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- Dried blood spots in clinical lipidomics: optimization and recent findingsPublication . Ferreira, Helena Beatriz ; Guerra, Inês M. S. ; Melo, Tânia ; Rocha, Hugo; Moreira, Ana S. P. ; Paiva, Artur ; Domingues, M. RosárioDried blood spots (DBS) are being considered as an alternative sampling method of blood collection that can be used in combination with lipidomic and other omic analysis. DBS are successfully used in the clinical context to collect samples for newborn screening for the measurement of specifc fatty acid derivatives, such as acylcarnitines, and lipids from whole blood for diagnostic purposes. However, DBS are scarcely used for lipidomic analysis and investigations. Lipidomic stud ies using DBS are starting to emerge as a powerful method for sampling and storage in clinical lipidomic analysis, but the major research work is being done in the pre- and analytical steps and procedures, and few in clinical applications. This review presents a description of the impact factors and variables that can afect DBS lipidomic analysis, such as the type of DBS card, haematocrit, homogeneity of the blood drop, matrix/chromatographic efects, and the chemical and physi cal properties of the analyte. Additionally, a brief overview of lipidomic studies using DBS to unveil their application in clinical scenarios is also presented, considering the studies of method development and validation and, to a less extent, for clinical diagnosis using clinical lipidomics. DBS combined with lipidomic approaches proved to be as efective as whole blood samples, achieving high levels of sensitivity and specifcity during MS and MS/MS analysis, which could be a useful tool for biomarker identifcation. Lipidomic profling using MS/MS platforms enables signifcant insights into physiological changes, which could be useful in precision medicine.
- Mitochondrial fatty acid β-oxidation disorders: from disease to lipidomic studies—a critical reviewPublication . Guerra, Inês M. S.; Ferreira, Helena B.; Melo, Tânia; Rocha, Hugo; Moreira, Sónia; Diogo, Luísa; Domingues, Maria Rosário; Moreira, Ana S. P.Fatty acid oxidation disorders (FAODs) are inborn errors of metabolism (IEMs) caused by defects in the fatty acid (FA) mitochondrial β-oxidation. The most common FAODs are characterized by the accumulation of medium-chain FAs and long-chain (3-hydroxy) FAs (and their carnitine derivatives), respectively. These deregulations are associated with lipotoxicity which affects several organs and potentially leads to life-threatening complications and comorbidities. Changes in the lipidome have been associated with several diseases, including some IEMs. In FAODs, the alteration of acylcarnitines (CARs) and FA profiles have been reported in patients and animal models, but changes in polar and neutral lipid profile are still scarcely studied. In this review, we present the main findings on FA and CAR profile changes associated with FAOD pathogenesis, their correlation with oxidative damage, and the consequent disturbance of mitochondrial homeostasis. Moreover, alterations in polar and neutral lipid classes and lipid species identified so far and their possible role in FAODs are discussed. We highlight the need of mass-spectrometry-based lipidomic studies to understand (epi)lipidome remodelling in FAODs, thus allowing to elucidate the pathophysiology and the identification of possible biomarkers for disease prognosis and an evaluation of therapeutic efficacy
- Cardiac molecular remodeling by anticancer drugs: Doxorubicin affects more metabolism while mitoxantrone impacts more autophagy in adult CD-1 male micePublication . Brandão, Sofia Reis; Reis-Mendes, Ana; Duarte-Araújo, Margarida; Neuparth, Maria João; Rocha, Hugo; Carvalho, Félix; Ferreira, Rita; Costa, Vera MarisaDoxorubicin (DOX) and mitoxantrone (MTX) are classical chemotherapeutic agents used in cancer that induce similar clinical cardiotoxic effects, although it is not clear if they share similar underlying molecular mechanisms. We aimed to assess the effects of DOX and MTX on the cardiac remodeling, focusing mainly on metabolism and autophagy. Adult male CD-1 mice received pharmacologically relevant cumulative doses of DOX (18 mg/kg) and MTX (6 mg/kg). Both DOX and MTX disturbed cardiac metabolism, decreasing glycolysis, and increasing the dependency on fatty acids (FA) oxidation, namely, through decreased AMP-activated protein kinase (AMPK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content and decreased free carnitine (C0) and increased acetylcarnitine (C2) concentration. Additionally, DOX heavily influenced glycolysis, oxidative metabolism, and amino acids turnover by exclusively decreasing phosphofructokinase (PFKM) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETFDH) content, and the concentration of several amino acids. Conversely, both drugs downregulated autophagy given by the decreased content of autophagy protein 5 (ATG5) and microtubule-associated protein light chain 3 (LC3B), with MTX having also an impact on Beclin1. These results emphasize that DOX and MTX modulate cardiac remodeling differently, despite their clinical similarities, which is of paramount importance for future treatments.
