A sensitive, accurate and precise method was developed for the quantification of a large number of organic acids in human urine by GC-MS/MS. The analytes were selected based on their role as key metabolic intermediates; intermediates of Krebs cycle, fatty acid oxidation, glycolysis, down-stream metabolites of neurotransmitter synthesis and degradation, metabolites indicative of nutritional deficiencies, byproducts of microbial activity in the gastrointestinal tract (GI) etc. The most efficient sample preparation protocol was selected based on tests for extraction with different solvents such as MTBE and ethyl acetate under acidic conditions, whereas finally a more general protocol was applied with methanol. Regarding derivatization, methoxyamine with MSTFA, 1% TMCS was applied. The method was extensively validated, including stability study, ensuring accurate determination of the studied organic acids in human urine. Proof of its utility was exhibited in a set of samples from human volunteers. The method can find wide applicability in the context of metabolomics for clinical or nutritional studies.
Diabetes mellitus (DM) and coronary artery disease (CAD) constitute inter-related clinical entities. Biomarker profiling emerges as a promising tool for the early diagnosis and risk stratification of either DM or CAD. However, studies assessing the predictive capacity of novel metabolomics biomarkers in coexistent CAD and DM are scarce.
This post-hoc analysis of the CorLipid trial (NCT04580173) included 316 patients with CAD and comorbid DM who underwent emergency or elective coronary angiography due to acute or chronic coronary syndrome. Cox regression analyses were performed to identify metabolomic predictors of the primary outcome, which was defined as the composite of major adverse cardiovascular or cerebrovascular events (MACCE: cardiovascular death, myocardial infarction, stroke, major bleeding), repeat unplanned revascularizations and cardiovascular hospitalizations. Linear regression analyses were also performed to detect significant predictors of CAD complexity, as assessed by the SYNTAX score.
After a median 2-year follow up period (IQR = 0.7 years), the primary outcome occurred in 69 (21.8%) of patients. Acylcarnitine ratio C4/C18:2, apolipoprotein (apo) B, history of heart failure (HF), age > 65 years and presence of acute coronary syndrome were independent predictors of the primary outcome in diabetic patients with CAD (aHR = 1.89 [1.09, 3.29]; 1.02 [1.01, 1.04]; 1.28 [1.01, 1.41]; 1.04 [1.01, 1.05]; and 1.12 [1.05–1.21], respectively). Higher levels of ceramide ratio C24:1/C24:0, acylcarnitine ratio C4/C18:2, age > 65 and peripheral artery disease were independent predictors of higher CAD complexity (adjusted β = 7.36 [5.74, 20.47]; 3.02 [0.09 to 6.06]; 3.02 [0.09, 6.06], respectively), while higher levels of apoA1 were independent predictors of lower complexity (adjusted β= − 0.65 [− 1.31, − 0.02]).
In patients with comorbid DM and CAD, novel metabolomic biomarkers and metabolomics-based prediction models could be recruited to predict clinical outcomes and assess the complexity of CAD, thereby enabling the integration of personalized medicine into routine clinical practice. These associations should be interpreted taking into account the observational nature of this study, and thus, larger trials are needed to confirm its results and validate them in different and larger diabetic populations.
ST-elevation myocardial infarction (STEMI) remains one of the leading causes of mortality worldwide. The identification of novel metabolic and imaging biomarkers could unveil key pathophysiological mechanisms at the molecular level and promote personalized care in patients with acute coronary syndromes. We studied 38 patients with STEMI who underwent primary percutaneous coronary intervention and thrombus aspiration. We sought to correlate serum ceramide levels with micro-CT quantified aspirated thrombus volume and relevant angiographic outcomes, including modified TIMI thrombus grade and pre- or post-procedural TIMI flow. Higher ceramide C16:0 levels were significantly but weakly correlated with larger aspirated thrombus volume (Spearman r = 0.326, p = 0.046), larger intracoronary thrombus burden (TB; p = 0.030) and worse pre- and post-procedural TIMI flow (p = 0.049 and p = 0.039, respectively). Ceramides C24:0 and C24:1 were also significantly associated with larger intracoronary TB (p = 0.008 and p = 0.001, respectively). Receiver operating characteristic analysis demonstrated that ceramides C24:0 and C24:1 could significantly predict higher intracoronary TB (area under the curve: 0.788, 95% CI: 0.629-0.946 and 0.846, 95% CI: 0.706-0.985, respectively). In conclusion, serum ceramide levels were higher among patients with larger intracoronary and aspirated TB. This suggests that quantification of serum ceramides might improve risk-stratification of patients with STEMI and facilitate an individualized approach in clinical practice.
