Background - The preservation of concentrated red blood cells (RBCs) is crucial for their vitality and functionality. During storage, various metabolic changes occur, such as the loss of ATP, diphosphoglycerate, and potassium, as well as oxidative damage to proteins, lipids, and carbohydrates. Lipids, the most abundant class of metabolites in cells, are essential for maintaining the integrity and function of the red blood cell membrane. This study aims to evaluate molecular changes and identify potential biomarkers to optimise RBC transfusion by analysing metabolic and lipid profiles of preserved RBC at different time points. Materials and methods - Thirty samples from 10 freshly collected RBC units from 10 donors, were collected at three time points: collection, after 20 days, and after 40 days, and stored properly. The RBCs were then centrifugated, and the pellet was stored at −80°C for the metabolomic and lipidomic analyses using liquid chromatography coupled with mass spectrometry. Results - Lipidomic analysis revealed a progressive reduction in major membrane phospholipid classes, including phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, and phosphatidic acids. At increased resolution, molecular-species-level analysis identified selective depletion of arachidonate-containing phosphatidic acid species alongside a concomitant increase in free arachidonic acid, indicating active phospholipid remodeling during storage. Metabolomic profiling confirmed previously described alterations in glycolysis, energy metabolism, and redox-related pathways and revealed coordinated changes in metabolites linked to phospholipid synthesis and turnover. Discussion - These findings confirm established features of RBC storage lesions while providing refined insight into membrane lipid remodeling at the molecular species and fatty acid building block levels. The integrated lipidomic and metabolomic approach highlights specific lipid signatures associated with RBC membrane aging, which may complement existing frameworks for studying storage-associated cellular changes and support future investigations linking molecular remodelling to functional RBC quality metrics.
Martina Nicolini
- Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy
Sandra Buratta
- Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy; Centro di Eccellenza Materiali Innovativi Nanostrutturati (CEMIN), University of Perugia, Perugia, Italy
https://orcid.org/0000-0002-9875-0138
Lorena Urbanelli
- Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy; Centro di Eccellenza Materiali Innovativi Nanostrutturati (CEMIN), University of Perugia, Perugia, Italy
https://orcid.org/0000-0003-0621-8476
Mauro Marchesi
- Trasfusional Unit, Azienda Ospedaliera di Perugia, Perugia, Italy
Laura Berchicci
- Trasfusional Unit, Azienda Ospedaliera di Perugia, Perugia, Italy
Anair Graciela Lema Fernandez
- Hematology and Bone Marrow Transplantation Unit, Laboratory of Molecular Medicine (CREO), Department of Medicine and Surgery, University of Perugia, Perugia, Italy
https://orcid.org/0000-0002-5653-8307
Carlotta Nardelli
- Hematology and Bone Marrow Transplantation Unit, Laboratory of Molecular Medicine (CREO), Depart-ment of Medicine and Surgery, University of Perugia, Perugia, Italy
https://orcid.org/0000-0002-3167-4177
Cristina Mecucci
- Hematology and Bone Marrow Transplantation Unit, Laboratory of Molecular Medicine (CREO), Depart-ment of Medicine and Surgery, University of Perugia, Perugia, Italy
Carla Emiliani
- Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy; Centro di Eccellenza Materiali Innovativi Nanostrutturati (CEMIN), University of Perugia, Perugia, Italy
Paolo Gorello
- Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy; Centro di Eccellenza Materiali Innovativi Nanostrutturati (CEMIN), University of Perugia, Perugia, Italy
https://orcid.org/0000-0003-2473-778X
, Francesco Arcioni
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