- Lipid profile variability in children at different ages measured in dried blood spotsPublication . Ferreira, Helena Beatriz; Melo, Tânia; Rocha, Hugo; Paiva, Artur; Domingues, Pedro; Domingues, M. RosárioDried blood spot (DBS) is a minimally invasive sampling technique that has several advantages over conventional venipuncture/arterial blood sampling. More recently, DBS has also been applied for lipidomics analysis, but this is an area that requires further research. The few works found in the literature on lipidomics of DBS samples performed the analysis in adult samples, leaving pediatric ages unmapped. The objective of this study was to assess the variability of the lipid profile (identified by high-resolution C18 RP-LC-MS/MS) of DBS at pediatric age (0-10 days, 2-18 months, and 3-13 years) and to identify age-related variations. The results revealed that the lipidomic signature of the three age groups is significantly different, especially for a few species of neutral lipids and phosphatidylcholines. The main contributors to the differentiation of the groups correspond to 3 carnitine (Car), 2 cholesteryl ester (CE), 2 diacylglycerol (DG), 2 triacylglycerol (TG), 3 phosphatidylcholine (PC), 1 ether-linked PC, 1 phosphatidylethanolamine (PE), 1 ether-linked PE and 1 phosphatidylinositol (PI) species, all with statistically significant differences. Additionally, lipid species containing linoleic acid (C18:2) were shown to have significantly lower levels in the 0-10 days group with a gradual increase in the 2-18 month, reaching the highest concentrations in the 3-13 year group. The results of this study highlighted the adaptations of the lipid profile at different pediatric ages. These results may help improve understanding of the evolution of lipid metabolism throughout childhood and should be investigated further.
- Lipidome plasticity in medium- and long-chain fatty acid oxidation disorders: Insights from dried blood spot lipidomicsPublication . Guerra, Inês M. S.; Rocha, Hugo; Moreira, Sónia; Gaspar, Ana; Ferreira, Ana C.; Santos, Helena; Rodrigues, Esmeralda; Chaves, Paulo Castro; Melo, Tânia; Goracci, Laura; Domingues, Pedro; Moreira, Ana S.P.; Domingues, M. Rosário; Carvalho de Azevedo Rocha, Hugo DanielFatty acid (FA) oxidation disorders (FAOD) are characterized by accumulation of specific acylcarnitines (CAR) and FA and can lead to potentially severe complications. In this study, dried blood spots (DBS) combined with LC-MS lipidomics analysis were used to assess lipidome plasticity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), compared to control (CT) individuals, for screening potential prognostic biomarkers. Statistically significant variations were found in CAR, biomarkers for FAOD diagnosis, but other lipid species showed variations depending on the FAOD. Common changes in all FAOD included a few phosphatidylcholine (PC) lipid species, notably an up-regulation of LPC 16:1, possibly associated with a higher risk of cardiovascular disease (CVD). In LCHADD and VLCADD, an up-regulation of odd-chain PC (PC 33:0, PC 35:4 and PC 37:4) was observed. VLCADD exhibited higher levels of odd-chain TG, while LCHADD showed an up-regulation of ceramide (Cer 41:2;O2). The increase in the Cer class has been found to be associated with neurodegeneration and may contribute to the risk of developing this condition in LCHADD. An upregulation of ether-linked PC lipid species, including plasmenyl (known as endogenous antioxidants), was observed in MCADD, possibly as a response to increased oxidative stress reported in this disorder. Overall, DBS combined with lipidomics effectively pinpoints the lipid plasticity in FAOD, highlighting potential specific biomarkers for disease prognosis that warrant further validation for their association with the development of FAOD comorbidities.