The paper reports the development of a multianalyte method and its application in metabolic profiling of biological fluids. The initial aim of the method was the quantification of metabolites existing in cell culture medium used in in-vitro fertilization (IVF) and in other biological fluids related to embryo growth. Since most of these analytes are polar primary metabolites a hydrophilic interaction liquid chromatography system was selected. The analytical system comprised Ultra-HPLC with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes. Mobile phase and gradient elution conditions were studied with the aim to achieve the highest coverage of metabolic space in a single injection namely the largest number of analytes that could be detected and quantified. The developed method provides absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, aminoacids, nucleotides, organic acids, and amines. Following validation, the method was applied for the metabolic profiling of hundreds of samples of spent culture medium originating from human IVF procedures and several hundreds of biological samples such as amniotic fluid, human urine and blood serum from pregnant women. The bioanalytical end-point was to provide assistance in the process of embryo transfer and improving IVF success rates but also to provide insight in complications related to the subsequent embryo growth during pregnancy.
A hydrophilic interaction liquid chromatography (HILIC)-MS/MS profiling method was developed for the efficient separation and quantification of small polar molecules, mostly primary metabolites. The method was based on an ultrahigh performance liquid chromatography (UHPLC) separation system coupled with ESI mass spectrometry on a triple quadrupole mass spectrometer, operating in both positive and negative ionisation mode using rapid polarity switching. With the developed method quantitation of 135 compounds belonging in four major classes of polar compounds (sugars, aminoacids, organic acids and amines) was achieved in a single run of 30 min. The method was applied to grape extracts from different varieties and provided information on primary metabolite content. Multivariate statistical analysis was applied using the concentrations found, with the aim of investigating the differences in metabolite profiles. Classification of grapes according to their skin colour was carried out using principle component analysis based on the concentration variation of a number of the metabolites studied.
Recent studies support that acylcarnitines exert a significant role in cardiovascular disease development and progression. The aim of this metabolomics-based study was to investigate the association of serum acylcarnitine levels with coronary artery disease (CAD) severity, as assessed via SYNTAX Score. Within the context of the prospective CorLipid trial (NCT04580173), the levels of 13 circulating acylcarnitines were accurately determined through a newly developed HILIC-MS/MS method in 958 patients undergoing coronary angiography in the AHEPA University Hospital of Thessaloniki, Greece. Patients presenting with acute coronary syndrome had significantly lower median acylcarnitine C8, C10, C16, C18:1 and C18:2 values, compared to patients with chronic coronary syndrome (p = 0.012, 0.007, 0.018, 0.011 and <0.001, respectively). Among CAD subgroups, median C5 levels were significantly decreased in unstable angina compared to STEMI (p = 0.026), while median C10, C16, C18:1 and C18:2 levels were higher in stable angina compared to STEMI (p = 0.019 p = 0.012, p = 0.013 and p < 0.001, respectively). Moreover, median C2, C3, C4 and C8 levels were significantly elevated in patients with diabetes mellitus (p < 0.001, [removed] 22 (p = 0.002, 0.024, 0.044 and 0.012, respectively), indicating a potential prognostic capability of those metabolites and of the ratio C4/C18:2 for the prediction of CAD severity. In conclusion, serum acylcarnitines could serve as clinically useful biomarkers leading to a more individualized management of patients with CAD, once further clinically oriented metabolomics-based studies provide similar evidence.