they can be a part of metabolome. Thus, it is essential to detect and identify all the possible acetylated products of α-amino acids for untargeted metabolomics studies. In this study, it is aimed to synthesize and characterize all acetylated products of natural α-amino acids. A total of 20 Nα -acetyl amino acids (1-20), six side-chain acetyl amino acids (21-26), and six diacetyl amino acids (27-32) were synthesized and characterized by liquid chromatography-electrospray ionizationtandem mass spectrometry (LC-ESI-MS/MS). The [M + H]+ ions of all the acetyl amino acids were subjected to MS/MS experiments to obtain their structural information. Apart from the expected loss of (H2 O + CO) (immonium ions), most of the acetyl amino acids specifically showed loss of H2 O and loss of a ketene (C2 H2 O) from [M+H]+ ions. The side-chain acetyl amino acids showed a clear-cut structure specific fragment ions that enabled easy differentiation from their isomeric Nα -acetyl amino acids. The other isomeric/isobaric acetyl amino acids could also be easily distinguished by their MS/MS spectra. The MS/MS of immonium ions of the acetyl amino acids were also studied, and they included characteristic products reflecting the structures of parent Nα -acetyl and side-chain acetyl amino acids.