- NMR-based urinary biomarkers in pediatric primary mitochondrial disorders and chronic kidney disease: shared mitochondrial dysfunction, diverging biosignaturesPublication . Coelho, Margarida Paiva; Rodrigues, João E.; Costa, Teresa; Dias, Aureliano; Graça, Inês C. R.; Rocha, Hugo; Vilarinho, Laura; Martins, Esmeralda; Gil, Ana M.; Carvalho de Azevedo Rocha, Hugo DanielRenal involvement is a recognized feature of primary mitochondrial disorders (PMD), either at presentation or during the disease course. Simultaneously, the metabolomic fingerprint of chronic kidney disease (CKD) is often associated with underlying mitochondrial dysfunction. This study aimed to characterize urinary metabolic signatures in genetically confirmed paediatric PMD without chronic kidney disease, comparing them to healthy controls, suspected (unconfirmed) mitochondrial disease (SMD), and non-mitochondrial CKD. We performed untargeted 1 H NMR metabolomic profiling of 76 urine samples from 51 paediatric patients and 10 healthy controls. PMD patients in acute decompensation or known CKD and statistical outlier samples were excluded. Final comparisons included genetically confirmed PMD without CKD (n=13), SMD (n=10), non-mitochondrial CKD (n=28; 17 at stages 1–2 and 9 at stages 3–5), and healthy controls (n=10). Spectral data were analyzed using multivariate statistical approaches—including principal component analysis (PCA) and partial least squares–discriminant analysis (PLS-DA)—as well as univariate methods with Mann-Whitney U for pairwise group metabolite comparison. Urinary metabolic profiles of PMD patients differed from healthy controls and CKD patients. Multivariate analysis revealed a strong discriminative ability between PMD and controls (Q² = 0.53) and advanced CKD (Q2=0.78). Compared to controls, PMD patients had increased levels of Krebs cycle intermediates (cis-aconitate, fumarate and succinate), creatine, tryptophan, homovanillate (HVA) and hypoxanthine, as well as decreased histidine. All, except fumarate and histidine, remained discriminative when comparing PMD to CKD. CKD patients showed a diverging metabolomic fingerprint with 1-methylnicotinamide (MNA) and 2-hydroxyisobutyrate emerging as potential CKD-specific biomarkers, effectively discriminating between CKD stage 3–5 from earlier stages and controls. A five-metabolite panel comprising cis-aconitate, fumarate, HVA, tryptophan and histidine achieved high diagnostic performance for identifying PMD, with an area under the curve (AUC) of 0.836 (PMD vs. controls) and AUC=0.783 across all groups. This biosignature integrates metabolites involved in distinct functional domains including energy metabolism, neurotransmitter turnover and amino acid metabolism and renal handling. Urinary metabolomic profiling by NMR revealed a distinct biosignature in pediatric PMD patients without renal involvement, characterized by elevated levels of tryptophan, HVA, and Krebs cycle intermediates, and diminished histidine. The divergent changes in tryptophan, histidine and HVA, suggest a mitochondria-specific metabolic phenotype in PMD. These findings support the use of urinary NMR metabolomics as a non-invasive tool for biomarker discovery in PMD and highlight the potential of integrated, multiparametric metabolic fingerprints for diagnostic refinement and patient stratification.