Valéria Cristina Nogueira1,2, Gisele Silvestre da Silva3, Valzimeire do Nascimento de Oliveira2, Maria Izabel Florindo Guedes2, Guilherme J. Zocolo4,5
Metabolomics is an emerging omics science that investigates the profile of small molecules in biological systems and has proven to be a promising tool for the discovery of biomarkers associated with several chronic diseases, including type 1 diabetes (T1D). T1D is a chronic autoimmune condition that promotes metabolic alterations, including dysregulation of lipid and glucose metabolism. This study aimed to characterize the urinary metabolomic profile of adult patients with T1D in comparison to healthy controls.
A total of 76 urine samples were collected—39 from adults with T1D and 37 from healthy individuals. Samples were analyzed using an untargeted metabolomics approach based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MSE). Statistical analyses included univariate tests (t-test), multivariate analysis (PCA and PLS-DA), fold change calculations, and pathway exploration using MetaboAnalyst and KEGG databases.
A total of 63 urinary metabolites were annotated, and 37 showed statistically significant differences between the groups. Among them, five metabolites stood out as the most discriminating: triacylglycerol (TAG 46:4), which was found at lower levels in T1D patients, and C4-acylcarnitine, C5-acylcarnitine, carnosine, and proline-betaine, which were found at elevated levels in the urine of patients with T1D. These compounds are associated with energy production, fatty acid oxidation, glucose metabolism, antioxidant activity, and dietary intake.
TAG 46:4 plays a central role in lipid storage and energy supply and was absent or significantly reduced in the urine of T1D patients. This finding may reflect metabolic adaptations resulting from healthier lifestyle habits adopted by the T1D group, including lower intake of calories, fats, and carbohydrates and higher fiber consumption. In contrast, increased urinary levels of acylcarnitines (C4 and C5) suggest intensified fatty acid β-oxidation and mitochondrial stress, which are commonly observed in individuals with impaired glucose homeostasis.
Carnosine, a bioactive dipeptide with antioxidant, antiglycation, and anti-inflammatory properties, was found in elevated urinary concentrations in T1D patients—marking, to our knowledge, the first report of this metabolite in urine from T1D adults. Proline-betaine, a dietary metabolite derived mainly from citrus fruits and legumes, also appeared at higher levels and is known to correlate with healthier dietary patterns and improved insulin sensitivity. Both carnosine and proline-betaine may serve as indicators of reduced risk for T1D complications and better metabolic control.
This study demonstrates, for the first time, increased urinary levels of carnosine and proline-betaine in adult T1D patients and identifies a set of metabolites related to lipid and glucose metabolism, oxidative stress, and dietary habits. The use of UPLC-QTOF-MSE allowed the precise detection of metabolite variations, and the use of urine as a non-invasive sample type proved valuable for monitoring systemic metabolic changes. These findings contribute to the understanding of T1D progression and support future research to validate these metabolites as potential non-invasive biomarkers for disease monitoring and early intervention.
Agradecimentos: We thank FIOCRUZ, EMBRAPA, CAPES, and CNPq for their essential support in the development of this project. Their contributions through infrastructure, scientific collaboration, and funding were fundamental to the execution of the analyses and the progress achieved in this study.