Measuring Vitamin D3 Metabolic Status, Comparison between Vitamin D Deficient and Sufficient Individuals

The main branch of vitamin D3 metabolism involves several hydroxylation reactions to obtain mono-, di- and trihydroxylated metabolites, including the circulating and active forms—25(OH)D3 and 1,25(OH)2D3, respectively. However, most clinical trials strictly target the determination of 25(OH)D3 to offer a view of the metabolic status of vitamin D3. Due to the growing interest in expanding this restricted view, we have developed a method for measuring vitamin D3 metabolism by determination of vitamin D3, 25(OH)D3, 24,25(OH)2D3, 1,25(OH)2D3 and 1,24,25(OH)3D3 in human plasma. The method was based on SPE–LC–MS/MS with a large volume injection of human plasma (240 µL). Detection of di- and trihydroxymetabolites, found at the picogram per milliliter level, was attained by the combined action of high preconcentration and clean-up effects. The method allows obtaining information about ratios such as the known vitamin D metabolite ratio (24,25(OH)2D3/25(OH)D3), which can provide complementary views of vitamin D3 metabolic status. The method was applied to a cohort of obese patients and a reference cohort of healthy volunteers to find metabolic correlations between target analytes as well as differences as a function of vitamin D levels within and between cohorts

Espectrometría de masas aplicada a la metabolómica clínica

Clinical metabolomics is a field of metabolomics research focused on the human metabolome study and its potential association to physiological status of the organism under study. Metabolomics has a great potential for clinical purposes such as diseases diagnosis and characterization. In fact, a global interest is recently emerging on the use of clinical metabolomics towards precision medicine. However, this area is still under study because each step from the analytical process can influence results reliability. Hence, the hypothesis preceding the establishment of objectives and the research planning was the existence of many challenges that need to be addressed. Some of those challenges are analytical methods development, its suitability for application to real clinical samples and generated results interpretation. Therefore, the global objective of this Doctoral Thesis research was to develop new analytical improvements to strengthen clinical metabolomics research. The proposals included in this Thesis research were based on liquid and gas chromatography (LC and GC) coupled to mass spectrometry (MS). In addition to all previously mentioned research developed by the student under the supervision of senior researchers with experience in the field, she received formation, participated in activities, and parallelly contributed to other investigation studies.La metabolómica clínica es un área de la metabolómica que se centra en el estudio del metaboloma humano y su posible asociación con el estado fisiológico del organismo en estudio. Por ello, esta subdisciplina tiene mucho potencial para el diagnóstico y la caracterización de enfermedades. De hecho, actualmente, está creciendo un interés global especial por la posible contribución de la metabolómica clínica a la medicina de precisión. Sin embargo, es un área aún en estudio puesto que todas las etapas del proceso analítico afectan en menor o mayor medida a la fiabilidad de los resultados obtenidos. De ahí que la motivación bajo la que se desarrolló esta Tesis Doctoral sea el gran número de desafíos que requieren abordarse en el área de la metabolómica clínica. Concretamente, el desarrollo metodológico de análisis, la viabilidad de su aplicación en muestras clínicas reales y la interpretación de los resultados generados. Por tanto, el objetivo general de esta Tesis Doctoral fue desarrollar nuevas mejoras analíticas que contribuyan a fortalecer la metabolómica clínica. Estas mejoras se focalizaron en las distintas etapas del proceso analítico y se basaron en el empleo de análisis metabolómico dirigido y no dirigido. Estas propuestas se aplicaron a muestras clínicas reales para garantizar su aplicabilidad y viabilidad. El factor común de estas propuestas es la técnica de detección empleada, espectrometría de masas (MS), en combinación con cromatografía de líquidos y gases (LC y GC, respectivamente). En general, el objetivo final de esta Tesis Doctoral fue la transferencia del conocimiento y la formación de la estudiante predoctoral durante su postgrado. Además de toda la investigación desarrollada durante este postgrado supervisada por investigadores con experiencia en el área, la estudiante ha participado en otras actividades formativas y contribuido en otras investigaciones paralelas a la Tesis Doctoral

Lipidomics signature in post-COVID patient sera and its influence on the prolonged inflammatory response

Background The ongoing issues with post-COVID conditions (PCC), where symptoms persist long after the initial infection, highlight the need for research into blood lipid changes in these patients. While most studies focus on the acute phase of COVID-19, there's a significant lack of information on the lipidomic changes that occur in the later stages of the disease. Addressing this knowledge gap is critical for understanding the long-term effects of COVID-19 and could be key to developing personalized treatments for those suffering from PCC. Methods We employed untargeted lipidomics to analyze plasma samples from 147 PCC patients, assessing nearly 400 polar lipids. Data mining (DM) and machine learning (ML) tools were utilized to decode the results and ascertain significant lipidomic patterns. Results The study uncovered substantial changes in various lipid subclasses, presenting a detailed profile of the polar lipid fraction in PCC patients. These alterations correlated with ongoing inflammation and immune response. Notably, there were elevated levels of lysophosphatidylglycerols (LPGs) and phosphatidylethanolamines (PEs), and reduced levels of lysophosphatidylcholines (LPCs), suggesting these as potential lipid biomarkers for PCC. The lipidomic signatures indicated specific anionic lipid changes, implicating antimicrobial peptides (AMPs) in inflammation. Associations between particular medications and symptoms were also suggested. Classification models, such as multinomial regression (MR) and random forest (RF), successfully differentiated between symptomatic and asymptomatic PCC groups using lipidomic profiles. Conclusions The study's groundbreaking discovery of specific lipidomic disruptions in PCC patients marks a significant stride in the quest to comprehend and combat this condition. The identified lipid biomarkers not only pave the way for novel diagnostic tools but also hold the promise to tailor individualized therapeutic strategies, potentially revolutionizing the clinical approach to managing PCC and improving patient care.This work was supported by the Spanish Agencia Estatal de Investigación (AEI) and the ERDF (RTI2018–098795-A-I00, PID2019–111327GB-I00, PID2019–111373RB-I00, PID2022-141534OB-I00 and PDC2022–133402-I00), by Xunta de Galicia and the ERDF (ED431F 2020/05, ED431B 2022/36 and Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09). The work of I.B. was financially supported in part by grants from the Departamento de Educación, Política Lingüística y Cultura del Gobierno Vasco [IT1456–22] and by the Ministry of Science and Innovation through BCAM Severo Ochoa accreditation [CEX2021–001142-S/MICIN/AEI/10.13039/501100011033] and through project [PID2020–115882RB-I00/AEI/10.13039/501100011033] funded by Agencia Estatal de Investigación and acronym “S3M1P4R" and also by the Basque Government through the BERC 2022–2025 program. We would like to thank Paula García Fandiño for her efficient and crucial work in gathering samples for this study